ADRENERGIC REGULATION OF ADIPOSE TISSUE LIPOLYSIS IN TRANSITION DAIRY CATTLE BASED ON GENETIC MERIT AND ENERGY INTAKE By SHAWNESE MARIE ROCCO A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF ANIMAL SCIENCE NUTRITION Washington State University Department of Animal Sciences AUGUST 2010
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ADRENERGIC REGULATION OF ADIPOSE TISSUE LIPOLYSIS IN
TRANSITION DAIRY CATTLE BASED ON GENETIC MERIT AND ENERGY
INTAKE
By
SHAWNESE MARIE ROCCO
A thesis submitted in partial fulfillment of the requirements for the degree of
MASTER OF ANIMAL SCIENCE NUTRITION
Washington State University Department of Animal Sciences
AUGUST 2010
ii
To the Faculty of Washington State University:
The members of the Committee appointed to examine the thesis of Shawnese
Marie Rocco find it satisfactory and recommend that it be accepted.
John P. McNamara, Ph.D., Chair Derek McLean, Ph.D. Joseph H. Harrison, Ph.D.
iii
ACKNOWLEDGMENT
I would like to thank the faculty and staff of the Department of Animal
Sciences for providing the facilities and supportive environment that made this
thesis possible. This department has been nothing but supportive.
I would also like to acknowledge and thank Dr. McNamara for being a
supportive and effective advisor. I am truly grateful for your dedication to my
success and for inspiring me to do better.
Thank you, as well, to Dr. Derek McLean and Dr. Joe Harrison for serving on
my committee. You were a great resource to turn to and I very much appreciate you
taking the time to help me finish this great project.
I would like to extend my deepest gratitude to my parents, my sister, my
boyfriend, and my closest friends for providing me the strength to finish this
accomplishment. Thank you for your unwavering support, advice, and love because
without it I would never have made it this far.
More specifically I need to thank Vanessa Michelizzi for never failing to help
me get through every rough patch I’ve encountered in college from the first day I
met her as my roommate freshman year, until today as a fellow graduate student
and neighbor. I would never have made it through if you hadn’t been there.
iv
ADRENERGIC REGULATION OF ADIPOSE TISSUE LIPOLYSIS IN
TRANSITION DAIRY CATTLE BASED ON GENETIC MERIT AND ENERGY
INTAKE
Abstract
by Shawnese Marie Rocco, M.S. Washington State University
August 2010 Chair: John P. McNamara
In lactating dairy cattle the adipose tissue stores energy as triacylglycerol
(TAG) that can be used during early lactation. Breakdown of TAG (lipolysis) is
regulated by stimulation of the beta-2 adrenergic receptors (β2
Cows were selected for genetic merit (high merit, HM; low merit, LM) based
on sire predicted transmitting ability of milk (PTAM) and fed to requirements (NE)
or to 90% of energy requirements (LE). We took adipose tissue biopsies at 21 and 7
days prepartum; and 7, 28, and 56 DIM to determine rates of lipogenesis and
lipolysis; and to measure gene expression of key lipolytic genes (β
AR) leading to
activation of hormone-sensitive lipase (HSL). It is not known whether control of
lipolysis is also a function of increased expression of mRNA for the ß2-adrenergic
receptor, HSL, and perilipin (PLIN). A decrease in rates of lipogenesis (fatty acid
synthesis) also occurs in early lactation. Therefore, objectives of this project were to
help define adipocyte responses to lactation and energy deficit, including changes in
expression of proteins known to control lipid metabolism.
2, HSL, and
v
PLIN). The cows on the LE diet consumed 12% less feed prepartum and 16% less
feed postpartum. Dietary energy restriction decreased milk production overall but
HM, LE fed animals produced more milk (P < 0.03).
Serum glucose was relatively unchanged and serum NEFA were highest at 7
DIM (P < 0.02). The slowest rates of lipogenesis occurred at 7 and 28 DIM (P <
0.001). HM cows had faster rates than LM cows (P < 0.04) and dietary restriction
further decreased (P < 0.05) lipogenesis in early lactation. Lipolysis increased (P <
0.03) in early lactation in a pattern consistent with differences in milk production.
The expression of β2
AR, HSL, and PLIN did not change expression in NE cows due
to lactation, but expression was decreased in early lactation by dietary restriction (P
< 0.05). Data from this experiment support the hypothesis that regulation of
adipose tissue metabolism in lactation is a function of diet and genetic merit and is
controlled by multiple mechanisms including gene transcription and post-
translational protein modifications.
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TABLE OF CONTENTS Page
ACKNOWLEDGMENT ..................................................................................... iii
ABSTRACT ....................................................................................................... iv
LIST OF FIGURES ......................................................................................... viii
LIST OF TABLES .............................................................................................. xi
1a. Feed intake of cows varying genetic merit fed normally or an energy restricted diet, effect of dietary energy intake over time ................................. 27 1b. Feed intake of cows varying genetic merit fed normally or an energy restricted diet, effect of genetic merit over time ............................................... 28 2a. Milk yield (kg/DM) of cows varying genetic merit fed normally or an energy restricted diet, effect of energy intake ............................................. 31 2b. Milk yield (kg/DM) of cows varying genetic merit fed normally or an energy restricted diet, effect of genetic merit .............................................. 32 3. Rate of lipogenesis in cows of varying genetic merit fed normally or an energy restricted diet, effect of dietary energy intake and genetic merit ...................................................................................................... 48 4. Rate of lipogenesis in cows of varying genetic merit fed normally or an energy restricted diet, effect of dietary energy intake and genetic merit ...................................................................................................... 49
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LIST OF TABLES
1. Dietary Ingredients and Chemical Composition .......................................... 22 2. RT-PCR Primers Used for Determining Gene Expression in Bovine Adipose Tissue ................................................................................................... 24 3. Feed intake of cows varying genetic merit fed normally or an energy restricted diet, effect of genetic merit, energy intake, and parity prepartum and Postpartum ................................................................................................. 26 4. Milk yield (kg/d) of cows varying genetic merit fed normally or an energy restricted diet, effect of genetic merit, energy intake, and parity ... ....................................................................................................... 30 5. Body weight (BW), body condition score (BCS), and empty body fat (EBF) of cows varying genetic merit fed normally or an energy restricted diet, interaction effect of diet and genetic merit ....................................................... 35 6. Serum glucose (mg/dL) of cows varying genetic merit fed normally or an energy restricted diet, interaction effect of diet and genetic merit across target days in Milk ................................................................................. 38 7. Serum NEFA (µM) concentration of cows varying genetic merit fed normally or an energy restricted diet, interaction effect of diet and genetic merit across target days in milk........................................................... 41 8. Estimated energy balance (Mcal/d) of cows varying genetic merit fed normally or an energy restricted diet, effect of diet and genetic merit interaction across target days in milk .............................................................. 42 9. Rates of lipogenesis of cows varying genetic merit fed normally or an energy restricted diet, overall diets and genetic merit; effect of parity .......... 44 10. Rates of lipogenesis of cows varying genetic merit fed normally or an energy restricted diet, effect of diet and genetic merit .................................... 46
x
11. Rates of lipolysis of cows varying genetic merit fed normally or an energy restricted diet, effect of parity ............................................................... 52 12. Rates of lipolysis of cows varying genetic merit fed normally or an energy restricted diet, effect of diet and genetic merit .................................... 55 13. Beta-2 adrenergic receptor gene expression of cows varying genetic merit fed normally or an energy restricted diet, effect of diet and genetic merit ...................................................................................................... 60
14. Hormone sensitive lipase gene expression of cows varying genetic merit fed normally or an energy restricted diet, effect of diet and genetic merit ...................................................................................................... 63 15. Perilipin gene expression of cows varying genetic merit fed normally or an energy restricted diet, effect of diet and genetic merit ........................... 67
1
Introduction
The transition period around parturition in dairy cattle is a time of increased
metabolic stress. Many cows, though not all, experience a negative energy balance
during this period that impacts metabolic efficiency. Ideally, cows need to not only
produce many pounds of milk but also maintain body condition and body fat
reserves to supply the demand of milk production and maintain health status.
Dairy managers can lose around $30,000 per 100 head (“Metritis: A Foul Disease
With Financial Costs”, 2010) annually because of metabolic disorders such as
metritis, displaced abomasum, milk fever, mastitis, and ketosis resulting from a
poor plane of nutrition during the transition period. Sumner and McNamara (2007)
and others have shown that in addition to parity and dietary energy intake, genetic
merit plays a tremendous role in metabolic status and thus efficiency during the
transition period.
Present research is integrating knowledge regarding the interaction of
genetics and nutritional status, commonly referred to as nutrigenetics or
nutrigenomics. The emerging field of nutrigenomics aims to study how diet affects
specific genes and nutrigenetics aims to determine how expression of genes affect
how individuals respond to specific nutrients (Mutch et al., 2005). Research is
needed to elucidate more specific integrated knowledge of metabolism, gene
expression, and overall production. One application of this systems approach could
be to select for higher genetic merit cows that will produce the same amount or
more milk by minimizing a negative energy balance and resulting loss of efficiency.
2
The overall hypothesis was that animals that vary in genetic merit for milk
production and in energy intake will have a different pattern of lipid metabolism in
the adipose tissue, including expression of key regulatory genes.
Thus, the objective of this experiment was to investigate the mechanisms
involved in lipid mobilization and utilization in adipose tissue as these mechanisms
relate to the interaction of genetics and diet. The experiment was designed to
determine whether or not specific genes known to control lipid metabolism are
altered in expression in adipose tissue of dairy cattle with varied genetic merit and
energy intake.
Lipolysis and Lipogenesis in Adipose Tissue
Review of Pertinent Literature
Lactation is considered one of the most versatile and important developments
in the evolution of mammals. Mammals are adapted to carry a food-producing
organ for their young that provides for greater efficiency and adaptability to
seasonal, predatory, or climatic changes to increase survival rates. Wild-type
mammals tend to produce just enough milk to nourish their offspring; however,
humans have domesticated animals and selected them for increased milk
production to provide milk for human consumption.
Even though we have selected for rapid rates of milk production and
extended lactation periods in dairy cattle, the underlying common patterns of
regulation of nutrient use are still present. All mammals generally undergo a
period of transient reduction in food intake near parturition, followed by a rapid
3
increase in mammary growth, milk secretion, and concomitant demand for
nutrients. In first lactation animals the mammary gland undergoes extensive
growth and development in addition to the demand for milk secretion. The female
often responds by quickly increasing feed intake. In mammals as diverse as the
order rodentia; and the families of canidae, felidae, bovidae, and suidae, feed intake
can double in one or two days and increase three to five times the normal level in
one to several weeks (Verstegen et al., 1985; Munday and Earle, 1991; Case, 1999;
Tyler and Ensminger, 2006; Peterson and Baumgardt, 1971).
A correlate of the evolution of lactation has been the evolution of adipose
tissue. In mammals, adipose has become a highly active and adaptable organ. In
wild-type mammals, adipose tissue stores energy dense triacylglycerols in periods of
nutritional abundance (as in spring, summer, and early fall) to provide energy when
nutrient supply is limited in winter. Interestingly, many thousands of years of
domestication have not altered the circadian nature of body fat storage and use; as
domesticated animals still demonstrate a seasonal cyclicity of body fat reserves
(Vernon et al., 1997; McNamara et al., 1986).
In addition to the seasonal cyclicity, mammals in their reproductive years
have an additional pattern superimposed on the functions and amounts of adipose
tissue. In early pregnancy, mammals store an increased amount of body fat even
when energy intake is moderately limited. Later in pregnancy this energy can be
used to support rapid fetal growth, and then at the initiation of lactation, the body
fat can be used to provide fatty acids and glycerol for milk fat and lactose synthesis
4
as well as for energy use in other organs. This pattern of adipose tissue use is
present in all mammals but has been finely manipulated and developed through
selection in domesticated mammals, especially the dairy cow.
Selection for rapid rates of milk production has resulted in a large proportion
of dairy cattle that do not increase their rate of nutrient intakes quickly enough to
avoid a period of nutrient deficit. For many years this period of nutritional
deficiency resulted in an increased incidence of metabolic diseases and reproductive
problems. In the population of lactating dairy cattle, regardless of parity, genetic
merit, and management intensity; there is wide variation in milk production,
increase in feed intake in response to higher demand for milk production, and an
increase in metabolic diseases due to the negative energy balance that high
producing dairy cows incur during lactation. The U.S. Holstein population is not a
genetically or environmentally homogenous population; so during the peri-
parturient period the herd and individual cows within the herd vary in how much
energy they need to support milk production and maintain normal body function.
During the late 20th century there was an increasing trend in dairy cattle to
develop metabolic diseases such as ketosis, milk fever, displaced abomasum,
retained placenta, bloat, acidosis, and fatty liver as a function of being in an intense
or constant negative energy balance (Drackley, 1999; Tyler and Ensminger, 2006).
However, with increased emphasis on nutritional and environmental management,
nutritionists now do a much better job of formulating and feeding rations balanced
for proper nutrient content which are prepared and fed to maximize intake. As a
5
result of better management practices the incidence of most metabolic diseases has
declined dramatically. Nevertheless, there remains a wide variation not only in
milk production but in voluntary feed intake regardless of milk production level in
cows in the same herd on the same rations. There are continued presences of
subclinical metabolic deficiencies in energy and other nutrients that diminish
overall efficiency (Mulligan and Doherty, 2008). One aspect of the control of feed
intake and therefore clinical diseases, subclinical diseases, and milk production is
the metabolism of lipid in the adipose tissue.
Cows mobilize fat reserves to support milk production and without adequate
fat reserves the cow is unable to meet her genetic potential for milk production
because they are in a negative energy balance. However, there are usually high
producing cows in a herd that eat substantially more than their herd mates and, at
the same productive level, have less or even no negative energy balance
(Schactshneider et al., 2009). While many cows will go into that negative energy
balance, there are some cows—that even on a restricted energy diet—will not go
into as negative of an energy balance and can still maintain high milk production
(McNamara and Valdez, 2005; Schactshneider et al., 2009; Sumner and McNamara,
2007). Thus, adipose tissue can be a limiting factor in milk production.
These high producing and feed efficient cows are those that producers need.
Improvement of selection criteria to increase the overall milk production yield, feed
efficiency, and economic return of the herd would be beneficial. Theoretically, if
producers can select for a cow that meets her milk production potential with
6
minimal negative energy balance and loss of body fat to produce a high milk yield,
we can produce more milk with fewer cows and the cost of feeding would decrease.
McNamara (1989) demonstrated that cows with an introduced energy
restriction of around 13%, due to increased forage content, showed a decrease in
milk production of about 7%. The cows that showed the highest weight gain were,
interestingly, low genetic merit cows fed a high energy diet, suggesting that lower
genetic merit cows will partition more energy toward weight gain on a diet
optimally formulated for energy. The cows that showed lower gains in weight
illustrate that, during lactation, nutrients are prioritized to the mammary gland for
milk synthesis before rebuilding body fat reserves (McNamara, 1989). What we
ideally want are animals that partition energy to both milk yield and body
maintenance or growth. By looking at genetic merit and energy intake we can
determine which cows express key genes indicative of high milk yield, high feed
efficiency, and a milder negative energy balance during the periparturient period.
A period of negative energy balance can lead to a decrease in pregnancy rates
(Tyler and Ensminger, 2006). Many cows will decrease feed intake around
parturition which increases fat mobilization and the risk of metabolic disorders; as
well as possibly decrease milk production in both the short term and long term
reproductive life of the cow (Roche, 2006). A loss in reproductive efficiency is also a
loss in productivity which ultimately causes a loss in profit. This presents the
problem of ensuring that cows achieve a positive energy balance more quickly to
maintain efficient fertility and decrease the susceptibility to metabolic disorders.
7
In general, high producing cows tend to be more susceptible to metabolic
disorders than low producing cows (Guo et al., 2007, Guo et al., 2008), which implies
that higher producing cows that do not consume enough energy will be forced into a
more negative energy balance than the lower producing cows. Guo et al. (2008)
have developed a model based on NEFA and insulin concentrations in peri-
parturient cows and heifers to assess and perhaps predict the occurrence or relative
risk of a cow or heifer developing ketosis. However, the metabolic pathways and
their regulation underlying the model need to be understood completely to define
the variation and reasons why these animals may develop a metabolic disorder.
The major pathways of lipid metabolism in white adipose tissue include
lipogenesis, lipolysis, and ß-oxidation. During the peri-parturient period, lipolysis
increases and stays elevated throughout lactation whereas lipogenesis (or fat
synthesis) decreases greatly during early lactation and increases during mid-
lactation (McNamara et al., 1986; Doris et al., 1996). Lipogenesis is the process of
free fatty acid conversion to triacylglyerol (TAG). During nutrient deprivation there
is an increase in lipolysis and ß-oxidation to utilize fat stores for energy. Lipolysis
is the breakdown of triglycerides into free fatty acids and glycerol to be used for
energy. The free fatty acids are then oxidized to CO2 in ß -oxidation for energy and
glycerol is recycled to form more triglycerides or is used in the synthesis of
phospholipids to maintain the lipid membrane. The primary purpose of lipolysis is
to provide fatty acids for milk fat synthesis as well as energy. However, it is often
overlooked that in the dairy cow the glycerol released from adipose tissue can
8
provide glucose for milk lactose synthesis, possibly as much as 15% of the total
(Hanigan et al., 2007).
Rates of adipose tissue lipolysis and lipogenesis differ in animals depending
on age, physiological state, and energy intake (McNamara, 1994). In the transition
period there is often a substantial increase in lipolysis. There is already a fairly
developed knowledge base on the control of lipid metabolism; therefore current
research is concentrating on the details of the multi-faceted mechanisms regulating
lipolysis, including the mechanisms of genetic and transcriptome regulation.
Regulation of Lipolysis
Adrenergic Stimulation
Due to its key role in energy balance, the regulation of lipolysis was studied
in great detail during the 1960s and 1970s, at which time researchers such as Metz
and Van den Bergh (1971), Yang and Baldwin (1973), and Guidicelli et al. (1974)
were able to begin to elucidate the metabolic control of lipolysis. Scientists observed
that in a starved state certain metabolic pathways such as the G-coupled protein
receptor pathway of ß-agonist receptors which, once activated, converts adenyl
cyclase to cyclic AMP, (Gorman et al., 1972 and 1973) signal the cascade of events
that lead to the breakdown of triglycerides. The early work on lipolysis led to
development of the ‘fight or flight response’. This response, activated by the binding
of the catecholamines (epinephrine or norepinephrine) to a ß-adrenergic receptor,
signals the cascade of events that lead to the release and breakdown of
triacylglycerols from the adipocyte for immediate energy (Burns et al., 1981;
9
Lefkowitz, 1974). Evolutionarily, this response allows an animal to escape a
dangerous situation or predator with a quick burst of energy for either fighting or
running away to enhance the chances of survival. In dairy cows, this catecholamine
mediated response is a survival mechanism to provide energy in times of nutrient
deprivation to be partitioned to milk production. Humans, by genetically selecting
for higher producing cows, have capitalized on this adrenaline response to increase
milk yield.
Adipocytes are in a constant flux of lipolysis and lipogenesis. During
lactation, the rate of lipolysis increases in response to the negative energy balance
(Bauman and Vernon, 1993). Hormonally, lipolysis is signaled by the
catecholamines epinephrine and norepinephrine that bind to the beta-adrenergic
receptor and activate a G-protein coupled receptor to convert adenyl cyclase to cyclic
AMP. Cyclic AMP activates protein kinase A (PKA) to phosphorylate hormone
sensitive lipase (HSL) to its active state. In addition, PKA phosphorylates the co-
factor protein perilipin (PLIN), which allows HSL to access the hydrophobic
triacylglycerol (TAG) droplet. Hormone sensitive lipase activates the breakdown of
TAGs into free fatty acids. Free fatty acids are then oxidized through ß-oxidation in
the mitochondria of various organs to CO2
The actions of catecholamines also inactivate acetyl CoA carboxylase to
decrease lipogenesis. Insulin elicits the opposite response of the catecholamines by
inactivating cyclic AMP and activating acetyl CoA carboxylase to increase
lipogenesis (Salway, 2004). The role of transcription in control of lipogenesis has
for energy.
10
been fairly well established. In lactating dairy cattle, lipolysis may be controlled by
increased expression of mRNA for the ß2-adrenergic receptor, HSL, and PLIN
during the transition period (Sumner and McNamara, 2007). However, the body of
knowledge on this process is limited and we need to determine more specifically the
quantitative complex of control of lipolysis.
Hormone Sensitive Lipase
Hormone sensitive lipase in the adipose tissue is an 86 kDa cytoplasmic
protein (Shen et al., 1999). Holm et al. (1988) showed that hormone sensitive lipase
responds negatively to insulin, positively to catecholamines, and has no sequence
homology to other lipases. There are both a long form and short form codes for a
protein isoform of hormone sensitive lipase that is involved in lipolysis while the
long form is important for steroidogenesis in the testes (Kraemer, 2002).
Perilipin
Perilipin A is a 57 kDa protein that coats lipid droplets to prevent them from
being hydrolyzed by lipases such as hormone sensitive lipases (Kern et al., 2004).
Perilipin A is the primary perilipin involved in adipocyte metabolism. During
lipolysis, perilipin is phosphorylated by protein kinase A (PKA) that causes a
conformational change in the protein coating to allow lipases access to the lipid
droplet contents (Kern et al., 2004). The triacylglcerols in the lipid droplet can then
be hydrolyzed to non-esterified fatty acids (NEFAs). Sumner and McNamara (2007)
showed that PLIN mRNA levels were very highly expressed in bovine adipocytes
and was increased in adipose tissue at 90 DIM. There was a much smaller increase
11
in PLIN mRNA levels during early lactation when there is greatly increased
lipolysis. It may be that by 90 DIM, when milk production is at maximal rates,
increased expression of PLIN is needed to make more of this protein to maintain
fast rates of lipolysis. It is as yet unclear if perilipin has any regulatory role in
control of lipolysis, or a constitutive permissive role.
ß -adrenergic receptors
There are three ß-adrenergic receptor subtypes known with a possible fourth
involved in cardiac muscle function (Galitsky et al., 1997; Chruscinski et al., 1999;
Kaumann et al., 1998; Grujic et al., 1997; Cao et al., 1998; McNeel and Mersmann,
1999; Pietri-Rouzel et al., 1995; Sillence and Matthews, 1994; Forrest and Hickford,
2000; Liang and Mills, 2002). Beta-adrenergic receptors are found in many cells
and are old proteins found in simple invertebrates as part of a rudimentary neural
response system (Stiles et al., 1984). In general, the expression of the ß-adrenergic
receptor subtypes is involved in adipose metabolism in rodents, pigs, and cattle
(Castiella et al., 1994; Galitsky et al., 1997; Cao et al., 1998; Chruscinski et al.,
1999; Kaumann et al., 1998; Grujic et al., 1997; McNeel and Mersmann, 1999;
Mersmann 1996; Mersmann et al., 1997). The ß2-adrenergic receptor subtype is
most involved in lipolysis in cattle, if not all ruminants (Sumner and McNamara,
2007; Chruscinski et al., 1999) whereas the ß3-adrenergic receptor subtype is the
primary adrenergic receptor for lipolysis in humans, rodents, and other non-
ruminant mammals (Chruscinski et al., 1999; Grujic et al., 1997; McNeel and
Mersmann, 1999; Mersmann, 1996; Mersmann et al., 1997). The ß1-adrenergic
12
receptor subtype, in dairy cattle, is the least expressed of the three known ß-
adrenergic receptor subtypes (Sumner and McNamara, 2007) while the ß-
adrenergic receptor subtype that induces lipolysis strongest is the ß2-adrenergic
receptor subtype.
Receptor Desensitization
Receptor activity and responsiveness can be decreased if exposed to chronic
stimulation over time, also tending to decrease the affinity for receptor agonists to
bind (Portillo et al., 1995). This desensitization is either a decrease in affinity for
receptor agonists or down regulation of receptor synthesis (Carpéné, 1992).
Beta adrenergic receptors are found within the G-coupled protein receptor
family and are characterized within the seven transmembrane g-coupled protein
receptor (7TMR) superfamily. A ligand binds to the 7TMR which causes a
conformational change in the receptor at the carboxyl group intracellularly. This
conformational change either promotes the activation of a second messenger
system, such as cyclic AMP (cAMP), or an inhibitory system through
phosphorylation of a G protein-coupled receptor kinase (GRK) that promotes the β-
arrestin adaptor protein to inhibit the second messenger activity (Rajagopal et al.,
2010). The β-arrestins desensitize the receptor by recruiting enzymes to degrade
the second messenger cAMP (Rajagopal et al., 2010). While desensitizing the
adrenergic receptors will reduce second messenger systems such as the cAMP
pathway that leads to lipolysis, the desensitization does not completely shut down
the entire system (Vicario et al., 1997). While this experiment did not focus on the
13
endocrinology of the 7-transmembrane receptor superfamily it is important to note
that current research in this area is finding that 7TMR and the ligands that bind
them have the ability to selectively recruit β-arrestins and or GRKs for regulating
the pathways activated by the ligands. For example, Β2AR ligands bind the
receptor, causing a conformational change that can allow the receptor to selectively
recruit β-arrestins (Rajagopal et al., 2010, Drake et al., 2008). These β-arrestins
recruit a signaling scaffold to recruit proteins that can internalize and essentially
desensitize the β2ARs (Drake et al., 2008, Willoughby et al., 2007). β-arrestins have
been shown to reduce the amplitude of cAMP signals by possibly recruiting
phosphodiesterase-4D to degrade cAMP, which leads to the attenuating β2AR
signals observed during β-arrestin recruitment (Willoughby et al., 2007).
Lipogenesis
Lipogenesis is the process of synthesizing triacylglycerol via esterification of
fatty acids and glycerol. Mature adipocytes in the adipose tissue are made up of a
lipid droplet containing primarily triacylglycerol. During homeostasis lipogenesis is
in simultaneous flux with lipolysis so that the animal generally is constantly
breaking down and rebuilding adipose stores. However, during lactation the
demand for energy increases and as a result lipogenesis is typically reduced to an
altered state of flux known as homeorhesis (Bauman and Currie, 1980). For
example, Tepperman and Tepperman (1970) discussed how animals in a starved
state will decrease rates of lipogenesis to mobilize body energy stores, typically in
the form of adipose. In contrast, when animals are refed, rates of lipogenesis
14
increased and surpassed rates of lipolysis to rebuild the energy stores lost to
starvation.
Glucose supply is the major driver of lipogenesis. Lipogenesis is a major
component of adipose tissue metabolism during the transition period in dairy cattle.
Rates of lipogenesis in relation to rates of lipolysis indicate the effect of negative
energy balance in a cow and perhaps how quickly the cow can reach a positive
energy balance. Research done in this laboratory demonstrated that lipogenesis is
highly sensitive to energy intake, falling quickly to zero as energy balances reduces
to zero (McNamara and Hillers, 1986). In addition, animals of higher genetic merit
for milk production have lower rates of lipogenesis even at the same energy intake.
However, it is clear that lipogenesis is much more responsive to diet than is
lipolysis; whereas lipolysis is a function of genetic differences. Herein is the crux of
nutrigenomics and nutrigenetics in the control of efficiency in dairy cattle: what is
the totality of the mechanisms that control lipid metabolism.
Acetyl Co A
Acetyl CoA Carboxylase is an enzyme responsible for regulating lipid
mobilization and malonyl coA synthesis—which is the substrate for fatty acid
synthesis (Wakil, 2008). Insulin upregulates Acetyl CoA Carboxylase in times of
exogenous energy availability to store excess energy as glycerol and triglycerides.
Acetyl CoA Carboxylase helps regulate lipid mobilization by acting as a product and
intermediate for β-oxidation.
Control of Lipolysis and Lipogenesis During Lactation
15
The mechanisms of control of lipogenesis by endogenous energy availability
and physiological state are well defined. The control of lipolysis is not as fully
understood. It is well known that lipolysis is controlled by the sympathetic nervous
system through binding of the β-adrenergic receptor and subsequent 2nd
In dairy cattle, genetic merit plays a role in determining or controlling rates
of lipogenesis and lipolysis, especially during lactation. For example, higher genetic
merit cows are able to more efficiently balance rates of lipolysis and lipogenesis to
respond to a decrease in energy balance.
messenger
cascade as defined above. However, it is not clear whether or to what extent control
of transcription affects lipolysis. In most situations lipogenesis and lipolysis are
controlled in a concerted balance to prevent the mobilization of excess energy or
unnecessary storage.
Milk production and feed efficiency are both a function of the rates of lipolysis
and lipogenesis. The cow tends to protect milk production by mobilizing body stores,
which is a function of both increased lipolysis and decreased rates of lipogenesis.
During physiological states of increased productivity the maintenance rates of
lipolysis and lipogenesis are altered to function in a different pattern, known as
homeorhesis. Usually during lactation lipogenesis decreases and lipolysis increases
to mobilize body fat to supply energy for milk production.
For example, during the transition period dairy cows will typically reduce
their feed intake around parturition which puts them at a decreased energy balance
that often becomes negative around lactation. Once the cow starts lactating she
16
will typically experience an increase in lipolysis and decrease in lipogenesis. The
cow will typically experience a decrease in empty body fat, serum glucose, insulin,
and acetyl coA carboxylase which attenuates the rate of lipogenesis; while
increasing transcription of HSL, PLIN, and β2-adrenergic receptors which enhances
lipolysis. This homeorhetic increase in lipolytic protein transcription is also closely
regulated by the sympathetic nervous system release of catecholamines and other
hormones that may enhance the rates of lipolysis.
Because of the complex nature of metabolic control, and the relative lack of
knowledge on mechanisms of control of lipolysis, we conducted an experiment to
attempt to define mechanisms of lipolysis by nutrigenomic and nutrigenetic
controls. How do animals of different genetic merit control lipolysis and lipogenesis
when faced with an energy restriction that is sufficient enough to alter energy
balance, but not severe enough to be out of a normal range for the average dairy
cow? Therefore, the overall hypothesis was that animals that vary in genetic merit
for milk production and in energy intake will have a different pattern of lipid
metabolism in the adipose tissue, including expression of key regulatory genes.
Thus, the objective of this experiment was to investigate the mechanisms
involved in lipid mobilization and utilization in adipose tissue as these mechanisms
relate to the interaction of genetics and diet. The experiment was designed to
determine whether or not specific genes known to control lipid metabolism are
altered in expression in adipose tissue of dairy cattle with varied genetic merit and
energy intake.
17
Methods and Materials
Animals and Treatment Protocol
Forty-eight Holstein cows from the Knott Dairy Herd (Pullman, WA) were
selected, blocked by parity (1st or 2nd) and by sire genetic merit as predicted
transmitting ability for milk (PTAM). There were 24 1st lactation and 24 2nd
lactation animals. Genetic Merit sire PTAM average was 1913 (High Genetic Merit,
HM) or 832 kg (Low Genetic Merit, LM) (SD 686). For 1st lactation animals sire
PTAM was 2072 (HM) and 787 kg (LM) and for 2nd lactation animals sire PTAM
was 1691 (HM) and 907 (LM). The 305ME for HM 2nd
Animals were fed once a day through Calan gates (American Calan, 1997;
Northwood, NH) between 10:00am and 11:00am. Animals were adapted to the
gates approximately 3 to 7 days prior to beginning dietary treatments which began
21 days prepartum and continued through 56 DIM. Normally fed (NE) animals
were fed to achieve 5 % ORTS, and LE animals were fed to achieve 90% of that
lactation animals was 30,582
kg and for LM it was 27,997 (SD 3,893), which places these animals in the top 10 %
of production in US Holsteins. Dietary treatments were either fed at TMR to
requirements (NRC, 2001; NE); or fed a TMR at 90 % of the intake of the NE group
based on intake as a percent of BW (LE) (Table 1). The LE diet was fortified with
10 % more protein as well as vitamin and mineral mixes so that was consistent
across groups, however other dietary compositions were not altered so that the
experimental model was a difference in overall energy intake regardless of energy
source.
18
intake as a % of BW. Intake as a % of BW was calculated daily for adjustments.
Dietary ingredients were sampled with each new batch; the TMR and orts were
sampled weekly and composited monthly for analysis at Kuo Labs (Othello, WA)
using AOAC methods for DM, ADF, NDF, CP, fat, Ca, and P (AOAC, 2000).
Samples and Measurements
Cows were milked twice a day and yield was measured daily. Milk
composition was determined approximately monthly by DHIA sampling using
infrared spectrophotometry at the regional DHIA laboratory in Burlington, WA
(AOAC, 2000). Body weight (BW) and body condition score (BCS) (Bernabucci et al.,
2005; Waltner et al., 1994) were assessed weekly. Body weight and BCS were used
to calculate body fat (Waltner et al., 1993). Blood was collected weekly via
venipuncture of the coxygeal vessel at 28, 21, 14, 11, 7, and 4 days prepartum and
then postpartum at days 1, 3, 7, and then weekly until week 8.
Subcutaneous adipose tissue biopsies were collected at 21 and 7 days
prepartum and at 7, 28, and 56 days postpartum, from the tail head region under
spinal anesthesia (Sumner and McNamara, 2007). Part of the sample was
immediately placed in Krebs/HEPES buffer (Sumner and McNamara, 2007) at 37°
C for tissue incubations to estimate rates of lipogenesis and lipolysis and part was
immediately homogenized in Qiazol reagent (Qiagen 75842, Valencia, CA; 91355)
and the homogenate frozen until extracted for RNA.
Analytical Methods Blood serum was collected and analyzed for non-esterified fatty acids (NEFA-
19
C kit; Wako Chemicals, Richmond, VA) with the modifications published previously
(McNamara and Hillers, 1986) and for glucose (Glucose (HK) Kit, Sigma-Aldrich;
St. Louis, MO).
Rates of lipogenesis were measured in vitro using adipose tissue incubated in
medium containing KREBS/HEPES buffer, 2% bovine serum albumin (fatty acid
free); 5 mM glucose and 0.5, 1, 2, 4, 8 mM acetate at 0.1 µCi/mM 2-C14
Incubations of adipose tissue were used to measure basal and stimulated
rates of lipolysis. Adipose tissue was sliced and pre-incubated in 2 ml of Krebs-
Hepes media containing 2 % bovine serum albumin (fatty acid free) for 20 min to
remove the effects of handling and slicing (McNamara and Hillers, 1986b;
McNamara and Valdez, 2005). The media was then removed and replaced with
fresh media and the tissue was incubated another 2 hours. Basal media had no
added stimulators. The response curve to beta-adrenergic receptor binding was
conducted using isoproterenol at 10
acetate, at
pH of 7.4 and 37º C for 2 hours in triplicate. The tissue was sliced to a thickness of
approximately 500 µm on a calibrated microtome (Etherton et al., 1977) and was
measured into approximately 80-100 mg slices for the triplicate incubations. After
incubation, samples were placed in DOLE’s reagent (Smith and Crouse, 1984) to be
extracted for total fatty acid synthesis. Rates were reported as mM acetate
converted to fatty acids per 2h/g tissue.
-8, 10-7, 10-6, 10-5, and 10-4 M. Adenosine
deaminase (6.6 U/ml; Calbiochem, #116880) and theophylline (1mM; Sigma-Aldrich
no. 200-305-7) were included to maximize response to isoproterenol. Rates of
20
lipolysis were expressed as glycerol release in nm/g tissue per 2h.
Gene Expression
Adipose tissue was saved for mRNA extraction in duplicate, each duplicate
was placed in 5 ml of Qiazol and RNA was immediately homogenized and chilled to
-20°C until extraction; then extracted using the RNA-easy midi-kit (Qiagen 75842,
Valencia, CA; 91355). The quality of the mRNA, once extracted, was determined by
re-suspending the RNA in RNAse free water and using the NanoDrop1000 (Thermo
Fischer Scientific; Wilmington, DE) spectrophotometer to estimate RNA purity via
the ratio of A260/A280. The absorbance for pure RNA should have an A260/A280
(RNA to Protein) ratio of between 1.9 and 2.1. For most samples purity was also
assessed on a 1.2% agarose gel to visualize quality of the RNA.
First strand cDNA synthesis was performed once quality and purity of RNA
was assessed and confirmed. A reverse transcriptase (RT) and a no RT control were
made from each set of cDNA to be run on the ICycler real time PCR machine
(BioRad, Hercules, CA). The primers in Table 2 were used to determine gene
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