glucose induces sensitivity to oxygen deprivation and alters

GLUCOSE INDUCES SENSITIVITY TO OXYGEN DEPRIVATION AND ALTERS

GENE EXPRESSION IN Caenorhabditis elegans

Anastacia M. Garcia, B.S.

Dissertation Prepared for the Degree of

DOCTOR OF PHILOSOPHY

UNIVERSITY OF NORTH TEXAS

August 2015

APPROVED:

Pamela A. Padilla, Major Professor Michael Allen, Committee Member Kent Chapman, Committee Member Shane L. Rea, Committee Member Aaron Roberts, Committee Member Art Goven, Chair of the Department of

Biological Sciences Costas Tsatsoulis, Interim Dean of the

Toulouse Graduate School

Garcia, Anastacia M., Glucose Induces Sensitivity to Oxygen Deprivation and

Alters Gene Expression in Caenorhabditis elegans. Doctor of Philosophy (Biology),

August 2015, 125 pp., 7 tables, 22 illustrations, references, 186 titles.

An organisms’ diet represents an exogenous influence that often yields colossal

effects on long-term health and disease risk. The overconsumption of dietary sugars for

example, has contributed to significant increases in obesity and type-2 diabetes; health

issues that are costly both economically and in terms of human life. Individuals who are

obese or are type-2 diabetic often have compromised oxygen delivery and an increased

vulnerability to oxygen-deprivation related complications, such as ischemic strokes,

peripheral arterial disease and myocardial infarction. Thus, it is of interest to identify the

molecular changes glucose supplementation or hyperglycemia can induce, which

ultimately compromise oxygen deprivation responses. By utilizing the Caenorhabditis

elegans genetic model system, which is anoxia tolerant, I determined that a glucose-

supplemented diet negatively impacts responses to anoxia and that the insulin-like

signaling pathway, through fatty acid and ceramide biosynthesis and antioxidant activity,

modulates anoxia survival. Additionally, a glucose-supplemented diet induces lipid

accumulation. Use of RNA-sequencing analysis to compare gene expression responses

in animals fed either a standard or glucose-supplemented diet revealed that glucose

impacts the expression of genes involved with multiple cellular processes including lipid

and carbohydrate metabolism, stress responses, cell division, and extracellular

functions. Several of the genes we identified are homologous to human genes that are

differentially regulated in response to metabolic diseases, suggesting that there may be

conserved gene expression responses between C. elegans supplemented with glucose

and a diabetic and/or obese state observed in humans. These findings support the utility

of C. elegans to model specific aspects of the T2D disease process (e.g., glucose-

induced sensitivity to oxygen deprivation) and identify potentially novel regulators of

common complications seen in hyperglycemic and T2D patients (e.g., macrovascular

complications).

Copyright 2015

by

Anastacia M. Garcia

ii

ACKNOWLEDGEMENTS

Many thanks to my primary investigator, Dr. Padilla for allowing me this

opportunity, and to Drs. A. Roberts, M. Allen, K. Chapman and S. L. Rea for serving on

my committee for all of these years. I’d also like to thank Drs. S. Brumbley, R.K. Azad

and B.K. McFarlin for valuable technical assistance and data analysis. I am also grateful

for Drs. J. Watts and S. J. Lee and the entire Caenorhabditis elegans Genetics Stock

Center (CGC), which is funded by National Institutes of Health Office of Research

Infrastructure Programs (P40 OD010440) for C. elegans or E. coli strains used in this

work. This work was supported by a grant from the National Science Foundation

(CAREER 0747391) to P.A. Padilla and by support from the University of North Texas

Office of Research and Economic Development, College of Arts and Sciences, and

Department of Biological Sciences to P.A. Padilla and R.K. Azad. I also greatly

appreciate the Toulouse Graduate School for awarding me with the Thesis and

Dissertation Fellowship, which has allowed me to complete my degree without the

added stress of teaching during my final year. Additionally, I am eternally grateful for the

many friends and colleagues that have encouraged me along the way, especially to my

colleagues in the lab, M.L. Ladage and S. D. King and principally L.S. Toni who has

kept me balanced. Ultimately I am indebted to my gracious family who has

supported and encouraged my pursuit of this dream. I love you Mom and Dad, to

the moon and back.

iii

TABLE OF CONTENTS

Page

CHAPTER 1 DIET, HUMAN HEALTH, OXYGEN DEPRIVATION AND Caenorhabditis

elegans ............................................................................................................................ 1

Diet and Human Health ............................................................................................... 1

Physiological and Metabolic Responses to Excess Sugar ........................................... 2

C. elegans as a Model to Study Oxygen Deprivation................................................... 9

History of C. elegans ................................................................................................ 9

C. elegans and Oxygen Deprivation ....................................................................... 11

Anoxia Tolerance in Wild-Type Adult C. elegans ................................................... 13

Genetic Factors Influence Anoxia Tolerance in Adult C. elegans........................... 15

Environmental Changes Influence Anoxia Tolerance in Adult C. elegans .............. 24

Research Focus......................................................................................................... 26

CHAPTER 2 GLUCOSE IMPACTS OXYGEN DEPRIVATION RESPONSE AND

SURVIVAL IN C. elegans .............................................................................................. 28

Introduction ................................................................................................................ 28

Glucose Supplementation in C. elegans ................................................................ 28

Glucose Supplementation and Oxygen Deprivation in C. elegans ......................... 30

Results ....................................................................................................................... 31

A Glucose Supplemented Diet Impacts Stress Responses .................................... 31

A Glucose Supplemented Diet Increases Whole Worm Glucose Concentrations .. 33

Glucose and Fructose Supplementation Induce Anoxia Sensitivity........................ 35

E. coli Food Source Impacts Glucose- Induced Anoxia Sensitivity ........................ 36

iv

Glucose Supplementation Induces Lipid Accumulation .......................................... 37

CHAPTER 3 GLUCOSE-INDUCED ANOXIA SENSITIVITY IS MODULATED VIA

INSULIN SIGNALING, LIPID BIOSYNTHESIS AND ANTIOXIDANT ACTIVITY ........... 39

Introduction ................................................................................................................ 39

Insulin Signaling ..................................................................................................... 39

Lipid Biosynthesis .................................................................................................. 40

ROS and Antioxidants ............................................................................................ 43

Results ....................................................................................................................... 45

Glucose-Induced Anoxia Sensitivity is Modulated by Insulin Signaling .................. 45

Glucose-Induced Anoxia Sensitivity is Modulated by Insulin Signaling via Lipid

Biosynthesis ........................................................................................................... 51

Glucose-Induced Anoxia Sensitivity is Modulated by Insulin Signaling Via

Antioxidant Activity ................................................................................................. 58

Glucose Supplementation Induces Lipid Accumulation .......................................... 58

CHAPTER 4 GLUCOSE INDUCES SIGNIFICANT GENE EXPRESSION CHANGES IN

C. elegans ..................................................................................................................... 61

Introduction ................................................................................................................ 61

Transcriptional Changes Associated With the Progression of Type 2 Diabetes ..... 61

Transcriptomics ...................................................................................................... 62

Results ....................................................................................................................... 63

Glucose Supplementation Induces Significant Transcriptional Changes ............... 63

CHAPTER 5 MATERIALS AND METHODS ................................................................. 79

C. elegans Strains and Culture Conditions ................................................................ 79

v

Bacterial Strains and Culture Conditions ................................................................... 80

Anoxia and Paraquat Exposure ................................................................................. 80

Oil Red O Staining ..................................................................................................... 81

Fatty Acid Supplementation ....................................................................................... 82

Protein Quantification ................................................................................................ 83

RNA Isolation ............................................................................................................. 83

RNA-Sequencing Analysis ......................................................................................... 84

Quantitative RT-PCR ................................................................................................. 86

Statistical Analysis ..................................................................................................... 87

CHAPTER 6 DISCUSSION ........................................................................................... 88

Glucose Induces Sensitivity to Oxygen Deprivation................................................... 89

Glucose-Induced Anoxia Sensitivity is Influenced by the Intestinal Microbiome .... 92

Glucose-Induced Anoxia Sensitivity is Modulated by Insulin Signaling via Multiple

Pathways ............................................................................................................... 94

Glucose Significantly Alters Gene Expression in C. elegans ..................................... 99

REFERENCES ............................................................................................................ 104

vi

CHAPTER 1

DIET, HUMAN HEALTH, OXYGEN DEPRIVATION AND Caenorhabditis elegans

Diet and Human Health

An organisms’ diet represents an exogenous influence that often yields colossal

effects on long-term health and disease risk. The overconsumption of dietary sugars

(and decreased caloric expenditure) that the United States has seen in recent years,

has contributed to significant increases in obesity and type-2 diabetes, health issues

that are costly both in terms of economics and human life (Koning and Malik 2011;

Ogden et al. 2014). To illustrate, approximately two-thirds of American adults are

considered overweight or obese (body mass index (BMI) of ≥ 25 or ≥30 kg/m2

respectively) and 16.9% of children and adolescents are considered obese (Frisbee

2007). Worldwide, the prevalence of obesity has nearly doubled since 1980, and in

2014, 11% of adult men and 15% of adult women were obese (World Health

Organization (WHO), Global status report on noncommunicable diseases 2014). The

accumulation of visceral adiposity is linked to several chronic health conditions such as

hyperglycemia, insulin-insensitivity and type-2 diabetes (T2D) that are becoming

increasingly more common. For example, T2D alone affects more than 29 million

Americans today, and contributes to over 230,000 deaths annually (Centers for Disease

Control (CDC), National Diabetes Statistics report 2014). This epidemic however is not

limited to the US, the WHO has recently defined T2D as a progressive worldwide

epidemic, and the global prevalence of diabetes in 2014 was estimated to be 9% (World

Health Organization (WHO), Global status report on noncommunicable diseases 2014).

Additionally, the risk of death in adults with T2D is 50% higher than in their non-diabetic

1

counterparts (CDC, 2014). In addition to the significant toll on human health and life

expectancy that T2D poses, there is also an associated economic burden. The total

estimated medical costs associated with T2D in the US for example, exceed 170 billion

USD in 2012, while indirect associated costs (disability, work loss and premature death)

totaled more than 60 billion USD (CDC 2014, American Diabetes Association (ADA)

2012). Thus, together T2D cost the US more than 245 billion dollars annually and is the

7th leading cause of death in the US (CDC 2014) and 8th leading cause of death in the

world (WHO 2014).

T2D is a well-known cause of premature death and disability, as patients with

T2D have a greater risk of vascular complications such as ischemic strokes, heart

disease, myocardial infarction and peripheral arterial diseases (Sonestedt et al. 2012;

Stratton et al. 2000). In fact, vascular diseases are the principal causes of death in

people with T2D. Moreover, the etiology of late-onset, chronic metabolic diseases such

as obesity and T2D are multifactorial and involve complex interactions between an

individual’s genetic makeup, dietary habits, activity level and fetal programming. Thus, it

is of interest to understand both the genetic and dietary factors that together, can

convert an individual’s healthy metabolism into a pathological one.

Physiological and Metabolic Responses to Excess Sugar

Metabolism involves a variety of chemical reactions that form an intricate network

of pathways and cycles necessary for an organism’s proper development, reproduction

and response to environmental cues. Metabolism generally encompasses virtually every

cellular process from DNA replication to transcription, translation and small molecule

2

reactions within each cell. The intermediate step within cells in which nutrients are

metabolized and converted into cellular energy and other vital cellular components

plays a central role in metabolic processes. The common dietary sugar, glucose for

example, is an essential component of intermediary metabolism and energy production,

and its intra- and extracellular levels are tightly regulated within an organism. In

humans, low blood glucose concentrations (hypoglycemia) can result in seizures,

weakness and death, while chronically high blood glucose levels can result in cardiac

and peripheral vascular disease. Thus, carefully regulated blood glucose levels are

optimally maintained within a narrow window, with optimal levels being at or near 90

mg/dL or 5 mM/L (Table 1.1) and seemingly minimal increases or decreases from this

optimal level can have dramatic impacts on physiology, phenotype and gene expression

(Szablewski 2011). Glucose enters the body in many forms, including

monosaccharaides, disaccharides, and polysaccharides and also via the breakdown or

of substrates stored within organs. Humans and other mammals can adjust glucose

levels by secreting two hormones, insulin and glucagon, that work in opposition to one

another. Glucose (and other monosaccharaides) are transported across the intestine

and into the liver and other organs, and pancreatic β-cells are induced to secrete

insulin. Insulin secretion in turn facilitates the import of glucose into other tissues

including muscle and adipose tissues. These simple sugars, can then be converted into

fatty acids, amino acids and glycogen, or can be oxidized by the various catabolic

pathways in cells, depending on the specific needs of an organism (Szablewski 2011;

Busik et al. 2002; Brownlee 2001).

3

A continual excess of glucose, however, can have deleterious effects on

metabolic functions, and impair the body’s ability to properly maintain glucose

homeostasis. Chronic disruptions in energy homeostasis and metabolic regulation can

elicit negative and life-long and phenotypic consequences in individuals (Brownlee

2001; Sonestedt et al. 2012). An excess of dietary sugars over time fore example, can

lead to impaired insulin secretion from pancreatic β-cells and/or a decreased biological

impact of insulin (insulin resistance) in peripheral tissues. Insulin resistance results in

decreased cellular glucose uptake, increased blood glucose levels (hyperglycemia) and

ultimately T2D (Table 1.1) (Brownlee 2001; Szablewski 2011).

Mean Blood

Glucose Normal Hyper Diabetic

mg/dL 80 115 150 180 215 250 280 315 350

mM/L 4.5 6.4 8.4 10 11.9 13.9 15.5 17.5 19.5

Table 1.1. Mean Blood Glucose Levels in Normal, Hyperglycemic (Hyper) and Diabetic Humans. Mean blood glucose levels are given in units of mg/dL of blood or mM/L of blood (American Diabetes Association).

Oxygen Deprivation

Chronic hyperglycemia and T2D often result in significant pathological changes

including vascular changes, that with time will induce kidney damage, coronary artery

disease, cerebral vascular disease, peripheral artery disease, blindness and poor

wound healing. These alterations in vascular homeostasis due to cellular dysfunction

are the main features of diabetic complications, and this pro-inflammatory state

ultimately leads to complications that over time can be life threatening (Giugliano et al.

4

1996; Creager et al. 2003; Beckman et al. 2013; Paneni et al. 2013; Stratton et al. 2000;

Vinik and Flemmer 2002; Nuzum and Merz 2009). A hallmark of diabetic macrovascular

disease is accelerated atherosclerosis (the narrowing of arterial walls) of the aorta and

other medium to large sized arteries, resulting in vascular abnormalities, vascular

dysfunction and ultimately the disruption of oxygenated blood flow to vital organs and

tissues throughout the body (Figure 1.1). Thus, the main adverse effect of vascular

disease on the body is oxygen deprivation.

It is thought that various metabolic abnormalities associated with diabetes

(hyperglycemia, increased free fatty acids and insulin resistance) further contribute to

macrovascular dysfunction. The effects of hyperglycemia on the vascular system occur

through multiple common mechanisms. For example, abnormal stimulation of the

hexosamine signaling (N-acetyl-glucosamine protein modification), protein kinase C and

polyl pathways, have been implicated in the promotion of cellular dysfunction and

damage as result of hyperglycemia. Additionally, the pathologic effects of advanced

glycation end product accumulation, nitric oxide inhibition and subsequent impaired

vasodilation, smooth muscle cell dysfunction, the overproduction of endothelial growth

factors, increased release of free fatty acids, chronic inflammation, enhanced platelet

aggregation (clotting), and enhanced reactive oxygen species generation have also

been shown to have a role in the progression of diabetic vascular disease (Cade 2008;

Yamagishi and Imaizumi 2005; Girach, Manner, and Porta 2006). Moreover, structural

changes in the vascular system also promote the progression of atherosclerosis. This

process begins with the progressive formation of plaques, via lipid accumulation.

Atherosclerosis is thought to occur more quickly as result of chronic inflammation and

5

injury to the arterial wall. In response to endothelial injury and inflammation, oxidized

lipids from low-density lipoproteins (LDLs) accumulate in the endothelial wall of

arteries. This accumulation of lipids results in a cascade of events, ultimately leading to

the formation of a lipid-rich atherosclerotic lesion with a tough fibrous cap. If a plaque

becomes unstable, it is susceptible to rupture and/or erosion, thus resulting in an acute

vascular event (Rader 2007). Thus, changes in specific genetic pathways as well as

changes in vascular structure and function contribute to the accelerated progression of

vascular disease in hyperglycemic and T2D patients.

While the progression of plaques begins early in life and precedes

hyperglycemia, T2D provides an environment particularly favorable to accelerated

atherosclerosis, given that its hallmarks include increased LDL, hypertriglyceridemia,

and reduced high-density lipoprotein (HDL) (Fowler 2008). In fact, vascular diseases

and their complications (vascular ischemic events) are the principal causes of death in

people with T2D (Figure 1.1). In T2D patients for example, macrovascular events such

as stroke, myocardial infarction, and peripheral arterial disease occur earlier than in

their non-diabetic counterparts and the underlying pathologies are often more severe

(Vinik and Flemmer 2002). Atherosclerotic peripheral arteries can compromise lower

limb function, often resulting in amputation (Frisbee 2007). In fact, 70% of all non-

traumatic lower limb amputations are in T2D patients (Jain et al. 2010). Moreover, while

hyperglycemia at the time of acute ischemia has been shown to adversely affect

prognosis, the mechanisms for the hyperglycemia-exacerbated damage are not

completely understood. Additionally, lowering the risk for macrovascular complications

is complex, and often involves more than simply decreasing glucose concentrations.

6

Given that compromised blood flow and oxygen deprivation are central to the pathology

of vascular disease, a more thorough understanding of the role metabolic abnormalities

have on oxygen deprivation responses is of interest.

Figure 1.1. Macrovascular Complications as a Result of Type-2 Diabetes. Various organs, tissues and limbs throughout the body are impacted by oxygen deprivation as a result of compromised blood flow. Compromised blood flow to the central nervous system can result in serious and sometimes fatal ischemic events. Compromised blood flow to the coronary arteries can result in chronic and severe heart conditions and compromised blood flow to peripheral arteries can result in compromised lower limb healing and function (ADA, Make the Link! Diabetes, Heart Disease and Stroke Initiative 2009).

Oxygen Deprivation in Non-Human Metazoans

While it is clear that oxygen availability is a critical factor for the survival of

humans, there are a few species that have adapted mechanisms that afford them the

ability to survive short or prolonged periods of oxygen deprivation, some at both

7

embryonic and adult stages. Some such organisms include, but are not limited to, the

brine shrimp (Artemia franciscana), turtle (Chrysemys picta belli), fruit fly (Drosophila

melanogaster), zebra fish (Danio rerio) soil nematode (Caenorhabditis elegans) and

killifish (Austrofundulus limnaeus) (Foe and Alberts 1985; Padilla et al. 2002; Clegg

1997; Podrabsky et al. 2007; Milton and Prentice 2007). The common responses to

oxygen deprivation among these species include a dramatic reduction in overall

metabolism, a reduction in developmental trajectory such as diapause, or entry into an

arrested state such as suspended animation.

In order to cope with low, or no oxygen (hypoxia or anoxia, respectively) in an

environment, each organism has adapted unique strategies within conserved

biochemical pathways, including a few basic adaptations that are common amongst

most oxygen-deprivation tolerant species. Physiologists Hochachka and Somero have

conducted extensive studies and outlined a biological framework for oxygen deprivation-

tolerant organisms and their conserved responses to hypoxia and anoxia at

physiological and cellular levels (Hochachka & Somero 2002). That is, initially, during

early, acute exposure to hypoxia there are three specific and profound physiological

alterations within an organism: first, a global decline in protein biosynthesis, a decline in

membrane permeability across tissues and declined firing frequency in the nervous

system, and finally, entrance into a state of altered metabolism (hypometabolic state)

allowing the demand and supply of ATP to remain in a low, steady state of flux. This is

then followed by a rescue phase in which only a subset of genes are expressed, and

others that are not involved directly with maintaining the physical integrity of the cell (i.e.

protein synthesis, anaerobic metabolism, glycogen storage, cell stabilization, hypoxia,

8

etc.) are down regulated. This model highlights the importance of a steady, low balance

of energy within an organism as well as maintenance at the cellular level of the genome,

proteome and general cell structure and function.

C. elegans as a Model to Study Oxygen Deprivation

The use of genetic model systems provides an in vivo means by which we can

experiment and elucidate specific biological phenomena that are conserved across

species, including phenomena associated with human diseases. The use of model

systems allows us to understand both normal and abnormal biological processes, and

to potentially improve the quality and duration of life in higher organisms. Model

organisms are powerful in that we can identify appropriate (not necessarily identical)

phenotypes of interest to study in a way that is ethical and feasible.

History of C. elegans

C. elegans was originally isolated in Bristol, England, and its use as a model

system was pioneered by scientist Sydney Brenner in the early 1970’s, while he

searched to find an appropriate and simple metazoan model system to study genetic

interactions, developmental biology, and neuroscience (Brenner 1974). Now, four

decades later, the microscopic nematode continues to serve as a valuable model

system and has proven useful for the examination of fundamental and complex

molecular and biological processes. Its usefulness has been thoroughly demonstrated

by numerous major discoveries and significant findings that have been the outcome of

C. elegans research, many of which resulted in Nobel Prize awards (6 C. elegans

9

related Nobel prizes were awarded in the last decade). C. elegans is a unique

multicellular model system equipped with a nearly invariable developmental program

and cell lineage (Boxem 2006). The relative timing of divisions, the orientation of

division axes, and the final cell fates are all highly reproducible, and the entire cell

lineage from a single cell embryo to the adult has been described. C. elegans was the

first metazoan model to have its genome sequenced, thus, a fully sequenced genome,

the capacity to use a wide range of molecular genetic techniques and the variety of

biological processes that can be studied has propelled the C. elegans model into the

forefront of biological research. To illustrate, many key pathways, biological phenomena

and molecular tools have been further elucidated using this model. Notably, RNA

interference to transiently knockdown gene products, the use of green fluorescent

protein to follow gene expression patterns in vivo, and discovery of the first ever

microRNA were all pioneered using C. elegans (Chalfie & Kain 2006; Fraser et al. 2000;

Jorgensen & Mango 2002; Timmons & Fire 1998). Research using this simple and well-

characterized model is convenient both in terms of time and associated cost. Wild-type

C. elegans have a short, 21-day mean lifespan, a 3-day reproductive cycle and large

brood size. They are reared in standardized and straightforward laboratory conditions

and many genetically manipulated and transgenic strains of C. elegans are available for

a nominal fee from the Caenorhabditis Genetics Center and other commercial

resources. Additionally, its transparent body allows morphological analysis of

abnormalities easily observable on a stereomicroscope, while visualization of

fluorescently tagged proteins is straightforward on a higher-powered compound

microscope.

10

C. elegans have been useful in biological research particularly because of the

overlap in conserved signaling pathways and stress response mechanisms with those in

higher organisms. The C. elegans genome for example, shares approximately 65%

identity to the human genome. C. elegans are also known to be particularly well

equipped to survive stress. In fact, C. elegans have an entire non-obligatory alternative

developmental stage called dauer, which can be induced via starvation, crowding and

other stressful conditions. Dauer larvae are characterized by a thickened cuticle, sealed

pharyngeal orifices, and resistance to desiccation, starvation and a multiple of other

stresses (Golden and Riddle 1984). Dauers enter into a reversible ageless state and

can survive for several months without food or water, and upon exposure to favorable

conditions, can exit dauer and resume normal development into adulthood virtually

indistinguishable from animals who never entered this alternative trajectory (Golden and

Riddle 1984; Cassada and Russell 1975).

C. elegans and Oxygen Deprivation

In humans and most other large animals, oxygen is delivered to cells via

oxygenated blood flow through a complex circulatory system. Thus, the oxygen

concentration at the tissue level is lower than ambient oxygen levels, and there exists

variability in oxygen levels between tissue types (Montgomery 1957; Dyson and Singer

2011). Fluctuations in the environmental availability of oxygen and the metabolic

demands of specific tissue stimulate responses to compensate and increase blood flow.

These mechanisms include vasodilation, increased respiration, and an increased

production of red blood cells. Thus it is difficult experimentally, to control the level of

11

oxygen deprivation in an intact organism. C. elegans on the other hand have several

unique characteristics, which make it a useful model in the investigation of oxygen

deprivation responses. C. elegans, for example do not have a circulatory system, and

instead they rely on diffusion for the delivery of oxygen into cells (Shen and Powell-

Coffman 2003). Therefore, using C. elegans we have a better ability to experimentally

control both genotype and cellular environment. It is important to note however, that the

specific amount of oxygen within the cells/tissues of C. elegans in hypoxic or anoxic

environments is not known, however due to the small size of the adult worm (~1mm),

the level of oxygen deprivation is likely to be more uniform, compared to more complex

vertebrates.

Additionally, C. elegans is a well-suited model for studying oxygen deprivation

since, due to its natural history it is often exposed to varying levels of oxygen. Though

often referred to as “soil-dwelling” nematodes, soil usually lacks enough organic matter

to support large, self-sustaining populations of C. elegans (Félix and Braendle 2010).

Rather, C. elegans recently have been found to prosper in rotting fruits and on rotting

herbaceous plant material in several locations in mainland France (Félix and Duveau

2012). Thus, they likely encounter microenvironments that are deprived of oxygen, and

are well adapted to survive oxygen deprivation. It has also been shown that many

mechanistic details of oxygen deprivation responses in C. elegans are common

amongst other metazoans, including humans (Powell-Coffman 2010).

In C. elegans, and in most other metazoans, a key to sensing low oxygen

concentrations within the environment is the highly conserved hypoxia-inducible factor,

HIF-1. Specific levels of oxygen in the environment however, dictate the exact

12

physiological responses to hypoxia and anoxia that the organism will utilize, and varying

oxygen levels often require specific subsets of fundamental metabolic and signaling

pathways to be active. For example, HIF-1 function in C. elegans is required for their

survival and normal development under specific hypoxic conditions (0.5% and 1%, or

0.5 and 2.0 kPa O2 respectively) however, HIF-1 is not required for the survival of anoxia

(Padilla et al. 2002). For the duration of this work I will focus on the mechanistic and

physiological responses of C. elegans to anoxia (anoxia, <0.001kPa O2, <0.0000075

mmHg).

Anoxia Tolerance in Wild-Type Adult C. elegans

Adult C. elegans, upon exposure to anoxia, enter into a reversible state of

suspended animation. In anoxia, development arrests, as does movement, eating and

reproductive processes. Wild-type (N2) C. elegans, at all stages of development, are

able to survive at least 24 hours of anoxia under standard laboratory conditions, with a

viability ≥90% (Van Voorhies & Ward 2000; Padilla et al. 2002; Hajeri et al. 2005). After

re-exposure to a normoxic environment, the nematode proceeds with normal

development, feeding behavior and reproduction and a significant majority display

normal movement and morphology, that is, they survive and recover in an unimpaired

state. Viability and recovery varies however, among developmental stages when anoxia

exposure is lengthened to 48 or 72 hours and beyond. Wild-type adult hermaphrodite

survival decreases to the point that they are almost completely non-viable when

exposed to long-term anoxia (LTA), defined as 72-hours or more of anoxia exposure

(Figure 1.3). Additionally recovery from LTA takes longer and not all physiological

13

processes appear to resume at the same rate. Moreover, animals that do survive LTA

exposure often display abnormal movement and/or morphology, that is, they survive but

display an impaired phenotype. Taken together, these data illustrate that while C.

elegans can survive some exposure to anoxia, there is an limitation to the duration in

which they can be exposed and remain viable (Padilla et al. 2002; Mendenhall et al.

2006; Mendenhall et al. 2009). Given this dose-dependent anoxia tolerance phenotype,

C. elegans can be useful in identifying factors that lead to both anoxia sensitivity

(inability to survive 24 hours of anoxia) and to anoxia tolerance (ability to survive LTA,

72 hours or more).

Figure 1.3 Viability of C. elegans exposed to anoxia. Survival of nematodes in anoxia for 24 hours (white bars), 48 hours (slashed bars), or 72 hours (black bars) was determined for all stages of development. L1 larvae were either starved or fed before placed into anoxia. Adult hermaphrodites were collected ∼24 hours after the L4 larvae stage. The data shown are representative of three independent experiments, with a total of more

14

than 400 nematodes for each of the postembryonic stages and more than 200 embryos. Error bar represents SD. All experiments were done at 20°C (Padilla et al. 2002).

Genetic Factors Influence Anoxia Tolerance in Adult C. elegans

Anoxia tolerance in C. elegans is multifactorial in nature, and to date, several

specific genes and pathways have been implicated in either conferring sensitivity to

anoxia, or as contributing to an enhanced anoxia tolerance phenotype. The pathway

best characterized in the study of anoxia tolerance in C. elegans is the insulin-signaling

pathway, a highly conserved and well-characterized signaling pathway in C. elegans

and in higher organisms.

Insulin-Signaling

Identification and characterization of the genes that function in the insulin-

signaling pathway have revealed its central roles in the regulation of metabolism,

lifespan, stress responses and dauer formation in C. elegans (Golden and Riddle 1984;

Kenyon et al. 1993; Gottlieb and Ruvkun 1994; Kimura 1997; Tissenbaum and Ruvkun

1998). Under standard laboratory conditions that support growth and development

(20°C, OP50 E. coli diet, non crowding), insulin-like ligands (e.g., ins genes & daf-28)

bind to and activate the insulin/IGF-1 receptor homolog DAF-2, which subsequently

activates a downstream AGE-1/PI3/AKT signaling cascade, which phosphorylates and

inhibits the FOXO transcription factor DAF-16 through its nuclear exclusion. In contrast,

under conditions of nutrient limitation or via a mutation in the insulin receptor daf-2,

there is an overall reduction in insulin-signaling activity, thus decreasing DAF-16

phosphorylation and allowing its translocation into the nucleus where it works together

with other nuclear factors to induce expression of a variety of genes which promote

15

dauer formation, longevity, fat metabolism, stress responses and innate immunity

(Figure 1.4) (Kenyon et al. 1993; McElwee et al. 2006; Hsu et al. 2003; Murphy et al.

2003; Oliveira et al. 2009). Reduction of insulin-signaling via a mutation in daf-2 (and

activation of DAF-16) results in several well-studied phenotypes such as increased

lifespan and resistance to heat and oxidative stress as well as resistance to a myriad of

other stresses (Gems et al. 1998). On the contrary, mutation of the transcription factor

daf-16 results in almost completely opposite phenotypes, including a shortened lifespan

and increased sensitivity to stress. It is important to note that while the human genome

encodes 10 insulin-like peptides, including insulin, insulin-like growth factors, and

relaxins, based upon bioinformatics analysis, C. elegans possess 38 genes predicted to

encode insulin-like peptides (daf-28, ins-1 through ins-37), many are divergent insulin

superfamily members, and many are clustered, indicating recent diversification of the

family (Pierce et al. 2001; Ritter et al. 2013; W. Li, Kennedy, and Ruvkun 2003; Claeys

et al. 2002). Based upon functional analysis, while the ins genes are primarily

expressed in neurons, many are also expressed in a variety of tissues including the

intestine, epidermis, vulva and pharynx. Additionally, it has been shown that some

function as daf-2 agonists (e.g., daf-28, ins-3, ins-6, ins-7, ins-33) while others function

as antagonists of daf-2 (e.g., ins-1, ins-8, ins-17, ins-18) and, based upon structural

predictions and predicted C-peptide cleavage sites typical of mammalian insulin, it is

suggested that ins-1 in C. elegans is most closely related to human insulin (Yutao Chen

and Baugh 2014; Pierce et al. 2001; Ritter et al. 2013; W. Li, Kennedy, and Ruvkun

2003; Murphy et al. 2003; Matsunaga, Nakajima, et al. 2012; Matsunaga, Gengyo-

Ando, et al. 2012; Kawano et al. 2006).

16

Figure 1.4 Insulin Signaling in C. elegans. Under standard conditions the insulin receptor DAF-2 interacts with insulin ligands and works though a conserved PI 3-kinase pathway to negatively regulate DAF-16 by inducing its phosphorylation and nuclear exclusion. Disruption of DAF-2 due to mutation results in alteration of the signaling cascade and translocation of DAF-16 into the nucleus to regulate hundreds of genes.

The reduction-of-function daf-2(e1370) allele that confers the most significant

extension of lifespan in C. elegans, also confers long-term anoxia tolerance

(Mendenhall et al. 2006). Both the extended lifespan and anoxia tolerant phenotypes

are dependent upon activation of daf-16. That is, the null daf-16(mu86) allele

suppresses the ability of daf-2(e1370) animals to survive exposure to LTA. Moreover,

5’- adenylic acid-activated protein kinase (AMPK) has been shown to modulate a

number of signaling pathways, including insulin-like signaling. AMPK functions as a

metabolic checkpoint that becomes activated when AMP levels within the cell are

heightened, a condition indicative of low nutrient levels (Hardie, Hawley, and Scott

2006). Aside from nutrient deprivation, additional stresses have been shown to trigger

17

AMPK activation, such as ischemia/oxygen deprivation, exercise and skeletal muscle

contraction. In C. elegans, aak-2 is one of 2 homologs of the catalytic alpha-subunit of

AMPK, which likely acts in parallel with DAF-16. While the overall rate of survival of

wild-type hermaphrodites and the anoxia-tolerance of daf-2(e1370) is not affected by

the knockdown of aak-2, daf-2(e1370) animals do display a significant increase in their

level of impairment following 72-hour anoxia exposure (LaRue & Padilla 2011). After 96-

hours of anoxia however, aak-2 knockdown suppresses the survival rate in both wild-

type animals grown at high temperature (28°C), and in daf-2(e1370) animals. These

data suggest that AMPK plays a role in anoxia tolerance and is necessary to preserve

an unimpaired, phenotype following exposure to long-term oxygen deprivation.

Metabolism

As previously mentioned, the daf-2(e1370) allele displays a LTA tolerant

phenotype, which is suppressed by mutations in daf-16 (Mendenhall et al. 2006). An

RNAi screen of genes known to be upregulated by DAF-16 led to the identification of

gpd-2 and gpd-3 as suppressors of the daf-2(e1370) anoxia tolerant phenotype. These

genes are 2 of 4 isoforms of the glycolytic enzyme glyceraldehyde-3-phosphate

dehydrogenase (GAPDH). The daf-2(e1370);gpd-2/3(RNAi) animal exposed to one day

of high-temperature anoxia (28°C) or 72 hours of long-term anoxia (at 20°C) have a

significantly reduced viability in comparison to daf-2(e1370) control animals.

Additionally, while the gpd-2/3(RNAi) animals survive short-term anoxia exposure, they

exhibit an impaired phenotype post-recovery. While a number of other glycolytic genes

were subjected to RNAi, no additional glycolytic genes resulted in anoxia sensitivity.

Together these data suggest that the anoxia sensitive phenotype induced via

18

knockdown of gpd-2/3 may be due to something other than merely a change in

glycolytic flux, and that the glycolytic enzymes gpd-2/3 are specifically required for long-

term anoxia tolerance in the context of reduced insulin signaling. It is difficult however to

assess the extent by which each glycolytic enzyme might be impacted via RNAi, thus

the specific aspects of glycolytic reduction and its impact on anoxia survival remain to

be determined.

Reproductive State/ Notch-Signaling

A number of C. elegans mutants that display an extension in lifespan also exhibit

altered rates of fecundity and increased resistance to stress (Gems et al. 1998;

Friedman & Johnson 1987). For example, a sterile germline-less genetic mutant, glp-

1(e2131), displays a significantly increased longevity phenotype. In C. elegans, glp-1

encodes an N-glycosylated transmembrane receptor within the LIN-12/Notch family.

While glp-1(e2141) mutants have a somatic gonad, they are incapable of producing

oocytes and sperm (Crittenden et al. 1994; Mendenhall, LaRue, and Padilla 2006b).

The sterile 1-day old adult glp-1(e2141) animals, similar to daf-2(e1370) animals, are

long-term anoxia tolerant, and display survival rate greater than 95% (Mendenhall et al.,

2009). Additionally, the glp-1(e2141);daf-16(mu86) double mutant survives long-term

anoxia at a high overall rate, suggesting that, unlike daf-2(e1370) animals, the glp-1

anoxia tolerant phenotype is independent of DAF-16 activity (LaRue & Padilla 2011).

However, the glp-1(e2141) tolerant phenotype can be partially suppressed when aak-2

was knocked down via RNAi in the glp-1(e2141);daf-16(mu86) double mutant, therefore

further suggesting that AMPK may play a role in anoxia tolerance, especially in the

context of insulin signaling.

19

Lipid Biosynthesis

Lipids are a diverse group of naturally occurring water-insoluble molecules. The

mammalian lipidome includes major lipid classes such as triacylglycerol, sphingolipids,

vitamins, steroids, ceramide, eicosanoids, phospholipids and glycolipids. The main

biological functions of these lipids include storing energy, signaling, and acting as

structural components of cell membranes. In C. elegans it has been shown in vitro, that

anoxia induces a significant increase in the activity of NADP-dependent isocitrate

dehydrogenase (HCDH; fatty acid oxidation). Given that The HCDH is located in the

mitochondria and is normally involved in the β-oxidation of fatty acids (an oxygen-

requiring process by which fatty acids are broken down), it was hypothesized that under

anoxic conditions, a reversal of β-oxidation results and excretory fatty acids are

synthesized to provide a sink for electrons to maintain proper redox balance for

continued glycolytic activity under anoxic conditions (Paul et al. 2000). Given that in

anoxia, C. elegans is a mixed acid fermenter, these fermentations must be redox

balanced, as their acid end products can build up over time (Butler et al. 2012). It was

later confirmed via GC-MS analysis of the C. elegans exometabolome in anoxia, that

indeed C. elegans employ short-chain unsaturated fatty acids (e.g., crotonyl-CoA, 3-

methylcrotonyl-CoA, methacryl-CoA and trans-2-methyl-but-2-enoyl-CoA) as terminal

electron acceptors to remove reducing equivalents in the form of volatile waste

products. Thus these data suggest that increased fatty acid synthesis in anoxia might

be important for anabolic activity and to regenerate reducing equivalents that are

required for continued glycolytic activity (Butler et al. 2012).

20

Moreover, in many models of anoxia and hypoxia, a link between chronic oxygen

deprivation and fat accumulation has been suggested. For example, oxygen-deprived

tumor cells display an enhanced level of de novo fatty acid biosynthesis and patients

suffering from sleep apnea or chronic cardio-pulmonary diseases (conditions in which

patients are chronically deprived of oxygen) display an increased risk for fat

accumulation (Romero-Garcia et al. 2014; Belamarich et al. 2000; Lam and Ip 2007).

Increases in fatty acid biosynthesis can impact fundamental cellular processes, signal

transduction pathways and gene expression, given the ability of lipid molecules to

modify cytosolic proteins, enzymes, and membrane proteins. Likewise, in C. elegans it

was determined that exposure to anoxia for a period of 4 or more hours, results in up

regulation of the sterol regulatory element-binding protein 1 (SREBP1) homolog sbp-1

(a transcription factor important for proper lipid homeostasis and fat accumulation).

Additionally it was shown that C. elegans display a significant accumulation of total lipid

content following a 24 hour recovery from anoxia, and this accumulation requires sbp-1

activity (Taghibiglou et al. 2009). These data suggest that SREBP1 may function as an

oxygen sensor in the regulation of lipid metabolism and likely plays a role in the

pathogenesis of lipid accumulation following oxygen deprivation. Also, it appears that

changes in lipid metabolism and lipid homeostasis are an essential component of the

response to anoxia.

Lipid Signaling

There is also evidence that lipid signaling has a critical role in oxygen deprivation

response and survival. A number of specific lipids function as known signaling

molecules, and have roles in a variety of signal transduction pathways. For example,

21

ceramide and its metabolites constitute a diverse group of lipids that have been

established as lipid second messengers or metabolic signals, and have known roles as

structural entities of biological membranes and regulators of cellular growth,

differentiation, insulin action and cell death (Goñi and Alonso 2006). Additionally,

imbalances in these lipid-signaling networks can contribute to the pathogenesis of

human diseases. Aligned with this idea is the fact that alterations of ceramide

biosynthesis in C. elegans result in altered anoxia tolerance (Menuz et al. 2009).

Moreover, in C. elegans it was recently demonstrated that worms defective in specific

ceramide and/or sphingomyelin biosynthesis are deficient in mitochondrial surveillance

(a mechanism which detects mitochondrial dysfunction), and the addition of a particular

ceramide species (C24) could rescue these defects (Liu et al. 2014a). Additionally Liu et

al. showed via immunostaining, that ceramide co-localizes with mitochondria, especially

during mitochondrial dysfunction. Thus it is likely that ceramides have a role in the early

detection of mitochondrial dysfunction and might act by marking specific dysfunctional

mitochondrial domains. While ceramides might act as either signaling molecules

themselves, or as components of cellular structures necessary for other signals to

emerge (e.g., components of lipid rafts), Liu et al. hypothesize that ceramides are likely

working as signaling molecules, since ceramide synthesis was induced by mitochondrial

insult, and ceramides were localized to the site of injury.

C. elegans possess 3 ceramide synthase genes, hyl-1, hyl-2 and lagr-1. A loss of

function hyl-2(gnv1) allele and the deletion hyl-2(tm2031) allele confer sensitivity to 48

hours of anoxia (Menuz et al. 2009). Mutation of the related ceramide synthase hyl-1,

on the other hand, confers increased LTA tolerance, and it was determined that hyl-1

22

deletion mutants display an increased level of hyl-2 mRNA (Menuz et al. 2009; Tedesco

et al. 2008b). It was also shown that HYL-1 is not a sufficient substitute for HYL-2 in hyl-

2 deficient worms, as hyl-2(gnv1);hyl-1::GFP animals remain sensitive to 48 hours of

anoxia. HYL-1 and HYL-2 have complementary specificities for fatty acyl chains, and C.

elegans lacking functional hyl-2 display fewer ceramide (Cer) and sphingomyelin (SM)

species with C20 to C22 fatty acyl chains, and more with C24 to C26 compared to wild-type

animals. By contrast hyl-1 deficient C. elegans expressed more C20 to C22 Cer and SMs

compared to wild-type worms, but a similar amount with C24 to C26 fatty acyl chains.

Thus, efficient synthesis of C20 to C22 Cer and SM species requires HYL-2 whereas

synthesis of species containing C16 to C18 and C24 to C26 fatty acid residues requires

HYL-1. Additionally, LAGR-1 (much like HYL-1) primarily contributes to the synthesis of

long-chain fatty acid containing SM and Cer species (Mosbech et al. 2013). These data

together suggest that anoxia tolerance likely requires one or more specific C20 to C22

ceramides and/or sphingomyelin species, that are either specifically or preferentially

synthesized by HYL-2 (Menuz et al. 2009). Although ceramides have been implicated in

ischemic responses in humans, the functional role that ceramides play in anoxia

survival is not well understood (Novgorodov et al. 2008). Ceramides have been reported

previously to be effectors of kinases and phosphatases in various biological processes.

It is likely that during anoxia, the role of key ceramide species is their interaction with

specific molecules integrated into other cellular pathways.

Moreover, Menuz et al. further demonstrated that hyl-2 and daf-2 might interact

genetically to influence anoxia survival. For example, daf-2(el370);hyl-2(gnv1) double

mutant worms survived 48 hours of anoxia significantly better than hyl-2(gnv1) controls,

23

but less than daf-2(e1370) controls. Thus, it appears that at least with respect to anoxia,

ceramide (HYL-2) and insulin signaling (DAF-2) are acting in parallel pathways that

mutually influence one another. Additionally, the interaction between ceramides and

insulin signaling appears to be conserved across species, given that in humans and

other mammals, it has been demonstrated that ceramides influence several distinct

intermediates in the insulin-signaling pathway and have a role in the induction of insulin

resistance (Boon et al. 2013; P. J. Larsen and Tennagels 2014; Xia, Morley, and

Scherer 2014; Holland et al. 2007; Lopez et al. 2013). Thus, the specific role of

ceramides in oxygen deprivation, mitochondrial dysfunction and insulin signaling is of

interest.

Environmental Changes Influence Anoxia Tolerance in Adult C. elegans

Among other physiological concerns, low oxygen conditions can limit available

energy stores that are necessary for metabolism and proper maintenance of

concentration gradients and critical cellular structures. In fact a great number of anoxia-

tolerant species display large reserves of glycogen that can be used during anaerobic

glycolysis for this type of maintenance. Using carminic acid, a fluorescent dye used to

detect glycogen and trehalose stores, La Rue et al. found that carbohydrate levels in C.

elegans correlated with varying levels of long-term anoxia tolerance. Additionally

animals displayed decreased carbohydrate levels following exposure to long-term

anoxia (LaRue & Padilla 2011). Thus, the level of available carbohydrates during anoxia

likely contributes to anoxia survival and post-anoxia recovery in C. elegans and these

stores are likely utilized during anoxia exposure. Along those same lines, LaRue et al.

24

provided evidence that the particular environmental conditions in which C. elegans are

exposed to preceding anoxia exposure, can precondition for an enhanced LTA survival

phenotype. For example, when animals were exposed to various environmental pre-

conditioning regimens their levels of stored carbohydrates and anoxia tolerance were

altered. Wild-type C. elegans cultured and fed under standard laboratory conditions (a

diet of OP50 E. coli and reared at 20°C) are sensitive to long-term anoxia, however,

when raised at 25°C and fed the HT115 strain of E. coli, there is a significant increase in

survival and an increase in survivors with an unimpaired phenotype. Although animals

grown at 25°C on the standard OP50 diet also survive long-term anoxia, they exhibit an

impaired phonotype upon recovery displaying both motility and tissue morphology

defects. Animals raised on HT115 bacteria at 25°C also displayed a much greater

intensity of carminic acid staining as compared to animals raised at either 20°C on

either food source or on OP50 at 25°C. Additionally, long term anoxia tolerant

genotypes, including daf-2(e1370) and glp-1(e2141), also displayed a high level of

carminic acid staining (regardless of rearing at 25°C or an HT115 food source) prior to

anoxia exposure, thus further suggesting a potential necessity of carbohydrate stores

and their utilization for proper physiological maintenance during anoxia exposure. RNAi

knockdown of a catalytic alpha-subunit of 5’-AMP-activated protein kinase (aak-2)

suppressed this high level of carbohydrate stores in daf-2(e1370) animals, and when

exposed to LTA they displayed an increased level of impaired motility compared to daf-

2(e1370) controls. Furthermore, aak-2 knockdown suppressed the daf-2(e1370) LTA

tolerant phenotype when exposed to extended anoxic stress (96 hours). These data

together suggest that the level of available carbohydrates during anoxia can influence

25

survival and recovery of normal morphology and motility. Additionally, preconditioning at

a higher temperature and feeding with HT115 E. coli may have equipped C. elegans to

better survive prolonged anoxic exposure, at least in part by increasing the amount of

stored carbohydrate available. Further evidence has been shown to support the idea

that metabolic stores are altered in C. elegans fed the HT115 E. coli strain since the

HT115 strain has a higher carbohydrate content than the OP50 strain (Brooks, Liang,

and Watts 2009). Thus, there appears to be a synergistic relationship between thermal

preconditioning and specific food source.

Research Focus

As discussed previously, individuals with obesity or T2D often have compromised

oxygen delivery and an increased vulnerability to oxygen-deprivation related vascular

complications, such as ischemic stroke, macrovascular disease and myocardial

infarction. Moreover, hyperglycemic patients are not only more susceptible to vascular

disease, damage and prognosis of patience following a vascular event is worse. It is not

well understood why, mechanistically, excess glucose increases sensitivity to ischemic

events. Thus, it is of interest to identify the molecular changes glucose supplementation

or hyperglycemia can induce, which in-turn compromise oxygen deprivation responses.

Additionally, given the implicated roles of lipid biosynthesis and signaling in the context

of oxygen deprivation and T2D, it is of particular interest to determine the interplay

between lipid homeostasis, insulin signaling and their contributions to oxygen

deprivation responses. My objective is to use the C. elegans model system to identify

genetic and cellular changes that modulate their response to glucose-supplementation,

26

oxygen deprivation and a combination of the two. To better understand the relationship

between glucose-supplementation on oxygen deprivation, I developed an assay to

analyze the impacts of glucose supplementation on anoxia response and survival in C.

elegans (Chapter 2). Additionally, in order to determine specific genetic pathways that

modulate the C. elegans response to glucose and anoxia, I evaluated the roles of

insulin signaling, fatty acid and ceramide biosynthesis as well as antioxidant activity

(Chapter 3). Finally, to determine how glucose supplementation, prior to anoxia, impacts

C. elegans, in a collaborative effort, I along with M.L. Ladage, R.K. Azad and P.A.

Padilla used RNA-sequencing to compare the gene expression profiles of wild-type

animals fed either a standard or a glucose-supplemented diet (Chapter 4). Together

these experiments suggest that C. elegans can be used as a model to identify specific

molecular mechanisms that modulate glucose-induced sensitivity to oxygen-deprivation.

27

CHAPTER 2

GLUCOSE IMPACTS OXYGEN DEPRIVATION RESPONSE AND SURVIVAL IN

C. elegans*

Introduction

Glucose Supplementation in C. elegans

Several studies have shown that a glucose supplemented diet impacts the

metabolism, development and stress responses of C. elegans, and that the underlying

identified pathways and cellular processes involving glucose metabolism are conserved

in metazoans (Choi 2011; Lee et al. 2009; Mondoux et al. 2011; Mendler et al. 2014;

Forsythe et al. 2006; Hashmi et al. 2013; Kitaoka et al. 2013). C. elegans recently has

been used as a model to evaluate the specific molecular targets affected by

pathological glucose concentrations and they have been implicated as a model to study

glucose toxicity, type-2 diabetes and obesity both inside and outside the context of

insulin signaling. For example, Lee et al. determined that supplementing C. elegans with

glucose at concentrations between 0.05 to 2.0% (2.775 to 111mM) is sufficient to

significantly decrease the lifespan of wild-type adults. They suggest that the lifespan

reduction is a result of FOXO transcription factor DAF-16 inhibition (Lee et al. 2009).

Additionally, Schlotterer et al. corroborated these data and further suggested that C.

elegans can be a suitable model to study glucose toxicity. It was determined that

*Parts of this chapter have been previously published, either in part or in full, from Garcia AM, Ladage ML, Dumesnil DR, Zaman K, Shulaev V, Azad RK, Padilla PA. Glucose Induces Sensitivity to Oxygen Deprivation and Modulates Insulin/IGF-1 Signaling and Lipid Biosynthesis in Caenorhabditis elegans. Genetics 2015. Reproduced with permission from the Genetics Society of America.

28

high glucose conditions also limit C. elegans lifespan by increasing reactive oxygen

species (ROS) formation and increasing the modification of mitochondrial proteins by

advanced glycation end products (AGEs), independent of insulin signaling. Additionally,

Schlotterer et al. found that glucose-induced lifespan reduction could be ameliorated via

up regulation of glyoxylase-1, which acts to detoxify methylglyoxal, and thus prevent

mitochondrial dysfunction (Schlotterer et al. 2009). In a later study, it was found that

human insulin was sufficient to ameliorate glucose-induced lifespan reduction in a

glyoxylase-1 dependent manner. Under high glucose conditions, human insulin reduced

the accumulation of glucose in C. elegans, reduced the formation of ROS and AGEs,

and increased the activity of superoxide dismutase (an antioxidant). The effects of

human insulin are, not surprisingly, mediated through the insulin signaling pathway and

require activity of the FOXO transcription factor daf-16 (Mendler et al. 2014). While

human insulin is an agonist of the mammalian insulin receptor, in C. elegans human

insulin has been only been shown as an antagonist of DAF-2 (Pierce et al. 2001).

Further, Mondoux et al. determined that O-linked-N-acetylglucosamine (O-GlcNAc)

cycling and insulin signaling are both essential components of the C. elegans response

to glucose toxicity under even higher concentrations of glucose (e.g., concentrations at

or above 250mM) (Mondoux et al. 2011c). It was determined that a number of insulin-

dependent processes are altered in response to high glucose, including, fertility,

reproductive timing, and dauer formation. Together these data indicate that the C.

elegans response to glucose includes mechanisms similar to those seen in mammalian

systems (e.g., involvement of the insulin signaling and O-GlcNAc cycling pathways,

induction of ROS and AGEs, and reduced lifespan and fertility), and that the C. elegans

29

model has the potential to aid in the identification of mechanistic details associated with

glucose-induced genetic, cellular and physiological changes. Additionally the potential

exists to provide a better understanding and treatment approach to type-2 diabetes and

its associated complications.

Glucose Supplementation and Oxygen Deprivation in C. elegans

To better understand the relationship between glucose-supplementation and

oxygen deprivation in C. elegans, I developed an assay to analyze the impacts of

glucose supplementation on anoxia response and survival. As discussed earlier, it is

known that hermaphrodite C. elegans at day one of adulthood are able to survive 24

hours of anoxia very well under standard laboratory conditions, 20°C, OP50 E. coli.

Thus, I was interested in determining the impact of glucose supplementation under the

same standard conditions (Figure 2.1). I opted to chronically supplement glucose from

embryo to adulthood, and during exposure to oxygen deprivation.

Figure 2.1. Assay to Analyze the Impacts of Glucose Supplementation on Oxygen-Deprivation Survival in C. elegans. Wild-type hermaphrodites are raised from embryo to day 1 of adulthood either on standard media with E. coli, or on glucose supplemented media with an E. coli. They are exposed to 24 hours of anoxia and given a 24-hour re-oxygenated recovery in normoxia before viability is scored.

30

Results

A Glucose Supplemented Diet Impacts Stress Responses

Glucose Induces Sensitivity to Oxygen Deprivation

Although it is known that both genotype and environment influence oxygen

deprivation responses, less is understood regarding the impact diet has on these

responses (LaRue and Padilla 2011; Mendenhall et al. 2009; Powell-Coffman 2010). I

determined that a glucose-supplemented diet reduces the ability of wild-type, 1 day old

adult C. elegans to survive anoxia (Figure 2.2A). Glucose induces anoxia sensitivity in a

dose dependent manner, as the concentration of glucose increases, the ability of C.

elegans to survive anoxia exposure is decreased (Figure 2.2A). While conducting the

anoxia sensitivity assays, I also examined whether the animal showed an impaired

phenotype (abnormal movement or morphology) after anoxia exposure. While a lower

concentration of glucose (0.0625%) did not reduce anoxia survival rate, it did increase

the level of impairment in animals following anoxia exposure. Since previous C. elegans

studies have shown that supplementation with 0.5% glucose (or 27.75mM) is sufficient

to significantly increase whole worm glucose concentrations and mimic human levels of

hyperglycemia, I chose to use this concentration for further analysis (Lee et al. 2009;

Schlotterer et al. 2009). Animals fed a 0.5% glucose-supplemented diet had a

decreased ability to survive even shorter bouts of anoxia (12-24 hours) (Figure 2.2B).

Glucose Induces Sensitivity to ROS

31

There is evidence that a high-glucose diet increases the level of ROS, and so we

wanted to determine if glucose supplementation increased sensitivity to paraquat-

induced oxidative stress (Schlotterer et al. 2009; Schulz et al. 2007; Mendler et al.

2014). Paraquat is a potent generator of intracellular superoxide, a reactive species of

oxygen (Hassan and Fridovich 1978). We found that adults raised on a 0.5% glucose

supplemented diet were more sensitive to paraquat (Figure 2.2C) indicating that anoxia

may not be the only stress response negatively affected by a glucose-supplemented

diet.

32

Figure 2.2. A Glucose Supplemented Diet Negatively Impacts C. elegans Ability to Survive Anoxia and Paraquat Exposure. (A) A diet supplemented with glucose (>.125%) reduces the ability of C. elegans to survive exposure to 1 day of anoxia. Bar indicates a significant decrease in the number of animals alive in comparison to animals not fed glucose prior to anoxia exposure. The * indicates that animals fed an OP50 only diet had a significant increase in unimpaired animals after anoxia treatment in comparison to all animals fed a glucose diet (1 way ANOVA, Bonferonni Multiple Comparisons, p <0.05; 3 independent experiments, with n ≥ 100, were conducted). (B) Animals fed a glucose-supplemented diet are sensitive to decreased anoxia exposures. The * indicates that there was a significant decrease in survivorship in animals fed a glucose diet prior to anoxia exposure in comparison to animals fed a glucose-supplemented diet and not exposed to anoxia. The bar indicates a significant decrease in unimpaired phenotype in comparison to control animals not fed a glucose-supplemented diet (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.05; 3 independent experiments, with n ≥ 100, were conducted). (C) Animals fed a glucose-supplemented diet are sensitive to the ROS generator paraquat. 1-day old adult animals were placed on OP50 E. coli food containing paraquat or .5% glucose and paraquat. Asterisk denotes a significant difference in percent survival (two-way ANOVA, p<001). For all experiments error bar equals standard deviation.

A Glucose Supplemented Diet Increases Whole Worm Glucose Concentrations

It was my aim to reach a glucose concentration in a C. elegans whole-body

extract of 10–20 mM/L (≥180 mg/dL), resembling the glucose concentrations in diabetic

patients under poor glucose control (Table 1.1). Therefore, given that the addition of just

0.5% glucose is sufficient to induce anoxia sensitivity in wild-type worms, and previous

studies have shown that supplementation with 0.5% glucose (or 27.75mM) is sufficient

to increase whole worm glucose concentrations, I chose to use this concentration for

further analysis. For 0.5% glucose supplementation conditions, 300 μl of glucose

solution at a concentration of 925mM was added to the agar of 10mL NGM plates

(50mg/plate) prior to the addition of E. coli. The glucose was allowed to diffuse for at

least 24 hours into the media, to reach a steady state as described (Schlotterer et al.

2009). Plates were seeded with 300μL of E. coli (OD600nm, 0.6≤0.9) that was spread

evenly across the plate’s entirety. The bacteria solution was allowed to dry completely

33

before animals were placed on the media. Freshly made plates were used within a 7-

day period. To confirm that 0.5% glucose supplementation was sufficient to increase

whole worm glucose concentrations to mimic those of T2D patients, an extract of C.

elegans was prepared by sonication and glucose concentration was analyzed using a

Chemwell-T autoanalyzer. Glucose concentrations of whole worm extracts were

normalized to the amount of total protein present in each sample analyzed. In all

subsequent experiments, C. elegans cultured under glucose supplementation

conditions refers to a glucose concentration of 0.5% in the agar (unless otherwise

stated), because it resulted in a glucose concentration ≥13mmol/L in the C.

elegans whole-body extract (Figure 2.3).

Figure 2.3. A Diet Supplemented With Glucose Increases the Glucose Concentration in Whole Worm Extracts. Animals were raised from embryo to day one of adulthood on NGM plates supplemented with 0.5% glucose and seeded with OP50 E. coli. Glucose was quantified in C. elegans whole-body extracts and normalized to total protein concentration. On average, under glucose supplementation conditions, the glucose concentration in whole-worm extracts was 14 mM/L (250 mg/dL) per mg protein, mimicking blood glucose levels in hyperglycemic humans. On average, C. elegans fed a standard diet had glucose levels below 10 mM/L (174 mg/dL) per mg protein, thus in the range below T2D. (Unpaired t test, p=0.1; 3 independent experiments, with n ≥ 300, were conducted)

34

Glucose and Fructose Supplementation Induce Anoxia Sensitivity

I also determined that C. elegans are sensitive to anoxia when fed a fructose-

supplemented diet, indicating that both glucose and fructose negatively impact oxygen

deprivation responses (Figure 2.4). Since it is possible that glucose is inducing anoxia

sensitivity by altering the osmotic environment in which C. elegans are exposed, I

examined whether or not other carbohydrate molecules that likely alter the osmotic

environment in the same manner, but cannot be readily metabolized by C. elegans,

would also alter anoxia survival. I fed animals a mannitol and a sucrose-supplemented

diet (at the same concentration as glucose and fructose) and determined that animals

fed these carbohydrates are able to survive 24 hours of anoxia similar to un-

supplemented controls. These data suggest that osmotic stress per se does not alter

anoxia survival in adult animals (Figure 2.4). The metabolism of sucrose requires an

enzyme to cleave sucrose into fructose and glucose, which may be rate limiting; thus it

is possible that this is the reason sucrose supplementation did not suppress anoxia

survival.

35

Figure 2.4. A diet supplemented with fructose reduces the ability of C. elegans to survive when exposed to 1 day of anoxia. A diet supplemented with fructose (0.5%) reduces the ability of C. elegans to survive exposure to 1 day of anoxia. Bar indicates a significant decrease in the number of animals alive in comparison to animals not fed glucose prior to anoxia exposure. (1 way ANOVA, Bonferonni Multiple Comparisons, p <0.05; 3 independent experiments, with n ≥ 100, were conducted).

E. coli Food Source Impacts Glucose- Induced Anoxia Sensitivity

The specific type of bacterial food source that they consume can regulate the

lifespan and metabolism of C. elegans. The two most common E. coli strains used

to feed C. elegans are the B-derived strain, OP50, and the K12-derived strain, HT115.

Each of these strains is known to have variations in nutrient composition (Reinke et al.

2010; Brooks et al. 2009). To assess if the commonly used OP50 bacteria’s capacity to

import glucose affected the response C. elegans had to a glucose-supplemented diet,

we used the OP50 E. coli glucose-transporter mutant strain ΔPTS-OP50. The anoxia

sensitivity was not as pronounced in the animals fed 0.5% glucose with the glucose-

transporter mutant strain of E. coli (ΔPTS-OP50), thus suggesting that the uptake

and/or metabolism of glucose by the bacteria also impacts anoxia survival (Figure 2.4).

There was however, a significant decrease in anoxia survival when the animals were

fed a ΔPTS-OP50 diet supplemented with higher concentrations of glucose (2%)

(Figure 2.5). Additionally I determined that glucose had a similarly affected anoxia

survival when animals are fed with the E. coli strains OP50 or with HT115 as the food

source, both strains are able to transport and metabolize glucose (Figure 2.5). These

data indicate that a glucose-supplemented diet alters anoxia survival and that the ability

of the E. coli to transport and metabolize glucose affects this response.

36

Figure 2.5. The E. coli strain and glucose impact anoxia survival. OP50 is the standard C. elegans food; HT115 is the parent strain used for RNAi and ΔPTS-OP50 has a mutation in a glucose transporter. The * indicates that there is a decrease in survival relative to control animals not fed a glucose-supplemented diet. The ** indicates a significant increase in impaired animals after anoxia exposure in comparison to animals not fed a glucose-supplemented diet (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.05; 3 independent experiments, with n ≥ 100, were conducted).

Glucose Supplementation Induces Lipid Accumulation

In C. elegans it has been demonstrated that a high-sugar diet increases lipid

stores, as detected by Nile Red staining (T. Nomura et al. 2010; Zheng et al. 2014).

Using Oil Red O staining to localize neutral lipids, I also observed that N2 wild-type

animals fed a 0.5% glucose-supplemented diet, relative to an OP50 only diet, display

increased lipid droplets throughout the body, and have a significant increase in lipid

droplets in the anterior (head and pharynx) region of the animal (Figure 2.6). These data

suggest that glucose is an obesity-mimetic that compromises stress resistance in C.

elegans.

37

Figure 2.6. A glucose-supplemented diet induces lipid accumulation. Oil Red O staining was used to localize lipids within the animal. (A) Wild-type animals fed a standard diet or a glucose-supplemented diet, were stained with Oil Red O. Representative images of whole animals are shown. (B) Enlarged image of the anterior region of the animals. Lipids can be detected in the intestine, oocyte/germline and pharynx regions. Arrow points to the posterior region of the terminal bulb of the pharynx, near the pharyngeal-intestinal valve. Note that glucose fed animals contain lipid droplets in the pharynx region. There is more Oil Red O staining in glucose-fed animals relative to control-fed animals. (C) The presence or absence of lipids within the anterior region (head and pharynx) of the animal was assayed. At least 10 animals from three independent experiments were randomly imaged and assayed for the presence of lipid droplets in the anterior and pharynx regions. Error bar equals standard deviation; bar indicates there was a significant difference in glucose-fed animals in comparison to OP50 fed control animals (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.05).

38

CHAPTER 3

GLUCOSE-INDUCED ANOXIA SENSITIVITY IS MODULATED VIA INSULIN

SIGNALING, LIPID BIOSYNTHESIS AND ANTIOXIDANT ACTIVITY†

Introduction

Insulin Signaling

Given the central role of insulin signaling in glucose homeostasis, I was

interested in examining the C. elegans insulin signaling pathway and its role in glucose-

induced anoxia sensitivity. As discussed previously, under conditions of nutrient

limitation or via a mutation in the insulin receptor daf-2, the transcription factor DAF-16

is activated and translocated into the nucleus where it works together with other nuclear

factors to induce expression of a variety of genes which promote stress tolerance,

longevity, fat metabolism, and innate immunity (Figure 1.4) (Kenyon et al. 1993;

McElwee et al. 2006; Hsu et al. 2003; Murphy et al. 2003; Oliveira et al. 2009;

Mendenhall et al. 2006). Insulin receptor mutants (daf-2 mutants) for example, display

several well-studied phenotypes, including increased tolerance to anoxia and oxidative

stress, higher amounts of TAG stores and a significantly increased lifespan. Additionally

it has been demonstrated that glucose shortens the lifespan of C. elegans via down

regulation of FOXO transcription factor DAF-16 (S. J. Lee, Murphy, and Kenyon 2009).

Thus, it is of interested to determine what role, if any, insulin signaling plays in the C.

elegans response to anoxia under glucose supplementation conditions.

† Parts of this chapter have been previously published, either in part or in full, from Garcia AM, Ladage ML, Dumesnil DR, Zaman K, Shulaev V, Azad RK, Padilla PA. Glucose Induces Sensitivity to Oxygen Deprivation and Modulates Insulin/IGF-1 Signaling and Lipid Biosynthesis in Caenorhabditis elegans. Genetics 2015. Reproduced with permission from the Genetics Society of America.

39

Lipid Biosynthesis

Fatty Acid Biosynthesis

Moreover, associated with the increased consumption of dietary fats and sugars

in the United States, is an increase in the rates of obesity and metabolic syndrome. Key

players in the regulation of lipid metabolism and fat storage are stearoyl-CoA

desaturases (SCD), also known as Δ9 desaturases. The Δ9 desaturase is a key

enzyme in the de novo lipid biosynthetic pathway, and human studies have identified

genetic variations in SCD1 that are associated with differential body fat distribution,

insulin sensitivity, and the development of metabolic syndrome (Gong et al. 2011;

Warensjö et al. 2007). Additionally, SCDs can also be regulated via dietary

carbohydrates and by hormones such as insulin and leptin (Ntambi 2004).

C. elegans possess three Δ9 desaturases, encoded by fat-5, fat-6 and fat-7, that

are responsible for the formation of monounsaturated fatty (MUFAs) acids from

saturated fatty acids (SFAs) (Figure 3.1) (Brock, Browse, and Watts 2007; J L Watts

and Browse 2000; Shi et al. 2013). MUFAs are the preferred substrates for the

synthesis of triacylglycerol (TAG), as well as for membrane phospholipids and

sphingolipids. Double-mutant strains that combine mutations in fat-5, fat-6, and fat-7,

display distinct fatty acid composition changes and reduced survival rates at low

temperatures. While all of the fat double mutant strains display some fatty acid

composition changes, the fat-6;fat-7 double mutants display the most severe defects in

the production of unsaturated fatty acids. The fat-6;fat-7 double mutants display

significantly reduced levels of TAG stores, significantly decreased levels of

polyunsaturated fatty acids (PUFAs) and increased levels of SFAs. Additionally in the

40

absence of fat-6 and fat-7, unusual C18 PUFAs are synthesized from palmitic acid by the

third C. elegans Δ9 desaturase fat-5. Given the previously identified roles of fatty acids

in anoxia (as terminal electron acceptors and potential signaling molecules) and their

regulation in response to dietary carbohydrates, it is of interest to determine whether or

not specific fatty acid desaturases and their products are involved in the C. elegans

response to anoxia under conditions of glucose supplementation.

Figure 3.1. Fatty Acid Biosynthesis in C. elegans. Arrows in this schematic diagram indicate successive steps in fatty acid biosynthesis by C. elegans, based on previously published data (Brock et al. 2007; Watts 2009). Enzyme activities ("Δn", "n-3" or "n-6" for desaturases) and the implicated genes are indicated beside each arrow, in blue font. Lipids increase in melting temperature with increasing chain length and/or saturation level, additionally, unlike mammals, which require dietary PUFAs to maintain health, C. elegans possess all of the enzymes necessary for their biosynthesis (e.g., omega-3 (n-3) desaturase enzymes), and thus their lipid composition depends on genetics and gene-expression as well as diet (Shmookler Reis et al. 2011).

41

Ceramide Biosynthesis

Further, imbalances in lipid-signaling networks have been implicated in the

pathogenesis of several human diseases, including T2D. In fact, recent studies have

indicated the involvement of ceramides in impaired insulin action, chronic oxidative

stress and the incidence of type 2 diabetes (Larsen and Tennagels 2014; Lopez et al.

2013; Xia et al. 2014; Fiedorowicz et al. 2014). Additionally, specific ceramide species

have been identified as protective during ischemic events (Bhuiyan et al. 2010;

Argraves et al. 2011; Chen et al. 2001). Aligned with this idea is the fact that alterations

of ceramide biosynthesis in C. elegans result in altered anoxia tolerance. As discussed

previously, C. elegans possess 3 ceramide synthase genes, hyl-1, hyl-2 and lagr-1

(Figure 3.2). The deletion hyl-2(tm2031) allele confers sensitivity to 48 hours of anoxia,

and these mutants lacking functional hyl-2 display fewer ceramide (Cer) and

sphingomyelin (SM) species with C20 to C22 fatty acyl chains. Thus, anoxia tolerance in

C. elegans likely requires one or more C20 to C22 of Cer or SM species (Menuz et al.

2009). Ceramides and their metabolites constitute a diverse group of lipids that have

been established as second messengers and metabolic signals, with known roles as

structural entities of biological membranes and regulators of insulin action and cell

death (Goñi and Alonso 2006). Given their roles in insulin resistance, type 2 diabetes

and oxygen deprivation responses, it is of interest to determine if C. elegans ceramide

synthases also have a role in glucose-induced anoxia sensitivity, especially in the

context of reduced insulin signaling.

42

Figure 3.2. Ceramide and Sphingolipid Biosynthesis in C. elegans. Arrows in this schematic diagram indicate successive steps in sphingolipid and ceramide metabolism by C. elegans, based on previously published data (Menuz et al. 2009; Mosbech et al. 2013; Liu et al. 2014a; Tedesco et al. 2008a; Y. Kim and Sun 2012; K. H. Nomura et al. 2011). The implicated proteins are indicated beside each arrow.

ROS and Antioxidants

Reactive oxygen species (ROS), such as the superoxide radical (O2−•), hydrogen

peroxide (H2O2), and the hydroxyl radical (OH•), are continuously generated via

43

metabolic reactions, and especially during mitochondrial energy production.

Additionally, many studies have shown that hyperglycemia and diabetes increase

oxidative stress, and in excess, ROS cause oxidative damage to DNA and to proteins

and have been implicated in the onset of insulin resistance, inflammation and

atherosclerosis (Giugliano et al. 1996; Jiang et al. 2013; Henry 1962; Paneni et al.

2013). Moreover, it is hypothesized that different susceptibility levels of diabetic

patients, to vascular complications, might actually be a function of their endogenous

antioxidant status.

As discussed previously, in C. elegans it was determined that

high glucose conditions limit lifespan, at least in part, by increasing ROS formation

(Schlotterer et al. 2009; Mendler et al. 2014). Additionally, under high glucose

conditions, human insulin reduces ROS formation in C. elegans, increases the activity

of superoxide dismutase, and rescues the decreased lifespan phenotype. Superoxide

dismutase (SOD) is a major antioxidant enzyme that protects against oxidative stress by

catalyzing the addition or removal of an electron from O2−•, and thus changing it into one

of two less damaging species, O2 or H2O2. In C. elegans, several genes encode SOD

enzymes, sod-1 encodes cytosolic Cu/Zn-SOD (which binds both copper and zinc), sod-

2 and sod-3 each encode mitochondrial Mn-SOD (which binds manganese; the

functional differences between these two Mn-SODs is not known), and sod-4 encodes

extracellular CuZn-SOD (Larsen 1993; Suzuki et al. 1996; Giglio et al. 1994; Hunter et

al. 1997; Fujii et al. 1998). Additionally, sod-3 mRNA levels are significantly increased in

daf-2 mutant animals, as compared to wild type, and this up regulation is dependent

upon activity of DAF-16. While daf-2 mutants are long lived and more resistant to

44

oxidative stress, it has been determined that double deletions of the two Mn-SOD

genes, sod-2 and sod-3, induce sensitivity to oxidative stress and modulate the

longevity phenotype in the daf-2 mutant background (Honda, et al. 2008). Moreover,

Honda et al. hypothesize that the Mn-SODs in C. elegans have a role in fine-tuning

insulin signaling, by acting not just as antioxidants, but as regulatory redox-signaling

molecules. Given the role of SODs in the modulation of insulin signaling and oxidative

stress, it is of interest to determine their role in anoxia with and without glucose

supplementation, especially in the context of reduced insulin signaling.

Results

Glucose-Induced Anoxia Sensitivity is Modulated by Insulin Signaling

I determined that the daf-2(e1370) allele that promotes enhanced anoxia

tolerance, also promotes anoxia tolerance in animals fed a glucose-supplemented diet.

The daf-2(e1370) animals have a higher anoxia survival rate in comparison to wild-type

animals, however their sensitivity to anoxia does increase when fed a higher

concentration of glucose (>1%) (Figure 3.3). Thus, modulation of the insulin-like

signaling pathway through the daf-2(e1370) mutation leads to anoxia resistance in

animals fed a high-glucose diet. Additionally, not surprisingly, I also determined that the

ability for daf-2(e1370) animals to survive anoxia, after being fed a glucose diet (0.5%),

is dependent upon activity of the transcription factor daf-16 (Figure 3.3).

Given that daf-2 mutant animals display increased tolerance to anoxia, I

examined whether or not general anoxia tolerance was sufficient to promote survival of

anoxia under glucose supplementation conditions. As discussed previously, the sterile

45

1-day old adult glp-1(e2141) animals, similar to daf-2(e1370) animals, are long-term

anoxia tolerant. However, their anoxia tolerance is independent of DAF-16 activity,

unlike that of daf-2(e1370) animals (LaRue and Padilla 2011; Mendenhall et al. 2009). I

determined that glp-1(e2141) animals are not resistant to anoxia when fed a glucose-

supplemented diet. Furthermore, the daf-2(e1370);glp-1(e131) double mutant fed a

glucose-supplemented diet did not have a significantly higher anoxia survival rate in

comparison to daf-2(e1370) animals (Figure 3.3). This data indicates that anoxia

resistance per se does not minimize the negative impact of a glucose-supplemented

diet.

Given the role of hypoxia-inducible factor, hif-1, in oxygen deprivation survival

across multiple species, I examined whether or not HIF-1 signaling had a role in

glucose-induced anoxia sensitivity. The highly conserved heterodimeric hypoxia-

inducible transcription factor HIF-1, regulates cellular and systemic responses to

hypoxia. When oxygen levels are normal, oxygen-dependent enzymes belonging to the

EGL-9/Prolyl hydroxylase superfamily, hydroxylate proline residues of HIF-1α. This

hydroxylation results in the targeted degradation of HIF-1 by the von Hippel-Lindau

tumor suppressor protein, VHL. In C. elegans the hif-1 and vhl-1 genes encode

homologs of the HIF-1α subunit and VHL respectively, while egl-9 encodes what is

thought to be the sole HIF prolyl hydroxylase. So, using oxygen as a substrate, EGL-9

hydroxylates HIF-1, increasing its affinity for VHL-1, which targets it for proteosomal

degradation. Thus, egl-9 deficient animals express constitutively high levels of HIF-1

(Shen and Powell-Coffman 2003; Jiang et al. 2001; Powell-Coffman 2010; Shao et al.

46

2009; Shen et al. 2005). I determined that HIF-1 stabilization via mutation of egl-9 did

not enhance anoxia survival of animals fed a glucose-supplemented diet (Figure 3.3).

Additionally it has been demonstrated that a high glucose diet (2%) shortens the

lifespan of C. elegans via ectopic apoptosis induction. Thus, I also examined whether or

not programmed cell death influences glucose-induced anoxia sensitivity. Caspases are

a family of cysteine proteases that are known to play essential roles in programmed cell

death (apoptosis) and necrosis. In C. elegans the proapoptotic caspase, CED-3, is

essential for the programmed death of all somatic cells (Kumar 2006; Mohapatra et al.

2011). The activator EGL-1 together with caspases CED-3 and CED-4 modulate all

developmentally programmed cell death in C. elegans. Thus, loss-of-function mutants

of egl-1, ced-3 and ced-4 result in the survival of cells that normally die (e.g., all 131

somatic cells that are destined to die, survive). I determined that ced-3 mutants display

anoxia survival levels similar to that of wild type under glucose supplementation

conditions (Figure 3.3). Thus, down regulation of programmed cell death is not sufficient

to suppress glucose-induced anoxia sensitivity in C. elegans. The role of apoptosis in

the context of reduced insulin signaling (in daf-2 mutants), remains to be determined.

Finally, a key sensor of nutritional status in most organisms is the hexosamine-

signaling pathway, which terminates in O-linked-N-acetylglucosamine (O-GlcNAc)

protein modification cycling. Two highly conserved enzymes regulate O-GlcNAc cycling,

O-GlcNAc transferase (OGT-1) and O-GlcNAcase (OGA-1). In C. elegans, OGT-1

catalyzes the addition of O-GlcNAc at Serine and Threonine residues of target proteins,

while OGA-1 catalyzes their removal (Hanover et al. 2005; Love and Hanover 2005;

Love et al. 2010; Mondoux et al. 2011; Mondoux et al. 2010; Forsythe et al. 2006).

47

Thus, animals with a mutation in oga-1 display increased levels of O-GlcNAc–modified

proteins, whereas null mutants of ogt-1 are unable to catalyze the modification and

display a lack of O-GlcNAc protein modification. It has been demonstrated that O-

GlcNAc cycling is genetically linked to insulin signaling in C. elegans. For example,

the oga-1(ok1207) null allele blunts signaling pathways that normally inhibit DAF-16

activity. Moreover, approximately 2–5% of all intracellular glucose is converted to UDP-

GlcNAc, the substrate for O-GlcNAc protein modification. Given that the substrate for

OGT-1, UDP-GlcNAc, is derived from glucose, the levels of glucose generally correlate

with the level of O-GlcNAc protein modification within an animal. Thus, in C. elegans,

under glucose supplementation conditions (glucose concentrations ≥250mM) it has

been demonstrated that the levels of O-GlcNAc–modified proteins are significantly

increased (Mondoux et al. 2011). Additionally it was shown that ogt-1 null mutants that

lack the ability to O-GlcNAcylate proteins are hypersensitive to glucose

supplementation, while oga-1 null mutants (with increased levels O-GlcNAc–modified

proteins) display phenotypes similar to that of wild-type animals under conditions of

glucose toxicity. Given the roles of O-GlcNAc cycling in nutrient sensing and insulin

signaling, I examined whether or not increased O-GlcNAc modification was sufficient to

rescue glucose- induced anoxia sensitivity. I determined that oga-1(ok1207) null

mutants displayed anoxia survival levels similar to that of wild type (Figure 3.3).

Additionally I assessed whether or not inhibition of O-GlcNAc protein modification was

sufficient to suppress the ability of daf-2(e1370) animals to survive anoxia under

conditions of glucose supplementation (0.5%). I determined that daf-2(e1370);ogt-

1(RNAi) animals display anoxia survival rates similar to that of daf-2(e1370) controls

48

under conditions of glucose supplementation, thus suggesting that O-GlcNAc cycling is

not sufficient to rescue glucose-induced anoxia sensitivity nor is it sufficient to suppress

the ability of daf-2 mutants to survive.

Together these data demonstrate that general anoxia tolerance does not

minimize the negative impact of a glucose-supplementation on anoxia responses.

Additionally, these data suggest that the insulin-signaling pathway, in a manner that is

independent of HIF-1, apoptosis and O-GlcNAc protein modification, modulates

glucose-induced anoxia sensitivity.

49

Figure 3.3. The insulin-signaling pathway modulates glucose-fed anoxia survival rate. (A) The daf-2(e1370) animals, in comparison to wild-type animals, survive anoxia better after being fed a glucose-supplemented diet. Bar indicates that there was a significant increase in survivorship for daf-2(e1370) animals in comparison to wild-type animals given the same diet and anoxia exposure (2 way ANOVA, Bonferonni Multiple Comparisons, p<0.05). (B) A mutation in daf-16 or knockdown via RNAi suppresses the ability of daf-2(e1370) animals to survive anoxia after being fed a glucose-supplemented diet. The * indicates a significant increase in survivorship for daf-2(e1370) animals fed a glucose-supplemented diet prior to anoxia exposure in comparison to other animals shown with the specified genotypes (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.05). (C) The long-term anoxia survival mutant glp-1(e2141) is sensitive to 1 day of anoxia exposure when fed a glucose-supplemented

50

diet; daf-2(e1370) suppresses the anoxia sensitivity observed in glp-1(e2141) mutants. Note that in order to conduct these experiments the animals had to be raised at 15ºC to the L3 stage and then transferred to 25ºC. Bar indicates a significant increase in survival in comparison to N2 animals (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.05). (D) The genotypes shown are not resistant to anoxia exposure when fed a glucose-supplemented diet. For all experiments shown, at least 3 independent experiments were conducted.

Glucose-Induced Anoxia Sensitivity is Modulated by Insulin Signaling via Lipid

Biosynthesis

The daf-2(e1370) allele confers increased anoxia resistance under conditions of

glucose supplementation in a daf-16 dependent manner. This suggests that glucose

induces anoxia sensitivity by preventing the FOXO transcription factor homolog DAF-16

from regulating the expression of specific target genes. To determine the specific DAF-

16 target genes that are required for glucose-fed anoxia resistance in daf-2 mutants, I

used RNA interference (RNAi) to screen through a subset of DAF-16 regulated genes. I

determined that knockdown of fat-7, which encodes a delta-9 fatty acid desaturase, was

sufficient to suppress the daf-2(e1370) glucose-fed anoxia resistance phenotype (Figure

3.4).

Fatty Acid Biosynthesis

The biosynthetic pathway responsible for the desaturation of short-chain fatty

acids to polyunsaturated fatty acids is known in C. elegans (Brock et al. 2007). By using

RNAi, I determined that knockdown of genes that encode any one of the fatty acid

desaturases or elongases in C. elegans was sufficient to suppress the daf-2(e1370)

glucose-fed anoxia resistance phenotype (Figure 3.4). Each of the three Δ9-

desaturases (fat-5, fat-6 and fat-7) have high sequence similarity, thus RNAi knockdown

for any one of them likely reduces function in the others. Similarly the elongases elo-1

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and elo-2 also share high sequence similarity. For further genetic analysis, I chose to

focus only on the Δ9 desaturase mutants (fat-5, fat-6 and fat-7). Given that the Δ9

desaturase are redundant under standard conditions, I tested double mutants (a fat-

5;fat-6;fat-7 triple mutant is lethal). The fat-5(tm420) and fat-6(tm331) are deletion

alleles that eliminate two of the conserved histidine-rich regions and two trans-

membrane domains, these are most likely null mutations. The fat-7(wa36) strain is a

single base pair mutation that leads to a premature stop codon that eliminates two

trans-membrane domains and one of the conserved histidine boxes, this allele is at

minimum a strong reduction-of-function allele (Brock et al. 2007). Aligned with the RNAi

data, I determined that the fat-5;fat-6 or fat-6;fat-7 double mutations also suppress the

ability of daf-2(e1370) animals to survive anoxia after being fed a glucose-

supplemented diet (Figure 3.4). However, it is important to note that the long-term

anoxia resistance phenotype observed in the daf-2(e1370) mutant (fed a standard diet)

is only slightly, but not significantly suppressed by mutations in fat-6;fat-7 or fat-5;fat-6,

suggesting that in the context of reduced insulin signaling, knockdown of delta-9 fatty

acid desaturases does not confer general sensitivity to anoxia (Figure 3.5).

Given the previously identified roles of fatty acids in anoxia, as terminal electron

acceptors and as potential signaling molecules, I examined whether or not specific fatty

acid biosynthesis mutants (in a wild-type background) had an overall sensitivity to

anoxia when fed a standard OP50 diet. I determined that with the exception of the fat-

6;fat-7 double mutant, all animals survive 24 hours of anoxia when fed a normal OP50

diet (Figure 3.5). That is, the fat-6;fat-7 mutants display increased sensitivity to anoxia

even in the absence of glucose supplementation. Thus, the proper synthesis of

52

monounsaturated fatty acids (and/or downstream polyunsaturated fatty acids) is

required for the survival of 24 hours of anoxia. To date, this is the only genetic mutation

identified in conferring sensitivity to 24 hours of anoxia.

Figure 3.4. The insulin-signaling pathway modulates glucose-fed anoxia survival via lipid biosynthesis and antioxidant activity. (A) RNAi of genes involved with lipid biosynthesis suppresses the ability of glucose-fed daf-2(e1370) animals to survive 1 day of anoxia (.5% glucose). Bar indicates that the number of animals alive is significantly different in comparison to daf-2(e1370) animals fed empty vector as a control (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.05). (B) Genetic mutations affecting lipid or ceramide biosynthesis suppress the ability of daf-2(e1370) animals to survive anoxia after being fed a glucose-supplemented diet. Bar indicates that the number of animals alive is significantly different in comparison to daf-2(e1370) animals fed empty vector as a control (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.05). For all experiments hermaphrodites were examined; error bar equals standard deviation; at least 3 independent experiments, with n ≥ 100, were conducted.

Ceramide Synthesis

As discussed previously, other studies have shown that a mutation in the hyl-2

gene, which encodes a ceramide synthase, affects anoxia survival (Menuz et al. 2009).

I replicated these data, and also determined that the hyl-2(tm2031) mutant, fed a

53

standard OP50 diet, is sensitive to two days of anoxia exposure, but this sensitivity to

anoxia is suppressed by daf-2(e1370) (Figure 3.5 B). Additionally, while the daf-

2(e1370) mutant can survive three days of anoxia, this phenotype is suppressed by

either hyl-2(tm2031) or a glucose-supplemented diet (Figure 3.5 B). Thus, these data

suggest that hyl-2 and daf-2 are acting in parallel pathways that mutually influence one

another to modulate the response to oxygen deprivation.

I also determined that the hyl-2(tm2031) mutation suppresses the ability of

glucose-fed daf-2(e1370) animals to survive anoxia (Figure 3.4). These results indicate

that modulation of ceramide synthesis influences anoxia survival in animals fed a

glucose-supplemented diet as well. Additionally, these data suggest that both diet and

ceramide biosynthesis can alter the ability of daf-2 animals to be resistant to long bouts

of anoxia treatment but are not essential for daf-2 animals to survive shorter bouts of

anoxia.

Further, given the role of hyl-2 in general anoxia tolerance and its ability to

suppress the survival of glucose-supplemented daf-2 mutants, I wanted to determine if

hyl-2(tm2031) animals are more sensitive than wild-type animals to the effects of

glucose supplementation. I compared the anoxia survival rates of 0.0625% to 0.5%

glucose-supplemented hyl-2(tm2031) animals, and determined that hyl-2 mutant

animals display a significantly reduced ability to survive 24 hours of anoxia, as

compared to wild-type, when supplemented with just 0.0635% glucose (Figure 3.5 C).

These data suggest that the hyl-2 mutation leads to an increased sensitivity to anoxia

under glucose supplementation conditions.

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Figure 3.5. Lipid biosynthesis impacts anoxia survival. (A) Animals of the specified genotype were fed a standard OP50 diet and exposed to 1 day of anoxia. The fat-6;fat-7 double mutant is sensitive to one day of anoxia exposure. The * indicates a significant decrease in survival compared to other genotypes (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.001). (B) Animals of the specified genotype were exposed to 2 or 3 days of anoxia. All animals were fed a standard OP50 diet with the exception of daf-2(e1370) as indicated. The * indicates that hyl-2(tm2031) animals exposed to 2 days of anoxia have a significant decrease in survival. The bar indicates that hyl-2(tm2031) or a glucose-supplemented diet suppresses the ability of daf-2(e1370) animals to survive 3 days of anoxia. (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.001). (C) The hyl-2(tm2031) animals, in comparison to wild-type animals, survive anoxia worse after

55

being fed a glucose-supplemented diet. Asterisk indicates that there was a significant decrease in survivorship for hyl-2(tm2031) animals in comparison to wild-type animals given the same diet and anoxia exposure (2 way ANOVA, Bonferonni Multiple Comparisons, p<0.05).

MUFA Supplementation

In order to determine if supplemental feeding of a specific fatty acid could rescue

the anoxia sensitivity induced by glucose-supplementation, animals were raised on

control media or on media supplemented with 0.3 mM oleic acid from the L1 larval stage

to 1-day old adults (Deline et al. 2013). I found that oleic acid supplementation was

sufficient to significantly rescue the anoxia sensitivity of daf-2;fat-6;fat-7 mutant animals,

and slightly but not significantly rescued the sensitivity in daf-2;fat-5;fat-6 mutants

(Figure 3.6). It has been shown previously that daf-2;fat-6;fat-7 mutant animals display

more extreme fatty acid composition changes as compared to daf-2;fat-5;fat-6 mutants,

that display relatively subtle fatty acid composition alterations under standard conditions

(Brock et al. 2007; Shi et al. 2013). Thus, I hypothesize that oleic acid or downstream

PUFAs are, at least in part, required for the anoxia survival of daf-2 mutants under

glucose supplementation conditions.

To improve uptake of the oleate, I opted to not chronically feed the animals both

glucose and oleate simultaneously, rather animals were raised on the fatty acid

supplemented media to maximize uptake, and then transferred as adults to 0.5%

glucose media and allowed to eat for 1 hour before being exposed to 24 hours of

anoxia. This difference in methodology may explain why the daf-2;fat mutants fed

glucose for one hour, as opposed to being raised on glucose, had a higher anoxia

survival rate (Figure 3.4 compared to Figure 3.6). The 1-hour feeding of glucose was

56

sufficient to induce the anoxia sensitivity in N2 and daf-2;hyl-2(tm2031) animals,

however, and the addition of oleic acid did not suppress this sensitivity (Figure 3.6). The

glucose-fed daf-2 mutant survives anoxia as expected and oleic acid supplementation

did not significantly alter survival rate (Figure 3.6). These data suggest that the hyl-2

mutation leads to an increased sensitivity to anoxia in glucose fed animals in

comparison to the fat mutants and that ceramide and fatty acid biosynthesis are likely

working in separate pathways to modulate anoxia survival in glucose-fed animals.

Figure 3.6. Fatty Acid Biosynthesis Impacts Anoxia Survival. Animals of the specified genotype were raised on a standard OP50 diet or a diet supplemented with oleate. Animals were then transferred to a glucose-supplemented diet, allowed to eat the glucose-supplemented diet for 1 hour and then exposed to 24 hours of anoxia. The * indicates a significant increase in anoxia survival when fed oleate (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.001). For all experiments hermaphrodites were examined; error bar equals standard deviation; at least 3 independent experiments, with n ≥ 100, were conducted.

57

Glucose-Induced Anoxia Sensitivity is Modulated by Insulin Signaling Via Antioxidant

Activity

Given the role of SODs in the modulation of insulin signaling and oxidative

stress, I wanted to determine if the ability of daf-2 mutant animals to survive anoxia was

dependent upon their ability to detoxify ROS. As discussed previously, daf-2(e1370)

animals display a significant increase in sod-3 mRNA and are more resistant to

oxidative stress. Thus, it is of interest to determine the role of mitochondrial SODs, sod-

2 and sod-3, in anoxia response, with and without glucose supplementation. I

determined that mutation of sod-2 and sod-3 was sufficient to suppress the anoxia

survival of daf-2(e1370) animals under glucose supplementation conditions (Figure 3.4).

Additionally, while mutation of sod-2;sod-3 increases sensitivity to oxidative stress and

hyperoxia in daf-2 mutant animals, I determined that this double mutation does not

impact the ability of daf-2 mutants to survive long-term anoxia under standard

conditions, suggesting that the combinatory stress of glucose-supplementation and

anoxia exposure likely elicits an increase in oxidative stress that requires more active

oxidative stress detoxification (Figure 3.5).

Glucose Supplementation Induces Lipid Accumulation

In the previous chapter I demonstrated, using Oil Red O staining to detect lipids,

that N2 wild-type animals fed a glucose-supplemented diet, relative to an OP50 only

diet, have a significant increase in lipid droplets in the anterior (head and pharynx)

region of the animal (Figure 2.6). Additionally, I assayed whether or not the same lipid

accumulation phenomenon could be observed in daf-2 and hyl-2 mutant animals,

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genotypes that are resistant and sensitive to anoxia, respectively. The hyl-2(tm2031)

animals fed a glucose-supplemented diet, compared to those fed a standard diet, did

not have a significant accumulation of lipid droplets in the head/pharynx region of the

animal (Figure 3.7). Also, compared to N2 animals, hyl-2(tm2031) animals appeared to

have an overall reduction in Oil Red O staining. As others have reported, daf-2(e1370)

animals have an increase in lipids (Figure 3.7). In some, but not all, of the animals

carrying the daf-2(e1370) allele and fed a standard OP50 diet, lipid droplets in the

head/pharynx region were observed (Figure 3.7). Together, these results further support

the idea that a glucose-supplemented diet acts as an obesity-mimetic in C. elegans and

that both diet and genotype affect lipid distribution within the animal. Additionally there is

a correlation between the level of fat stores and the ability of the animals to survive

prolonged anoxia exposure under standard conditions. For example, daf-2(e1370)

animals display high levels of lipid stores while hyl-2(tm2031) animals display lower

amounts. However, given that glucose significantly increases the amount of lipid

accumulation, but sensitizes animals to anoxia, it appears likely that the specific types

of lipids present in animals will have a role in determining their ability to survive anoxia.

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Figure 3.7. Glucose supplementation induces lipid accumulation. Oil Red O staining was used to localize lipids within the animal. (A) Animals of specified genotypes fed a standard diet or a glucose-supplemented diet were stained. Representative images of whole animals are shown. (B) Enlarged image of the anterior region. Lipids can be detected in the intestine, oocyte/germline and pharynx. Arrow points to the posterior region of the terminal bulb of the pharynx. Note that glucose fed animals (N2, daf-2, daf-2;hyl-2) contain lipid droplets in the pharynx region. For each genotype, there is more staining in glucose-fed animals relative to controls. (C) The presence or absence of lipids within the anterior region of the animal was assayed. At least 10 animals from three independent experiments were randomly imaged and assayed for the presence of lipid droplets, in the anterior (head and pharynx) regions. Error bar equals standard deviation; bar indicates there was a significant difference in glucose-fed animals in comparison to controls (1 way ANOVA, Bonferonni Multiple Comparisons, p<0.05).

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CHAPTER 4

GLUCOSE INDUCES SIGNIFICANT GENE EXPRESSION CHANGES IN C. elegans‡

Introduction

Transcriptional Changes Associated With the Progression of Type 2 Diabetes

Given the delayed onset of symptoms, and the fact that symptoms can remain

mild and are often ignored by sufferers for some time, type-2 diabetes often remains

undetected in patients for several years. Additionally, the diagnosis of type-2 diabetes is

often made incidentally via abnormally high blood or urine glucose concentrations when

tested. Consequently, at the time of detection, the disease is often at more advanced

stages, and vascular complications have already begun to occur. Moreover, in many

cases, the detrimental effects of glucose can occur, even when blood glucose levels are

still below the threshold level for the diagnosis of diabetes. Mild hyperglycemia and

fluctuations in glucose levels can have a role in the functional and structural alterations

of vessel walls, ultimately resulting in diabetic vascular complications (Paneni et al.

2013; Beckman et al. 2013). Thus, a more thorough understanding of the underlying

mechanisms in diabetic vascular disease is necessary to provide potential novel

approaches that may prevent, delay the progression of, or rescue these

complications. While multiple pathways have been implicated in the pathophysiology of

diabetes and its associated complications, and a number of studies have identified

specific gene expression signatures related to these diseases states in rodents and

‡ Parts of this chapter have been previously published, either in part or in full, from Garcia AM, Ladage ML, Dumesnil DR, Zaman K, Shulaev V, Azad RK, Padilla PA. Glucose Induces Sensitivity to Oxygen Deprivation and Modulates Insulin/IGF-1 Signaling and Lipid Biosynthesis in Caenorhabditis elegans. Genetics 2015. Reproduced with permission from the Genetics Society of America.

61

humans, there remains questions regarding the biological relevance and interactions of

each of these identified genes.

Transcriptomics

Many of the implicated genetic factors associated with type-2 diabetes and its

complications were found through genome wide association studies (GWAS) on human

subjects. While GWAS can be a powerful tool to identify genetic factors associated with

a particular trait/disease, they can also be problematic. For example, limited sample

numbers, high stochastic variation and/or patterns of correlation that lead to indirect

associations between genetic markers and traits where no causal relation actually exists

(Platt et al. 2010). In more recent years, transcriptomic approaches, such as next

generation sequencing (sometimes in conjunction with GWAS studies), have gained

traction as another tool to investigate the associations between genetic factors and

disease states. Early transcriptomic studies however, often relied on hybridization-

based microarrays that frequently were limited in their ability to fully catalog and quantify

the diversity of RNA molecules expressed. More recently, the introduction of high-

throughput next-generation sequencing technologies has transformed and

revolutionized transcriptomics approaches, by allowing RNA transcript analysis through

large-scale cDNA sequencing (Ozsolak and Milos 2011). For example, RNA sequencing

has advanced our ability to characterize transcriptomes, across multiple species, at

specific stages of development and/or under specific physiological conditions. RNA

sequencing provides an ability analyze RNA transcripts (often including mRNA, tRNA,

rRNA and other non-coding RNAs) in a specific cell type, tissue or whole animal. This

62

approach focuses on gene expression patterns at the RNA level (at the level of

transcription) and offers genome-wide information and insight into molecular

mechanisms that are involved in the regulation of specific biological processes. Thus,

using such an approach, there lies potential to better establish meaningful genetic

associations, which have causative roles in the pathology of disease states.

Results

Glucose Supplementation Induces Significant Transcriptional Changes

To identify gene expression changes associated with a glucose-supplemented

diet in C. elegans, we used RNA sequencing (RNA-Seq) to compare the transcript

profile of wild-type adults (young, non-gravid) raised on a glucose-supplemented diet

relative to those raised on an OP50 only diet (Figure 4.1). Glucose supplementation

significantly impacted the gene expression of 2,370 of the 20,375 genes analyzed;

1,850 genes were up regulated and 520 genes were downregulated (Figure 4.2, Table

4.1). The fold-increase in expression values (normalized read count, FPKM; see

Methods) for significantly upregulated genes in animals fed a glucose-supplemented

diet ranged from 1.34 to 136.99. On the other hand, the fold-decrease in expression of

downregulated genes in animals fed a glucose diet ranged from 1.34 to 9.63 (Table

4.1).

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Figure 4.1. Workflow of RNA Sequencing Analysis. The Padilla laboratory conducted the wet lab protocol and subsequent categorization, confirmation and biological interpretation.

Genes differentially regulated by a glucose diet were classified and evaluated

using PANTHER (http://www.pantherdb.org), Wormbase annotation

(http://www.wormbase.org), and Database for Annotation and Visualization and

Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov/home.jsp) (Dennis et al.

2003; Mi et al. 2013). The PANTHER classification system combines gene function,

ontology, and pathways to analyze large data sets (Mi et al. 2013). This classification

system can classify genes into categories based on their biological process, molecular

function, cellular component or protein class.

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Table 4.1 Overview of Genes Regulated by a Glucose-Supplemented Diet

Glucose Induced

Number, (Percent*)

Glucose Reduced Number, (Percent *)

Genes Altered 1,850 520

>50 fold Change 22 (1.19%) 0

25>50 Fold Change 41 (2.21%) 0

10>25 Fold Change 85 (4.59%) 0

5>10 Fold Change 522 (28.22%) 5 (.96%)

<5 Fold Change 1,147 (62.0%) 511 (98.27%)

Undetectable in OP50 only 33 (1.78%) -

Undetectable in Glucose - 4 (.77%)

Analyzed using GO/Panther 1,823 513

Genes Not Analyzed using GO 27 7

20,375 transcripts were analyzed 11.63% of the genes analyzed responded to a glucose diet 9.08% of genes were upregulated and 2.55% were downregulated * The percent is of total changed for that category

Genes Upregulated in Response to Glucose

Based on the biological processes classification, the most abundant category

(category with the highest number of all significantly upregulated genes) was the

metabolic process category (Figure 4.2, Table 4.2). Moreover, cellular process and

localization categories were the second and third most abundant categories,

respectively (Table 4.2).

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Figure 4.2. RNA-Sequencing analysis reveals differentially regulated genes in response to a glucose-supplemented diet. (A) Scatter plot shows expression values (normalized read counts, FPKM; see Methods) for transcripts in animals fed a standard OP50 E. coli diet (X-axis) versus those in a glucose-supplemented diet (Y-axis); data are shown on log scale. Transcripts that were upregulated or downregulated in response to a glucose diet are shown. (B) and (C) Genes were categorized based on Gene ontology (GO)

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annotations for biological functions; the proportion for each category is displayed in the pie-chart; (B) shows the gene expression profile for those upregulated in glucose condition, and (C) shows the gene expression profile for those downregulated in glucose condition. For both (B) and (C), the GO classification analysis was performed using PANTHER.

Of the genes listed in the category of metabolic processes, 83% of them are

involved in primary metabolic processes (Table 4.2). Upon further sub-categorization, it

was determined that genes involved in either carbohydrate or lipid metabolism comprise

almost half of the genes involved in primary metabolic processes (lipid and

carbohydrate metabolism make up 35% of the 83% of genes involved in primary

metabolic processes, while 40% are involved in protein metabolism/modification) (Table

4.2). To illustrate, several genes that are known or predicted to be involved with lipid

biology were upregulated in response to glucose supplementation; these genes

included those that are involved with lipid processes, lipid binding or lipid transport. This

list includes genes predicted to encode stearoyl-CoA desaturase (delta-9 desaturase)

(fat-5), long-chain acyl-CoA synthetase (acs-2, acs-15, acs-18) and ceramide glycosyl

transferase (cgt-1) indicating that a glucose-supplemented diet induces genes involved

with lipid biosynthesis and modification. We found that there were 112 genes (6% of all

significantly upregulated genes) that were classified by PANTHER to be involved with

some aspect of lipid metabolism. Additionally, Zhang et al, documented a more

comprehensive list of lipid metabolic genes present in the C. elegans genome (Zhang et

al. 2013). So, we used the Gene List Venn Diagram program

(http://genevenn.sourceforge.net) to compare the genes we found upregulated by

glucose to the data set from Zhang et al and identified 57 of their documented 471

genes to be upregulated by glucose (11.9%). Additionally, I went on to compare the

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PANTHER generated list of genes involved in lipid metabolism, to the Zhang et al. 2013

study, and compare those together with our list of glucose- induced genes, to get a

more comprehensive idea of glucose-induced genes involved in lipid metabolism.

Together, of all known C. elegans genes involved in lipid metabolism, a total of 137

(7.41% of all significantly upregulated genes) were found to be significantly upregulated

in response to glucose supplementation (Figure 4.3).

Figure 4.3. Glucose Induced the Expression of Genes Involved in Lipid Metabolism. Using the PANTHER classification of lipid metabolic genes, and a comprehensive list of C. elegans lipid genes generated through comparative genomics and functional analysis by Zhang et al. 2013, we identified that 137 of all genes found to be upregulated (7.4%) were involved in some aspect of lipid metabolism.

Further, in the same general category of metabolic processes, there were a

number of genes that are known or predicted to be involved with carbohydrate

metabolism. Not surprisingly, out of the 1,850 genes significantly induced by glucose,

91 of them (5% of all significantly upregulated genes) were involved in the metabolism

of carbohydrates. For example, glycolytic genes gpd-2, gpd-3, gluconeogenesis genes

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PEPCK pck-1, pck-3 and carbohydrate transport (sugar transporter swt-1) were

upregulated in response to glucose supplementation.

Table 4.2 Overview of Upregulated Genes Classified by PANTHER

Upregulated (Glucose Induced)

Biological Process

Molecular Function

Metabolic Process 21%

• 83% Primary Metabolic Process Protein metabolism/modification Lipid metabolism Carbohydrate metabolism

Catalytic Activity 36%

• 69% Hydrolase & Transferase Activity

Cellular Process 16%

• 72% Cell Communication & Cell Cycle

Transporter Activity 16%

• 84% Transmembrane Transporter Activity

Localization 12%

• 99% Transport

Structural Molecule Activity 16%

• 99% Cytoskeleton & Extracellular Matrix Molecule Activity

Additionally when PANTHER categorical analysis was based on molecular

function rather than biological process, the top 3 activity categories most upregulated

include catalytic, transporter and structural molecule activity (Table 4.2). For example, a

number of upregulated genes that are predicted to be involved with the transfer of

glycosyl groups, in addition to several other genes that have catalytic activities (e.g. acyl

transfer, oxidoreductase, hydrolase) were significantly upregulated. Finally, the idea that

a glucose-supplemented diet is stressful for the animal is supported by the fact that

stress response genes were induced (e.g. chaperones, antioxidants, apoptotic

processes, cellular defense, toxic substance response). In total, 78 (4.2% of all

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significantly upregulated genes) stress response genes were found to be upregulated in

response to glucose supplementation.

Genes Downregulated in Response to Glucose

There are a fewer number of genes that were downregulated in response to a

glucose-supplemented diet, and a much small number of these genes are predicted to

be involved with lipid or carbohydrate biology (Table 4.1, Table 4.3). Although, two of

the genes that code for the glycolytic enzyme GPD (gpd-2, gpd-3) were upregulated in

the glucose-fed animals, the other two genes that code for the glycolytic enzyme GPD

(gpd-1; gpd-4) were downregulated in response to glucose. The mRNA expression of

daf-16 itself was not differentially regulated by a glucose diet. Additionally, genes (sod-

2/3, hyl-2) that suppress the daf-2(e1370) glucose-fed anoxia resistant phenotype were

not differentially regulated by a glucose-diet. However, there was a down-regulation of

several genes (daf-18, ins-8, daf-15, dct-16 and smg-1) involved with the insulin-like

signaling pathway indicating that a glucose diet can impact the mRNA expression of

genes involved with insulin signaling. There are many labs that have identified genes

that are potentially regulated by DAF-16 (Lee et al. 2009; Murphy 2006; Murphy et al.

2003; Riedel et al. 2013). Given that daf-2(e1370) animals survive anoxia and N2

animals do not survive anoxia, when fed a glucose- supplemented diet, it is of interest to

identify genes that are upregulated in daf-2(e1370) and downregulated by a glucose

diet. We identified 12 DAF-16 target genes to be downregulated in N2 in response to

glucose supplementation (Table 4.4).

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Table 4.3 Overview of Downregulated Genes Classified by PANTHER

Upregulated (Glucose Induced)

Biological Process

Molecular Function

Metabolic Process 37%

• 82% Primary Metabolic Process Nucleobase-containing- compound metabolic process

Catalytic Activity 38%

• 63% Hydrolase & Transferase Activity

Cellular Process 21%

• 76% Cell Cycle & Cell Communication

Binding Activity 38%

• 87% Nucleic Acid & Protein Binding Activity

Localization 12%

• 100% Transport

Structural Molecule Activity 6%

• 99% Cytoskeleton, Extracellular Matrix & Ribosome Structural Molecule Activity

Moreover, using PANTHER to classify downregulated genes based upon

biological process, the top three most abundant categories include metabolic

processes, cellular processes and localization, respectively (Table 4.3). For example,

more than half (58%) of the genes classified as being involved in metabolic processes

were classified as nucleobase-containing compound associated with metabolic process,

this category contains genes predicted to be involved with DNA or RNA metabolic

processes (Table 4.3). Examples of genes within this classification include those known

or predicted to be involved with DNA repair, DNA replication, DNA recombination, RNA

splicing, RNA transcription, RNA processing or nucleobase metabolism. Additionally,

the most abundant category in of cellular processes were genes known or predicted to

be involved with the cell cycle (Table 4.3); many of these genes are also classified as

being in the DNA or chromosome binding protein class. Furthermore, if one looks at the

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genes that are classified by protein class, the largest grouping was for the nucleic acid

binding protein class. Moreover, classification based upon molecular function identified

the top three most abundant functional categories as catalytic activities, binding

activities and structural molecule activities (Table 4.3).

Together, these data indicate that glucose supplementation significantly alters

many biological processes and is likely a stress for the animal. I used quantitative RT-

PCR to verify a few genes identified by the RNA-Seq approach as confirmation and

validation of the RNA sequencing data (Figure 4.3). All of the genes were similarly up or

downregulated between the two approaches.

Figure 4.3. Validation of RNA-Seq analysis by qRT-PCR for two targets that showed an up-regulation (cgt-1, ttr-22) or down-regulation (CO7G1.7, ins-8) in animals fed a glucose diet relative to a standard OP50 diet. Shown is the fold change in N2 animals fed a glucose (0.5%) diet relative to N2 animals fed a standard diet. Data shown are from 3 independent experiments. Error bars represent standard deviations. The * indicates p<.05; **** indicates p<.0001, 2 way ANOVA, Bonferroni multiple comparisons.

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We used BLASTp analysis to identify the human proteins that have high identity

and query coverage in local alignment to the most differentially expressed C. elegans

genes (>25 fold upregulated or >2.5 fold downregulated) in glucose-fed animals. With

this classification there are 63 genes that are upregulated >25X fold; 31 of these genes

(49%) are predicted or known to encode a collagen protein. Several of these genes

(17/63; 27%) did not have a significant similarity to any human genes. The other genes

were known or predicted to be involved with various processes including metabolism or

stress responses. There were 20 genes that were >2.5X downregulated in glucose fed

animals; 4 of these genes (20%) did not have a significant similarity to any human

genes.

There were two cytochrome P450 genes (cyp-25A1 and cyp-35D1) that were

downregulated in response to a glucose diet. Evaluation of differentially expressed

genes indicates that five other cytochrome P450 genes were actually upregulated (cyp-

13B1, cyp-33C5, cyp-32A1, cyp-13A12, cyp-33C8); cyp-13A12 has been implicated in

responses to O2 deprivation and reoxygenation (Ma et al. 2013). The gene T19H12.6

that was downregulated has not been studied in C. elegans but is predicted to encode a

protein that is part of the gamma glutamyl transferase/peptidase (GGT) family and

shows identity to human GGT1. The GGT proteins cleave γ-glutamyl peptide bonds in

glutathione and transfer the γ-glutamyl moiety to acceptors; they are associated with

diseases such as diabetes and metabolic syndrome (Hong et al. 2014). A positive

association exists between insulin and GGT and GGT1 was found to be downregulated

in whole blood gene expression analysis of obese subject with type 2 diabetes after

bariatric surgery or weight loss (Kónya et al. 1990; Berisha et al. 2011). This suggests

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that there may be some conserved genes that respond to a glucose-supplemented diet

in C. elegans and genes that are associated with metabolic diseases in humans.

Table 4.4 DAF-16 Target Genes Downregulated by Glucose

Gene/Sequence Gene Class Description

Comparison of glucose downregulated genes with genes upregulated in daf-2 mutants compared to daf-2;daf-16 double mutants (Lee et al. 2009)

spp-17 SaPosin-like Protein family cyp-25A1 CYtochrome P450 family Y4C6B.6 beta-GlucocereBrosidAse

Comparison of glucose downregulated genes with genes that code for proteins that Co-Purifying with DAF-16::GFP (Riedel et al. 2013)

cku-80 Caenorhabditis KU sas-5 Spindle ASsembly abnormal lys-7 LYSozyme npp-9 Nuclear Pore complex Protein pqn-70 Prion-like-(Q/N-rich)-domain-bearing protein

Comparison of glucose downregulated genes with genes upregualted in daf-2(RNAi) and in daf-2 mutants and downregulated in daf-16(RNAi) (Murphy et al. 2003)

lys-7 LYSozyme D1086.3 not known T23G7.3 not known

Y32H12A.8 not known

Others used microarray analysis to identify genes that are differentially regulated

when fed a 2% glucose diet (Lee et al. 2009). Of the 40 genes identified in their study,

13 of the genes were also identified by our RNA-seq analysis (Table 4.5). The

experimental procedure between our study and the Lee et al study were not identical

which may contribute to the differences in the genes identified. While Lee et al.,

identified a much smaller set of glucose responsive genes, our results share 35%

similarity, even despite different experimental conditions. In terms of experimental

conditions, our transcriptome analysis was conducted using RNA from young (non-

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gravid) adult, wild-type worms supplemented with 0.5% glucose while their analysis was

conducted with gravid day-1 adult, wild-type worms supplemented with 2.0% glucose

(1.5x more glucose). Additionally, to reduce the potential for upregulated stress

response genes not related to glucose supplementation we chose to synchronize our

populations via egg lay rather than hypochlorite treatment before RNA isolation. Thus,

the differences in gene expression changes in response to glucose between each group

can likely be attributed to different experimental procedures, including different

transcriptome analysis procedures (microarray vs. RNAseq) different glucose

concentrations (2% vs 0.5%), different adult stages analyzed and different

synchronization methods.

Table 4.5 Genes differentially regulated by glucose- identified by two independent studies*

Gene (Sequence) Gene Class Description

Upregulated: Unknown (C53A3.2) Not known aqp-8 (K02G10.7) Aquaporin or aquaglyceroporin related comt-4 (Y40B10A.6) Catechol-O-MethylTransferase family ugt-41 (F10D2.11) UDP-glucoronosyl/glucosyl transferase col-12 (F15H10.1) Collagen nhr-43 (C29E6.5) Nuclear hormone receptor family ptr-22 (Y80D3A.7) Patched Related family Unknown (F44G3.2) Not known far-3 (F15B9.1) Fatty acid/retinol binding protein cdr-2 (C54D10.1) Cadmium responsive amt-1 (C05E11.4) Ammonium Transporter homolog Downregulated: gba-4 (Y4C6B.6) Beta-glucocerebrosidase spp-5 (T08A9.9) Saposin-like protein family *Identified by this study and (Lee et al. 2009).

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We used BLASTp and HomoloGene analysis to identify other C. elegans genes

that are differentially regulated by a glucose diet and have similarity to human genes

that are known or thought to be involved with metabolic syndrome, obesity and/or type 2

diabetes or obesity (Table 4.6). We identified genes involved with cellular energy

metabolism, insulin or glucose regulation, and lipid biosynthesis. Additionally, we

identified human homologs, that are differentially regulated in a diabetic or obese

physiological state, for C. elegans genes that were upregulated in animals fed a

glucose-supplemented diet (e.g., asm-2; UGCG ceramide glucosyltransferase)

(Błachnio-Zabielska et al. 2012; Boini et al. 2010; X. Li, Gulbins, and Zhang 2012;

Schuchman et al. 1992). Our findings from this study indicate that there are potentially

conserved gene expression responses between C. elegans fed a glucose-

supplemented diet and a diabetic and/or obesity state observed in humans.

Table 4.6. Examples of C. elegans Glucose Responsive Genes that are Relevant to Human Metabolic Diseases

Gene Human Gene (% Identity) Brief Description Associated Disease State

T19H12.6 GGT1 (28%)

-glutamyl traspeptidase

Associated with type 2 diabetes and obesity (Berisha et al. 2011; Kónya et al. 1990).

ant-1.4 SLC25A6/ANT3 (71%)

ADP/ADT Translocator

ADP/ATP translocase family that plays a fundamental role in cellular energy metabolism; Interacts with SIRT4 to regulate insulin secretion in response to glucose; differentially expressed in diabetic mice (Ahuja et al. 2007; H. Lu et al. 2008).

aqp-7 AQP10 (36%) Aquaglyceroporin

AQP10 is expressed in human adipose tissue, functions to regulate glycerol efflux and thus a normal adiposity (protection from obesity) (Laforenza et al. 2013).

lbp-6 FABP4 (48%)

Fatty acid binding protein

FABP4 functions as an adipokine, regulates insulin secretion during obesity; associated with pathogenesis of atherosclerosis, type 2 diabetes,

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metabolic and vascular disease (Engeli et al. 2013; Kim et al. 2014; Kralisch and Fasshauer 2013; Lamounier-Zepter et al. 2013; Wu et al. 2014).

C54E4.5 MIOX (51%) Inositol Oxygenase

Increase in MIOX activity is proportional to serum glucose concentrations; diabetic mice display increased MIOX in the cortex of the kidney (Y. Lu et al. 2009; Nayak et al. 2005; Yang et al. 2010).

poml-3 PON2 (30%) Paraxonase

encode high density lipoprotein (HDL)-related glycoproteins; PON2 has a significant protective role against macrophage triglyceride accumulation, macrophage TG biosynthesis, microsomal DGAT1 activity and macrophage oxidative stress, under high glucose concentrations in mice (Meilin et al. 2010).

pkc-2 PRKCA (69%) Protein Kinase C

type 2 diabetic rats display persistent translocation and activation of PKC in soleus muscles, and this persistent PKC activation may contribute to impaired glycogen synthesis and insulin resistance (Avignon et al. 1996).

asm-2 SMPD1 (37%)

Acid Spingomyelinase

Stress is thought to activate sphingomyelinase to generate ceramide, which serves as a second messenger in initiating the apoptotic response; Expression higher in obese diabetic patients (Błachnio-Zabielska et al. 2012; Boini et al. 2010; X. Li, Gulbins, and Zhang 2012; Schuchman et al. 1992).

cgt-1 UGCG (40%)

Ceramide glucosyltransferase

catalyzes the first glycosylation step in glycosphingolipid biosynthesis; lipid/sugar membrane components; synthase in central nervous system regulates body weight and energy homeostasis (Nordström et al. 2013).

R11F4.1 GK (50%) Glycerol Kinase

glycerol kinase deficiency has been implicated in in insulin resistance and type 2 diabetes mellitus; Enhanced adipose glycerol kinase enzymatic activity in aquaporin 7 deficient obese mice (Hibuse et al. 2005; Rahib et al. 2007).

ech-7 ECHS1 (56%)

Enoyl-Coenzye A Hydratase

catalyzes the second step in mitochondrial fatty acid beta-oxidation; downregulated in WT mouse heart tissue in response to ischemic post-conditioning following ischemia-reperfusion. This effect is not observed in diabetic mouse heart tissue (Zhu et al. 2012).

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acs-15 ACSL1 (36%)

Long-chain fatty acid CoA ligase

key role in both the synthesis of cellular lipids and the degradation of fatty acids; increased expression is associated with an inflammatory macrophage phenotype and atherosclerosis in type 1 diabetes (Kanter et al. 2012).

fat-5 SCD (47%)

Stearoyl-CoA desaturase

delta-5 desaturase activity is altered in type 2 diabetes patients; Role in the onset of diet-induced peatic insulin resistance (Imamura et al. 2014; Gutiérrez-Juárez et al. 2015).

ins-37; ins-8 insulin insulin-related

peptide beta type Insulin regulates glucose uptake; insulin resistance results in diabetes

ugt-62 glucagon Glucagon preprotein Control of carbohydrate metabolism (Bataille and Dalle 2014; Cho et al. 2014).

fut-2

FUT1 (23%) fucosyltransferase

FUT1; Fucosyl transferase; increased activity in human serum of type 2 diabetes; indicator of liver disease; FUT7 mutation in humans associated with noninsulin dependent diabetes (Bengtson et al. 2001).

wrt-4 DHH, IHH, SHH Hedgehog Hedgehog signaling is implicated in protecting beta-cells from cytokine-induced cytotoxicity (Puri et al. 2013; Teperino et al. 2014).

clec-70 CRD-4 (28%)

C-type carbohydrate recognition domain

C-type Lectin; binds sugar; implicated in innate immunity response (Feinberg et al. 2000; Hitchen et al.1998).

col-60 COL6A3 (46%) Collagen Collagen; deposition in obesity patients

(McCulloch et al. 2015)

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CHAPTER 5

MATERIALS AND METHODS

C. elegans Strains and Culture Conditions

The wild-type Bristol strain (N2) and mutant strains were cultured using

nematode growth media (NGM) plates (3g NaCl, 20.0g agar, 2.5g peptone, 975mL

diH2O, 1mL of 1M CaCl2, 1mL of 2mg/mL uracil, 1mL of 5mg/mL cholesterol, 25mL of

1M PPB, 1mL of 1M MgSO4) seeded with E. coli (OP50, HT115 or ΔPTS-OP50, as

noted in each experiment) and raised at 20ºC as described (Brenner 1974). Animals

were maintained on OP50 unless otherwise noted. Genetic mutants used for these

studies include: daf-2(e1370), daf-16(mu86), daf-2(e1370);daf-16(mu86), fat-

5(tm420);fat-6(tm331), daf-2(e1370);fat-5(tm420);fat-6(tm331), fat-6(tm331);fat-

7(wa36), daf-2(e1370);fat-6(tm331);fat-7(wa36), hyl-2(tm2031), daf-2(e1370);hyl-

2(tm2031), daf-2(e1370);sod-2(sj173);sod-3(sj134), glp-1(e2141), oga-1(ok1207), ced-

3(n717), hif-1(ia4) and egl-9(sa307). Adult hermaphrodites were allowed to lay eggs on

a plate and the subsequent offspring developed on the specified diet. Anatomical

markers used to identify developmental stage include gonad morphology or hours post

L4-adult molt. Adult animals used in the assays were collected 22-26 hours post the L4-

adult molt. To feed animals a sugar-supplemented diet, NGM plates were made

(60mmx15mm petri dishes filled with 10mL NGM); the plates were allowed to solidify for

at least one day before being seeded with 300μL of appropriate glucose (D-(+)-Glucose,

Sigma) stock solution, prepared in diH2O, to reach the final desired glucose

concentration. The concentration of glucose used depended on the specific experiment.

For example, to make the 0.5% glucose plates, 300μl of a 925 mM stock was used,

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(50mg/plate). Glucose solution is spread evenly, fully covering the entire plate, and

allowed to dry and reach equilibrium in the media for at least 24 hours. Plates were

seeded with 300μL of E. coli (OD600nm, 0.6≤0.9) that was spread evenly, covering the

plate. The bacteria solution was allowed to dry completely before animals were placed

on the media. Freshly made plates were used within a 7-day period. It is important to

note that the bacterial lawn must be seeded to cover the entirety of the NGM plate, as

results vary when the bacterial lawn is placed to only partially cover the NGM.

Bacterial Strains and Culture Conditions

Overnight cultures of all E. coli strains (OP50, HT115 or ΔPTS-OP50, as noted in

each experiment) were grown in LB (10g Bacto-tryptone, 5g Bacto-yeast, 5g NaCl, H2 O

to 1 liter, pH to 7.0 using 1M NaOH) at 37°C (OD600nm, 0.6≤0.9). E. coli OP50 is a uracil

auxotroph whose growth is limited on NGM plates. ΔPTS-OP50 does not grow in

minimal media with glucose as the sole carbon source, but grows similarly to OP50 and

HT115 E. coli strains in an overnight culture grown in LB at 37°C. For all experiments,

bacterial strains from overnight cultures that reached an OD600nm of 0.6≤0.9 were used

for seeding NGM plates.

Anoxia and Paraquat Exposure

Animals were placed into anoxia in BD Biobag type A anaerobic environmental

chambers (BD Biosciences, Rockville MD) at 20ºC (Padilla et al. 2002; LaRue and

Padilla 2011). Anoxic conditions were verified using resazurin indicators (<.001kPa of

O2 detection limit). Any trials that failed to reach anoxic conditions within 1.5 hours

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were nulled. After anoxia treatment, animals were allowed to recover for 1 day and the

surviving animals were examined for an unimpaired or impaired (abnormal movement or

morphology) phenotype. For oxidative stress assays using paraquat, 200 µl of a 150

mM of freshly made aqueous paraquat solution (Sigma-Aldrich) was distributed onto

small NGM breeder plates seeded with OP50 E. coli. The plates contained either 0.5%

glucose or no glucose. Media was allowed to dry before 10 synchronized 1-day old

adult hermaphrodites were transferred to each plate and kept at 20ºC. Animals were

assayed on a daily basis to examine survival rate. Five independent plates were

assayed.

Oil Red O Staining

To detect lipid levels in C. elegans of the specified genotypes, we used the Oil

Red O method as described (O’Rourke et al. 2009). Briefly, approximately 200-500

synchronized 1-day old adult animals were collected and washed 3 times with 1x PBS.

To permeabilize the cuticle, worms were resuspended in 120µL of 1X PBS to which an

equal volume of 2X MRWB buffer containing 2% paraformaldehyde was added.

Samples were rocked for 1 hour at room temperature. Animals were then allowed to

settle by gravity, supernatant was removed and animals were washed with 1X PBS to

remove paraformaldehyde. Worms were resuspended in 60% isopropanol and rocked

for 15 minutes at room temperature to dehydrate. Animals were then allowed to settle,

supernatant was removed and 1mL of 60% Oil Red O dye was added. Animals were

rocked overnight at room temperature in the dye. After allowing worms to settle, dye

was removed, samples were washed 1 time with 1x PBS and then resuspended in 200

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µL of 1x PBS to which 0.01% Triton X-100 was added. Animals were mounted on 2%

agarose slides and imaged with a Zeiss microscope and color camera. The total

number of animals stained was N ≥200 for each of 3 independent experiments. At least

10 animals per experiment and genotype were randomly imaged and assayed for the

presence of Oil Red O in the pharynx region.

Fatty Acid Supplementation

Fatty acid supplementation was conducted as described (Deline, Vrablik, and

Watts 2013). Briefly, NGM containing 0.1% Tergitol (NP-40) was autoclaved and cooled

to 55C, after which oleic acid sodium salt (Sodium Oleate, Nu-Chek Prep, Inc) was

added to a final concentration of 300uM. The supplemented plates are allowed to dry in

the dark for 2-4 days and then seeded with 300μL of OP50 E. coli (OD600nm, 0.6≤0.9)

that was allowed to dry in the dark for an additional 1-2 day(s). Embryos were then

added to supplemented plates and incubated at 20C until they reached day-1 of

adulthood. On day-1 of adulthood, animals were transferred to glucose-supplemented

plates for 1 hour before being exposed to 24 hours of anoxia while on the glucose-

supplemented plates.

Glucose Quantification

C. elegans were raised from embryo to day one of adulthood under standard, or

under glucose supplemented conditions. For each treatment, approximately 300-2000

synchronized 1-day old adult animals were collected from 3 large NGM plates, and

washed 3 times with M9 to remove bacteria. Animals were then allowed to settle by

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gravity, supernatant was removed, and 200uL of worm suspension was kept. A 50uL

aliquot of worm suspension was removed and frozen immediately for subsequent

protein quantification. The remaining 150uL worm suspension was sonicated on ice for

1 minute at medium speed. Glucose in lysed worm extracts was quantified using a

glucose hexokinase assay on a Unitech Chemwell-T autoanalyzer. Glucose

concentrations were normalized to the amount of total protein (mg) in each sample.

Protein Quantification

Approximately 300-2000 synchronized 1-day old adult animals were collected

from 3 large NGM plates, and washed 3 times with M9 to remove bacteria. Animals

were then allowed to settle by gravity, supernatant was removed, and 200uL of worm

suspension was kept. A 50uL aliquot of suspension was removed for protein

quantification. 50ul of tissue extraction buffer containing complete protease inhibitor was

added to the 50uL of worm suspension. Suspension was sonicated for 1-2 minutes on

ice at medium speed. Following sonication, 50uL of tissue extraction buffer with

protease inhibitor was added. The total volume of suspension was then centrifuged at

13,000 RPM for 12 minutes at 4C to remove debris. Approximately 80-100uL of

supernatant was removed for protein quantification. Protein was quantified using a

Millipore Direct Detect Spectrometer.

RNA Isolation

Young non-gravid adults were collected for mRNA isolation. We opted to collect

animals by an egg laying procedure rather than a hypochlorite protocol in order to

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minimize potential exposure to stress (L1 starvation) or reactive oxygen species (ROS)

induction by hypochlorite solution. Thus, to grow a large population of animals, gravid

adults were allowed to lay eggs on several plates for 2-4 hours. All adults were washed

off with M9 and the embryos, which remained on the plate, were allowed to hatch. L1

animals were collected and placed on respective media plates (OP50 only or OP50

supplemented with 0.5% glucose). Approximately 10,000 animals (per trial) were grown

to young adulthood, past the L4 molt but prior to egg production, before collection by

gravity separation with M9 in 15ml conical tubes. The animals were washed 3x with M9

and allowed to settle by gravity separation to remove bacteria. Excess M9 was

removed, and 4 volumes of Trizol per volume of animals were added. The tubes were

immediately frozen in liquid nitrogen, thawed and briefly vortexed; this step was

repeated and then followed by a 5-minute incubation at room temperature. To each tube

0.2 ml of chloroform per 1 ml of Trizol was added, mixed gently, for 15 seconds,

incubated at room temperature for 2-3 minutes, and then centrifuged at 12,000g for 15

minutes at 4ºC. The colorless upper phase was transferred to an RNase free tube. RNA

was then purified using an Ambion PureLink RNA Mini Kit (Cat#12183018A). Following

RNA purification, the RNA was treated with DNase I (Life Technologies Cat#

12185010), quantified using a NanoDrop Spectrophotometer and stored at -80ºC. RNA

was isolated from three independent experiments.

RNA-Sequencing Analysis

We performed next-generation RNA sequencing (RNA-Seq) experiments and

analysis to decipher the expression pattern of genes differentially regulated by glucose

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in wild-type C. elegans. For a reliable statistical analysis, RNA sampling was done in

three biological replicates both for wild type and glucose-fed C. elegans. The RNA

sequencing was done at the University of Texas Southwestern Genomics Core facility,

which resulted in generation of approximately 33.5M, 30.9M and 33.9M single-end

reads of length 50 bp for the three wild-type replicates (mean 32.8 million reads), and

34M, 32.5M and 32.1M single-end reads of length 50 bp for the three glucose-fed

replicates (mean 32.9 million reads). These deep sequencing raw transcriptome data

were then used for a comprehensive quantitative analysis of differential gene regulation

in glucose-fed C. elegans. We employed the frequently used, publicly available Tuxedo

suite of programs for RNA-Seq analysis, which includes the software Bowtie, Tophat

and Cufflinks (Langmead et al. 2009; Trapnell et al. 2009; Trapnell et al. 2010). Bowtie

is an ultra high-throughput sequence read aligner that takes the FASTQ files of

sequence reads as input and performs alignment of reads onto the reference genome.

Tophat utilizes the services of Bowtie to first obtain the read alignment and then parse

the alignment to infer the exon-exon splice junctions. A database of putative junctions is

built by examining the “islands” of reads aligning contiguously onto exonic locations.

The splice junctions are confirmed by aligning the reads against this database; the two

fragments of “junction” reads aligning to different genome locations provide the hints of

5’ and 3’ splice sites. Cufflinks utilizes the read alignment provided by Tophat to perform

the differential expression analysis of transcripts. We used the C. elegans genome build

WS195 at NCBI as the reference genome for obtaining the read alignment using Bowtie

and the splice junctions using Tophat. Cufflinks was used to identify the genes in

glucose-fed C. elegans with significant alteration in their expressions compared to wild-

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type C. elegans. This is accomplished by examining the difference in their abundance

under the two conditions. The abundance of a transcript is measured in terms of

“Fragments Per Kilobase of transcript per Million fragments mapped” (FPKM),

normalized for the transcript length and total number of cDNA fragments for a sample

replicate (Trapnell et al. 2010).The difference in expression is obtained as the logarithm

of fold change in abundance between the two conditions. Cufflinks performs a statistical

significance test for differential expression of each transcript based on a negative

binomial model estimated from the data (Trapnell et al. 2010). The transcripts passing

the significance test (False discovery rate (FDR) adjusted p-value to be less than 0.05,)

were examined further for their abundance fold change between the conditions

(Trapnell et al. 2010). Custom programs were developed for further parsing of this data

and classifying the differentially expressed genes based on functional annotation at

Wormbase database (http://www.wormbase.org/). The differentially expressing genes

were further grouped into different functional classes using gene ontology based

hierarchical functional classification and the Database for Annotation, Visualization and

Integrated Discovery (DAVID) functional classification tool

http://david.abcc.ncifcrf.gov/home.jsp). We used the Gene List Venn Diagram program

(http://genevenn.sourceforge.net) to compare our RNA-seq data set with other gene

expression datasets.

Quantitative RT-PCR

Animals were collected at day-1 of adulthood for mRNA isolation. RNA extraction was

performed as described above. Reverse transcription to generate cDNA was performed

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using SuperScript III first-strand synthesis system for RT-PCR (Invitrogen, cat #18080-

051). Quantitative RT-PCR was carried out using a ViiaTM7 Real-Time PCR system

(Applied Biosystems) and following primer validation, results were analyzed using the

comparative Ct method (Applied Biosystems ViiaTM7 Real-Time PCR system Booklet 3).

As per Hoogewijs et al. mRNA levels of Y45F10D.4 were used as an endogenous

control for normalization (Hoogewijs et al. 2008). The average of at least 2 technical

replicates was used for each independent experiment.

Statistical Analysis

All statistical analyses were performed using GraphPad Prism for Macintosh. A

one-way or two-way ANOVA followed by either a Bonferroni or Tukey’s Multiple

Comparisons Test was used to determine significance between 2 or more groups. A T-

test was used to determine significance between 2 groups. For all statistical analysis the

significance level was set at p<0.05.

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CHAPTER 6

DISCUSSION

An individuals diet can dramatically influence one’s phenotype, physiology, long-

term health and disease risk. A prime example of such a case is the continual increase

in sugar consumption within the human diet and the associated increases in chronic and

severe health issues such as obesity and T2D. Additionally, accompanying these

chronic illnesses are often severe metabolic and vascular dysfunctions (e.g.

cardiovascular disease, insulin resistance, hyperglycemia, hypertriglyceridemia) that

over time lead to detrimental and often fatal complications. Dietary-induced

hyperglycemia can disrupt the auto-regulation of blood flow resulting in oxygen

deprivation in different organs and tissues, and can also result in a decreased ability for

the body to effectively respond to ischemic events (e.g. stroke and myocardial

infarction) (Trayhurn 2013). Furthermore, microvascular and macrovascular dysfunction

can lead to multi-organ and tissue damage, blindness and amputations (Beckman et al.

2013; Creager et al. 2003; Paneni et al. 2013). In fact, as discussed in chapter 1,

vascular diseases are the principal causes of death in people with T2D, and 70% of all

lower limb amputations are a result of T2D complications (Frisbee; Jain et al. 2010;

Vinik and Flemmer 2002). Therefore, a thorough understanding of the impact that

metabolic abnormalities associated with T2D can have on oxygen deprivation

responses is necessary for a more complete understanding of the T2D disease process.

It is not well understood why chronic hyperglycemia (as is the case in T2D patients)

increases sensitivity to ischemic events.

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Glucose Induces Sensitivity to Oxygen Deprivation

Oxygen deprivation occurs when oxygen levels drop below what is considered as

a normal concentration for the organism, and in many cases is detrimental to survival.

However, some organisms are able to survive and mitigate the temporary loss of normal

oxygen tension however. Many hypoxia and anoxia tolerant organisms utilize

mechanisms such as a dramatic reduction in metabolism (hypometabolism), increased

buffering capacity, excess macromolecule stores and up-regulation of specific genes to

promote survival upon exposure to oxygen deprivation. The vast majority of vertebrates,

however, lack the ability to utilize such mechanisms, and thus are less tolerant of

oxygen deprivation stress. For these organisms survival in the face of oxygen

deprivation often comes in the form of stress-response proteins (heat shock proteins,

antioxidants, cytochrome P40s, etc.) or transcription factors (FOXO, HIF-1, etc.), which

can temporarily promote survival under such stress. Humans with compromised

metabolic homeostasis however, are more susceptible to ischemic events and these

events often lead to significant morbidity and mortality in T2D patients. Thus, their

capacity to tolerate even transient ischemia is significantly impacted. While many of the

signaling cascades and structural changes that promote the early onset of vascular

diseases are well characterized, the mechanisms regulating the increased trauma and

decreased survival seen in T2D patients following an ischemic event are not well

understood.

Given the relative ease of environmental and genetic manipulation, the C.

elegans model is frequently used to explore how exogenous factors influence

phenotypes. Thus, using this well characterized and simple genetic model system that

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is tolerant of oxygen deprivation exposure, we set out to identify genetic pathways by

which added glucose might impact oxygen deprivation responses. Glucose-

supplementation negatively impacts oxygen deprivation survival in wild-type C. elegans,

increases the amount of stored lipids, and increases sensitivity to the induction of

oxidative stress. Additionally, supplementation with 0.5% (27.75mM) glucose is

sufficient to increase whole worm glucose levels comparable to those seen in T2D

patients. While the difference in whole worm glucose levels between non-supplemented

and 0.5% supplemented C. elegans failed to meet statistical significance (p=0.1), it is

important to note that in mammalian systems, the levels of glucose are maintained

within a narrow window, and seemingly minimal increases or decreases from this

optimal blood glucose level (90 mg/dL or 5mM/L) can have dramatic impacts on

physiology, phenotype and gene expression (Table 1.1) (Szablewski 2011). Since

glucose concentration was measured in whole worm extracts, it remains to be

determined where in the body of C. elegans the excess glucose is stored (e.g., in

specific tissues, cell types or intracellular vs. extracellular). Furthermore, the specific

effects of glucose supplementation on oxygen deprivation response and survival are

partially modulated by other dietary factors (e.g., the specific bacterial food source).

Together these glucose-induced phenotypes demonstrated in C. elegans closely mimic

those seen in hyperglycemic mammalian models. Thus, these data further suggest that

C. elegans can be used to gain mechanistic insight regarding the regulation of the

combinatory stress of glucose supplementation and oxygen deprivation.

Moreover, while some of the mechanisms by which C. elegans survive anoxia

are well characterized (e.g., hypometabolism, fermentation, alternative electron

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acceptors, etc.) a complete understanding of what is required for proper cellular

maintenance during prolonged anoxia exposure is lacking. N2 animals fed a glucose

diet can survive 6 hours of anoxia, but not 24 hours of anoxia. This suggests that these

anoxia sensitive animals arrest properly (e.g., enter into suspended animation), but

likely cannot properly maintain an anoxia resistant phenotype over time. As discussed in

chapter 1, conserved mechanisms for the survival of oxygen deprivation across species

include decreased protein biosynthesis, a decline in membrane permeability across

tissues, declined firing frequency in the nervous system, and entrance into a state

hypometabolism, which allows the demand and supply of ATP to remain in a low,

steady state of flux. Thus, survival of oxygen deprivation requires not only a steady, low

balance of energy, but also the right cellular milieu of proteins to maintain proper cell

structure and function. During anoxia, for example, it has been shown that the

metabolism of C. elegans is depressed to just 3–4% of aerobic values (Föll et al. 1999).

Thus, it is plausible that glucose supplementation or a hyperglycemic state disrupts the

ability of cells to properly maintain this steady state of hypometabolism. Additionally

proper cellular maintenance during oxygen deprivation requires a specific subset of

genes to be expressed (e.g., genes required for anaerobic metabolism, cell stabilization,

oxygen sensing, etc.) while others that are not involved directly with maintaining the

physical integrity of the cell are typically downregulated. Thus, it is also likely that

glucose might impact the expression of genes required to maintain a hypometabolic

state or to maintain cellular integrity. For example, the accumulation of advanced

glycation end products, ROS and post-translationally modified proteins (hallmarks of

hyperglycemia and T2D) can alter the expression and/or functional activity of such

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required genes or proteins. Moreover the metabolism of glucose immediately preceding

anoxia exposure, and its abundance during anoxia might alter the ability of C. elegans

to re-route metabolites and properly utilize non-oxygen requiring metabolic pathways of

energy productions such as fermentation reactions.

Glucose-Induced Anoxia Sensitivity is Influenced by the Intestinal Microbiome

Given the results obtained by feeding C. elegans either OP50 or the ΔPTS-OP50

E. coli strain, I hypothesize that both glucose and the metabolism of glucose by the

bacteria impact the stress responses of C. elegans and that the specific repertoire of gut

bacteria in the worm during anoxia is likely important. While glucose induces sensitivity

to anoxia in all cases, the specific concentration of glucose required to elicit significantly

reduced survival is dependent upon the ability of the bacteria to transport and

metabolize glucose (Figure 2.5). In recent years, the nematode has emerged as a

proposed model for understanding, at least in part, the influence of the gut microbiome

on host metabolism, health and ageing (Cabreiro et al. 2013; Cabreiro and Gems 2013).

In a controlled laboratory setting, C. elegans are seemingly germ-free, apart from the

bacteria on which they feed (Montalvo-Katz et al. 2013). In the lab, C. elegans are

reared most often on a single bacterial source, the OP50 strain of E. coli (a uracil

auxotroph). C. elegans crush bacteria with their pharyngeal grinder, and are known to

crush the standard OP50 E. coli strain very efficiently (Avery and Thomas 1997). Adult

C. elegans contain a gut composed of 20 epithelial cells, arranged to form a tube with a

central lumen, thus they can (and have been shown to) harbor fairly large bacterial

populations (Portal-Celhay and Blaser 2012). Considering the natural habitat of

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C.elegans, their evolution likely occurred in the constant presence of intestinal

microbes, thus it is plausible that many aspects of biological function in the worm

evolved to operate in conjunction with and assisted by gut microbes (Cabreiro and

Gems 2013; Félix and Braendle 2010). For example, a variety of bacterial strains, used

as food sources, have been implicated to have a causal role in C. elegans lifespan,

development, gene expression, fat storage and stress response (MacNeil et al. 2013;

Brooks, Liang, and Watts 2009; Watson et al. 2013; Reinke et al. 2010; LaRue and

Padilla 2011). Additionally, similar to higher-organisms, even in a laboratory setting C.

elegans rely on their gut microbiota to provide essential micronutrients (e.g., folates)

and adult worms harbor 10 times more bacterial cells than their own somatic cells

(Nguyen and Clarke 2012; Cabreiro and Gems 2013). These findings underscore the

idea that bacteria can modulate C. elegans physiology and that the bacteria might

function as more than just a food source. Moreover, it has been shown that gut

microbiota in humans can impact metabolism and energy storage and is likely involved

in the development and control of obesity and inflammation, conditions strongly

associated with T2D (Patrice D. Cani and Delzenne 2009; van Olden, Groen, and

Nieuwdorp 2015; Hartstra et al. 2014; Delzenne and Cani 2010; Allin, Nielsen, and

Pedersen 2014; Cabreiro et al. 2013). Additionally, changes in diet can dramatically

impact gut microbiota and these changes in bacterial populations can impact the

permeability of the gut (Cani et al. 2008). Decreased membrane permeability is an

important feature of anoxia tolerance. Thus, it is likely that the ability of OP50 E. coli to

metabolize glucose might impact the microbiome of C. elegans in such a way that it is

detrimental under anoxic conditions. It is also worth noting that it was determined that E.

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coli is at least partly alive in the gut of the animals during anoxia exposure (Föll et al.

1999). It is also likely that the potentially toxic byproducts produced by the E. coli play a

role in the induction of anoxia sensitivity in glucose-supplemented animals. E. coli

(including OP50 and HT115 derivatives) for example preferentially utilize glucose as a

carbon source, and E. coli cells produce acetate as an extracellular byproduct of

aerobic cultivations under excess-glucose conditions (De Mey et al. 2007). While high

quantities of acetate have been shown to significantly alter pH, retard growth and inhibit

protein synthesis, its affect on C. elegans physiology and stress responses remains to

be determined. It is also important to note that in anoxia, C. elegans utilized mixed acid

fermentation, which produces multiple acidic end products. Thus, the combination and

possible over-accumulation of acidic byproducts from both E. coli and C. elegans might

be detrimental. Targeted manipulation of the gut microbiota (in humans and in simple

model organisms) could be an interesting approach in understanding the complex

relationship between diet, metabolic disease and stress sensitivity.

Glucose-Induced Anoxia Sensitivity is Modulated by Insulin Signaling via Multiple

Pathways

I determined that the insulin-signaling pathway modulates glucose-induced

anoxia sensitivity in C. elegans, and that daf-2(e1370) resistance under these

conditions requires the activity of multiple pathways. A mutation in the daf-2 insulin/IGF-

1 receptor led to anoxia resistance even when fed a glucose-supplemented diet, which

is not surprising given that the daf-2 animal is known to be resistant to stress in general.

Many of the phenotypes associated with the daf-2(e1370) mutant are mediated through

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the activation of the FOXO/forkhead transcription factor DAF-16; and a mutation in daf-

16 did indeed suppress the daf-2(e1370) glucose-fed anoxia survival phenotype. Using

a genetic approach I identified some downstream targets of DAF-16 (fat and sod genes)

and a DAF-16 independent gene (hyl-2) that are required for the glucose-fed anoxia

resistant phenotype exhibited by the daf-2(e1370) mutant (summarized in Figures 6.1

and 6.2).

Since excess glucose is thought to increase ROS levels, it seems appropriate

that antioxidant genes such as sod-2/3, which are activated in the daf-2(e1370) animal,

will protect against the effects of a glucose-supplemented diet. Moreover, the response

of mitochondria to oxygen deprivation is particularly critical given their role in energy

production. Additionally, it has been demonstrated that C. elegans mitochondrial (Mit)

mutants are resistant to oxygen deprivation. These mutants utilize a novel metabolism

that allows supplementation of their mitochondrial ETC with non-oxygen requiring

energy generating pathways, and thus they can prolong anoxia tolerance (Butler et al.

2010). It has also been recently demonstrated in C. elegans that cells adapts to oxygen

deprivation, at least in part, via regulated mitochondrial dynamics (Ghose et al. 2013).

Thus, there is a clear role for proper mitochondrial function as a profound effector of

anoxia response and tolerance.

It is not clear what the protective role fatty acid and ceramide biosynthesis plays

in the daf-2(e1370) animal fed a glucose-supplemented diet and exposed to anoxia.

However, consideration of the phenotypes exhibited by the mutations that suppress the

daf-2(e1370) glucose-fed anoxia resistant phenotype may shed some light on the

mechanisms required for anoxia survival. The fat and elo mutants have distinct fatty

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acid composition changes, with the fat-6;fat-7 double mutant displaying the most

significant alterations in the production of unsaturated fatty acids (Brock, Browse, and

Watts 2007; Shi et al. 2013). Moreover, all of the Δ9 fatty acid desaturase mutants

display decreased cold tolerance and increased endurance to heat stress (Horikawa

and Sakamoto 2009; Savory et al. 2011; Horikawa et al. 2008). The preservation of cell

membrane fluidity is thought to be important for cold tolerance in some organisms, as

an increase in temperature leads to weakened interactions among acyl chains, and

membranes tend to prefer a more fluid state. Thus it is not surprising that fatty acid

biosynthesis in C. elegans has an important role in the alteration of membrane

phospholipids to promote survival during cold stress (unsaturated fatty acids are

required to increase membrane fluidity). Moreover, as phospholipid bilayers experience

environmental fluctuations rendering them more fluid, changes in membrane

permeability occur (increased leackage) (Hochachka & Somero 2001). Thus, given the

importance of membrane permeability in terms of anoxia survival, a decreased

abundance of unsaturated fatty acids might be detrimental to anoxia survival by altering

the rigidity, and thus permeability of membranes in such a way that is not conducive for

the survival of oxygen deprivation. Further, since fat-2, fat-6 and fat-7 mutants display

strong sensitivity to oxidative stress, it seems likely that fatty acids in C. elegans are

important for the survival of oxidative stress, however the mechanistic action of these

fatty acids during oxidative stress is not known (Horikawa and Sakamoto 2009). It is

also thought that one of the primary causes of anoxia- induced death in mammals is

brain dysfunction and cardiac arrhythmias due to a loss of ionic integrity of cell

membranes (Boutilier 2001). Taken together these data highlight the importance of

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membrane stability, fluidity and permeability in the survival of stress. It appears likely

that alterations in fatty acid composition can negatively impact the adaptability of

membranes under stressful conditions, and thus compromise an organism’s ability to

survive. Additionally, while fatty acids are important structural components of cell

membranes, they also have functional roles in energy storage and as signaling

molecules. It has been determined that dietary fats influence the onset of insulin

resistance through an unknown mechanism, and hyperglycemia-induced increases in

circulating free fatty acids can lead to their accumulation in specific cell types (e.g., lipid

accumulation inside the muscle cell) which in turn can promote insulin resistance by

reducing insulin stimulated glucose uptake (Medina-Urrutia et al. 2015). Further, C.

elegans utilize mixed acid fermentation during anoxia, and these reactions are typically

redox balanced by short-chain unsaturated fatty acids (Butler et al. 2012). Thus, it is

plausible that alterations in fatty acid biosynthesis might negatively impact the ability of

C. elegans to properly make these particular short-chain fatty acids that are likely

important for their ability to maintain an anoxia tolerant state (e.g., continue fermentation

and glycolysis reactions by utilizing these alternative electron acceptors). While it

remains to be determined what specific role(s) fatty acid molecules play in terms of

anoxia survival in C. elegans, it is possible that in the daf-2(e1370) mutant the lipid

profile is altered in such a way that is conducive for maintenance of cellular structures

and function during anoxia stress, and that knock-down of genes involved with lipid

biosynthesis disrupts this lipid profile. For example, it is possible that a change in the

fatty acid composition of cell membranes influences receptor binding or activity, as well

as ion permeability and cell signaling. Additionally, fatty acids have the potential to act

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as ligands and can modulate the expression of downstream targets. It will be interesting

to tease apart the specific protective role of fatty acids in oxygen deprivation stress,

especially since dietary supplementation of the polyunsaturated fatty acid, oleic acid, is

sufficient to rescue glucose-induced anoxia sensitivity in daf-2;fat-6;fat-7 mutants.

It has recently been shown that ceramides are involved with a surveillance

system that detects mitochondrial defects thus, it is possible that the daf-2(e1370)

animal requires specific ceramides to aid in the defense against the toxic effects that

glucose supplementation has on mitochondria (Liu et al. 2014b). It remains to be

determined if the sod-2/3 antioxidant system will function independently or in

conjunction with ceramide and fatty acid molecules in promoting glucose-fed anoxia

survival in the daf-2(e1370) animal. The recent finding that specific ceramide species

are associated with type 2 diabetes, further implicates the role these lipid molecules

may have in metabolic dysfunction (Lopez et al. 2013; Larsen and Tennagels 2014; Xia

et al. 2014; Boon et al. 2013). Together these data suggest that a combination of

cellular changes ultimately contributes to anoxia sensitivity. We hypothesize that a

disruption in ceramide and fatty acid homeostasis might result in aberrations in

metabolic processes and/or stress response pathways that are required for the proper

maintenance of suspended animation, and thus survival of oxygen deprivation

(Summarized in Figure 6.2).

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Figure 6.1. Interactions Between Glucose Supplementation and Oxygen Deprivation. My work (highlighted in red) and work from others indicates that both genotype and glucose supplementation impacts C. elegans response to anoxia. Genetic analysis has revealed specific genes that have a role in either resistance or sensitivity to anoxia with and without glucose supplementation.

Glucose Significantly Alters Gene Expression in C. elegans

Using RNAseq, our goal was to focus on examining how a glucose-

supplemented diet impacts wild-type C. elegans prior to oxygen deprivation, by

comparing the gene expression profiles between animals fed a glucose-supplemented

diet to those fed a standard diet. Our findings along with what others have seen indicate

that a glucose-supplemented diet is a stress for C. elegans. We found that glucose

alters the expression of several genes classified as stress response genes. However,

this response does not enhance the capacity to survive additional stresses (e.g., anoxia,

paraquat-induced oxidative stress). This is aligned with the idea that glucose is a stress

and that an additional stress (such as that of anoxia), which can require overlapping and

99

distinct mechanisms for survival, overwhelms the system and leads to compromised

viability.

The differential expression of specific genes, in response to different

environments, does not necessarily imply functional relevance or essential function of

the genes relative to the environmental change. However, aligned with the genetic

mutant analysis data, we did identify differentially expressed genes that are compatible

with the notion that a glucose-supplemented diet alters many cellular functions, notably

metabolism (glycolysis, gluconeogenesis, lipid biosynthesis), stress responses

(chaperons, cytochrome P450 genes), protein modifications (protein glycosylation) and

cellular structure (collagen). Tightly aligned with the findings that glucose induces

significant lipid accumulation in C. elegans, a glucose-supplemented diet also induced

gene expression changes in many genes predicted to be involved with lipid metabolism

or function.

In wild-type animals, the mRNA expression of daf-16 or sod-2/3 was not

differentially regulated by a glucose-supplemented diet. However, there was a down-

regulation of daf-18, ins-8, and daf-15 which are involved in the insulin/insulin-like

signaling pathway but not necessarily directly regulated by DAF-16 (Murphy and Hu

2013). We identified 11 genes that are downregulated by a glucose diet and found by

others to be regulated in a DAF-16 dependent manner (Lee et al. 2009). Further

analysis of these genes would be helpful to determine if they have a role in the daf-

2(e1370) glucose-fed anoxia resistance phenotype. The idea that a glucose-

supplemented diet impacts insulin signaling is in line with the finding by others that

dauer formation (which is regulated by insulin signaling) is suppressed in animals fed a

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glucose diet (Mondoux et al. 2011). Regulation of the insulin-signaling pathway is

mediated through DAF-2 and DAF-18 with a down-stream affect being the activation or

deactivation of DAF-16 via post-translational phosphorylation/dephosphorylation. DAF-

18, the human PTEN tumor suppressor homolog, negatively regulates insulin-like

signaling by dephosphorylation of AGE-1-generated PIP3 which impacts AKT-1/AKT-2

kinase activity thus regulating DAF-16 (Ogg and Ruvkun 1998). Mutations in daf-18 can

suppress the dauer constitutive and longevity phenotype in daf-2 mutants. However, it is

important to recognize that a daf-18 allele may not impact an organism in the same

manner as a decrease in the expression of daf-18.

Moreover, to our surprise, RNAseq analysis revealed that nearly half (49%) of

the most differentially upregulated C. elegans genes (>25 fold upregulated) in glucose-

fed animals are predicted or known to encode a collagen protein. Interestingly, one

common mechanistic link between hyperglycemia and macrovascular disease is the

modification of collagen (thickening) and the endothelium via AGE induced protein

modification (Stitt, Jenkins, and Cooper 2002). Further, a recent study demonstrated an

important role for collagens in the increased lifespan phenotype of daf-2 mutant

animals. These mutants display significant upregulation of many collagens, some of

which are important for lifespan extension (Ewald et al. 2014). While there is some

overlap between these collagens and the ones identified by our RNAseq experiment,

particularly col-10, col-13 and col-120, we did not conduct RNAseq on daf-2 mutants,

and thus do not know if these collagens are altered in response to glucose feeding in

these mutants. Glucose- induced upregulation of these collagens in wild type animals

however, is not sufficient to confer anoxia survival. It will be interesting to investigate the

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role of specific collagens and extracellular matrix remodeling in glucose-induced anoxia

sensitivity, especially in the context of reduced insulin signaling.

Together these data show that a glucose-supplemented diet negatively impacts

oxygen deprivation survival and significantly alters gene expression profiles in C.

elegans. Additionally these data suggest that C. elegans share conserved biological

mechanisms in response to glucose- supplementation as those seen in higher

organisms. Thus, we hypothesize that by using C. elegans we can identify novel

mediators in hyperglycemia induced oxygen deprivation sensitivity and further elucidate

the mechanistic action of known mediators. For example, genetic suppression analysis

of the insulin signaling pathway (via mutation of daf-2) revealed that antioxidant activity,

fatty acid and ceramide biosynthesis are essential for the survival of anoxia during

conditions of glucose supplementation. Since, in wild-type animals, many genes were

upregulated in response to a glucose-supplemented diet and a large number of these

genes are known or predicted to be involved with metabolism, we suggest that many

processes (e.g. metabolism, cell signaling, stress responses) are altered in animals fed

a high-glucose diet and that central to glucose-induced anoxia sensitivity is

mitochondrial dysfunction via the disruption of lipid metabolism, ceramide homeostasis,

protein modifications and changes in ROS (summarized in Figure 6.2).

Lastly, it is important to note that while the overall applicability of the study of

complex diseases such as T2D using C. elegans is sometimes limited due to their lack

of complex organs and a circulatory system, studies such as this, utilizing C. elegans to

study specific events associated with such a disease is feasible. The utilization of high-

throughput sequencing, for example, can provide an important foundation for the

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discovery of potential therapeutic targets or dietary supplements that are conserved

between worms and humans. Additionally, using gene-silencing approaches (such as

RNAi) to explore the biological role of candidate genes that have been identified is more

straightforward and less costly to carry out in C. elegans compared with mammalian

models. Thus, while C. elegans may not be the best model to study T2D per se, we can

model specific aspects of the disease process (e.g., glucose-induced sensitivity to

oxygen deprivation) and identify potentially novel regulators of common complications

seen in hyperglycemic and T2D patients (e.g., macrovascular complications, ischemia).

Figure 6.2. Potential Impacts of Pathways Implicated in Glucose-Induced Anoxia Sensitivity. Genes involved in fatty acid (fat-5, fat-6, fat-7) and ceramide biosynthesis (hyl-2) and antioxidant activity (sod-2, sod-3) is required for resistance to glucose-induced anoxia sensitivity in the context of reduced insulin signaling (daf-2 mutants). Glucose-induced alterations in these implicated pathways (and possibly other pathways) can potentially impact mitochondrial function, gene expression, protein function and membrane fluidity, stability and permeability, thus resulting in a reduced ability to maintain an anoxia tolerant state.

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