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Metabolic Reprogramming by Hexosamine Biosynthetic and
Golgi N-Glycan Branching Pathways
By
Michael Christopher Ryczko
A thesis submitted in conformity with the requirements
Additional intramolecular functions of glycans include regulation and monitoring of folding in the
ER, and maintenance of three-dimensional conformation (Ohtsubo & Marth, 2006).
Glycan complexity increases with vertebrate phylogeny, suggesting involvement in
multicellular development and morphogenesis, i.e. the flow and exchange of information required
to transform a collection of cells into a coherent society of different interacting components
(Ohtsubo & Marth, 2006). A large-scale N-glycoproteomic study revealed that more than 10% of
the mouse proteome is N-glycosylated (Zielinska et al, 2010). Intracellularly, glycans within the
secretory pathway regulate protein maturation, quality control, turnover, and trafficking of
molecules to organelles (Ohtsubo & Marth, 2006). The abundance, diversity and ubiquity of N-
glycan structures at the cell surface suggest a significant role for encoded information that is
distinct from the genome. Cell surface N-glycans on glycoproteins serve as ligands for a number
of evolutionarily conserved carbohydrate-binding protein families, such as C-type lectins,
galectins, and siglecs, whose function is to decipher biological information conveyed by the vast
array of N-glycans (Dennis et al, 2009). Functional interactions of galectins with cell surface
glycoconjugates can modulate cellular functions such as cell signaling and cell adhesion (Dennis
et al, 2009). Located at the interface of the inside and outside of the cell, N-glycans are perfectly
positioned to interact with the extracellular environment of the cell, and to mediate a variety of
recognition events and biological processes in health and disease through protein-glycan
interactions (Dennis et al, 2009). Changes in patters of cell surface N-glycans often accompany
development of cancer and metastasis. In fact, altered glycosylation, either increase or decrease of
specific N-glycans, or the appearance of glycans normally restricted to embryonic expression, is
frequently observed in tumours (Dennis et al, 2009). These structural alterations are often the result
of changes in expression and activity of glycosyltransferases, and their substrate availability.
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1.1.3 N-Glycosylation
N-linked glycosylation occurs through co- and post-translational modification of
membrane and secreted glycoproteins in eukaryotic cells. N-glycans are attached to the nitrogen
atom of asparagine (Asn) residues in the peptide consensus sequon Asn-X-Ser/Thr, where X
corresponds to any amino acid except Pro (Dennis et al, 2009). The presence of a sequon is
necessary but not sufficient for N-glycosylation to occur, as some sequons are not glycosylated
(Zielinska et al, 2010). Thus, when Asn-X-Ser/Thr motifs are present in the amino acid sequence
of a protein they are not identified categorically as N-glycan sites, but rather are referred to as
potential N-glycan sites. The enzyme oligosaccharyltransferase initiates protein N-glycosylation
by transferring a pre-assembled sugar oligosaccharide from dolichol to a nascent protein. Factors
influencing oligosaccharyltransferase catalysis include availability of precursors, enzyme activity,
the number of sequons in a glycopeptide, and their conformational accessibility (Schachter, 1986).
N-glycosylation is catalyzed by a series of enzymes functioning in a sequential and
competitive manner in the rough ER (rER) and Golgi apparatus, where it assumes an assembly-
line style of manufacturing (Schachter, 2010). Different glycosyltransferases reside in different
compartments of the Golgi, where they act in a specific order during glycoprotein transit through
the secretory pathway (Dennis et al, 2009). Moreover, many glycosyltransferases are expressed in
a tissue and time specific manner (Ohtsubo & Marth, 2006). Glycosyltransferases are single-pass
type II integral membrane proteins with a short cytoplasmic amino terminal domain, a
transmembrane anchor domain, a Proline-rich neck or stem region, and carboxyl terminal catalytic
domain (Schachter, 2010). Glycosyltransferases use activated high-energy sugar-nucleotides as
donor substrates, attaching them to polypeptides or to the growing glycan chain (Ohtsubo & Marth,
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2006). N-linked glycosylation is required for proper function of numerous glycoproteins. For
instance, cell surface delivery and retention of receptors and nutrient transporters depends on their
N-glycan branching, which is both under genetic and metabolic control (Dennis et al, 2009). The
extent of N-glycan branching is dependent on the expression and kinetics of medial Golgi enzymes
- the mannosidases and acetylglucosaminyltransferases, the metabolic flux through the
hexosamine biosynthetic pathway (HBP) to generate the high-energy sugar-nucleotide donor
uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc), as well as protein synthesis rate and
availability of glycoprotein acceptors (Dennis et al, 2009).
N-glycosylation of proteins starts with an en block transfer of pre-assembled lipid-carrier-
linked Glc3Man9GlcNAc2 donor to Asn residue in receptive Asn-X-Ser/Thr sequons of nascent
proteins by oligosaccharyltransferase in the lumen of rER (Schachter, 2010). The transferred pre-
assembled Asn-linked glycan is then extensively remodeled by removal and addition of various
monosaccharides during transition through the rER and Golgi apparatus en route to the cell surface
(Dennis et al, 2009). Remodeling, or lack thereof, results in formation of three main types of N-
linked glycans on mature glycoproteins: high-mannose, hybrid, and complex (Schachter, 2010).
All N-glycans contain a single conserved core structure consisting of two GlcNAc residues
(chitobiose core), and three Man residues (tri-mannosyl core), forming a common penta-saccharide
(Man3GlcNAc2) core structure (Schachter, 2010). This core is further linked to other sugars to
form a variety of different branched N-glycans. UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-
phosphate transferase (GNPTA/DPAGT) is the enzyme in the first committed step of the dolichol-
linked oligosaccharide pathway for N-glycan biosynthesis to utilize UDP-GlcNAc as a substrate
(Schachter, 2010). This initial step can be blocked by the drug tunicamycin, an analog of UDP-
GlcNAc that inhibits GNPTA (Schachter, 2010).
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In metazoans, trimming is performed by glucosidases and mannosidases, while extension
is carried out by Golgi N-acetylglucosaminyltransferases (Mgats) (Dennis et al, 2009). The high-
mannose structures are formed from the oligosaccharide precursor by α-mannosidase-mediated
cleavage of Man residues, without addition of any other monosaccharides (Schachter, 2010).
Complex-type N-glycans are formed by successive removal and addition of monosaccharides, and
are characterized according to the number of GlcNAc branches attached to terminal Man residues
on the core penta-saccharide structure (Taniguchi & Korekane, 2011). Depending on the number
of branches attached, complex-type N-glycans are subdivided into bi-, tri-, tetra-, and penta-
antennary structures. The hybrid-type N-glycans share structural features found in both high-
mannose and complex-type N-glycans (Schachter, 2010).
1.1.3.1 N-Glycan Branching and Glycan-Galectin Lattice
Biosynthesis and branching of complex N-glycans proceeds via linkage of GlcNAc by
mannosyl glycoprotein N-acetylglucosaminyltransferase (Mgat) enzymes, also known as GlcNAc-
transferases, to the conserved core Man residues (Dennis et al, 2009). Each Mgat transfers GlcNAc
in a specific linkage (Taniguchi & Korekane, 2011) (Figure 1.1). GlcNAc branches in turn are
further extended through sequential addition of Gal and other monosaccharides to complete
elongation of glycan antennas that act as ligands for galectins (Dennis et al, 2009) (Figure 1.2).
Galectins are a family of secreted proteins. Most of them are bivalent or multivalent with regard
to their carbohydrate-binding activities (Dennis et al, 2009). Membrane glycoproteins carrying N-
glycan branches offer binding sites for galectins to facilitate transmembrane glycoprotein cross-
linking, thus forming a glycan-galectin lattice that regulates cell surface residency and activity of
numerous receptors, cell adhesion proteins, and solute transporters (Dennis et al, 2009)
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Figure 1.1 Mgats and Their N-Glycan Branches
An idealized N-glycan showing different possible branches, with specific linkages and the glycosyltransferase enzymes (Mgat1, Mgat2, Mgat3, Mgat4, Mgat5 and Mgat6) responsible for their formation. Each GlcNAc branch may be elongated with galactose, poly-N-acetyllactosamine, sialic acid and fucose (Taniguchi & Korekane, 2011).
Figure 1.2 Mgat5 in N-Glycan Branching
The branched N-glycans attached at N-X-S/T sequons in mammalian glycoproteins. Mono, bi, tri, and tetra refer to the number of branches in an N-glycan. The glycosyltransferase Mgat5 catalyses the addition of a β1,6-linked GlcNAc branch (green arrows) to form tri- or tetra-antennary N-glycans, the most complex types of branched N-glycans in mammals. In general, the more GlcNAc branches per N-glycan, the more Gal residues are added and elongated to form poly-N-lactosamine [Galβ1,4GlcNAc]n. Galectins bind Gal and form specific cross-linked lattices with glycoproteins, which increases their cell surface expression (Stanley, 2007).
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(Figure 1.3). Galectins are N-acetyllactosamine (Gal and GlcNAc) binding proteins whose major
ligands are Golgi-remodeled N-glycans common to cell surface glycoproteins (Dennis et al, 2009).
Poly-N-acetyllactosamine glycan structures serve as high affinity ligands for galectins, which bind
to N-glycans of glycoproteins with affinities proportional to GlcNAc content (Dennis et al, 2009).
Glycoproteins with N-acetyllactosamine glycans interact with galectins in a cross-linking manner,
which leads to their retention at the cell surface by slowing later mobility, and delaying loss by
constitutive endocytosis (Dennis et al, 2009). Increased cell surface residency in turn leads to
greater sensitivity to extracellular cues and promotes receptor mediated signaling (Johswich et al,
2014; Lau et al, 2007; Mendelsohn et al, 2007). Sustained surface exposure and clustering of
signaling receptors creates a platform to multiply ligand-induced signal intensity.
1.1.3.2 Golgi N-Glycan Branching Pathway
1.1.3.2.1 UDP-GlcNAc
UDP-GlcNAc is an essential common donor substrate required by all Mgat enzymes. UDP-
GlcNAc is synthesized in the cytosol by the HBP and transported through sugar-nucleotide
transporters into the medial Golgi apparatus (Dennis et al, 2009) (Figure 1.4). The Golgi N-glycan
branching pathway is characterized by multistep ultrasensitivity to UDP-GlcNAc for branching
enzymes Mgat1, Mgat2, Mgat4, and Mgat5 (Dennis et al, 2009). The relative affinity of branching
enzymes for UDP-GlcNAc declines sequentially by ~300 fold (0.04 to 10 mM) moving down the
N-glycan branching pathway from Mgat1 to Mgat5, while this trend is reversed for their respective
glycoprotein N-glycan acceptors (Dennis et al, 2009). Thus, activities of enzymes Mgat1 and
Mgat2 are limited by low affinity for acceptor glycoproteins, while activities of enzymes Mgat4
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Figure 1.3 Glycan-Galectin Lattice Dynamics
The glycocalyx is the thick carbohydrate layer surrounding the cell. Glycan structures generated in the Golgi differ in affinities for galectins. Galectins cross-link glycoprotein receptors and oppose (1) loss of EGFR to Caveolin 1-positive microdomains, (2) coated-pit endocytosis, (3) precocious clustering of receptors, and (4) F-actin-mediated entry of T-cell receptor into and exit of CD45 from ganglioside GM1-positive microdomains (blue). (5) Nutrient supply and growth signaling increase membrane remodeling, regulate metabolite flux through the hexosamine biosynthetic pathway to UDP-GlcNAc and Golgi N-glycan branching on receptors and transporters to promote surface retention by the glycan-galectin lattice (Dennis et al, 2009).
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Figure 1.4 N-Glycan Branching Pathway
Oligosaccharyltransterase (OST) utilizes the preassembled donor Glc3Man9GlcNAc2-pp-dolichol to transfer the glycan to N-X-S/T sequons on glycoproteins in the ER. In the secretory pathway, glycoproteins transit from the ER to cis, medial, and trans Golgi apparatus, en route to the cell surface. The N-acetylglucosaminyltransferases enzymes, designated by their gene names (Mgat1, Mgat2, Mgat4, and Mgat5) generate branched N-glycans that display a range of affinities for galectins. The Km values for Mgat1, Mgat2, Mgat4, and Mgat5 are indicated as measured in vitro for UDP-GlcNAc and acceptor glycoproteins. The Golgi UDP-GlcNAc antiporter exchanges uridine monophosphate (UMP) for UDP-GlcNAc and establishes the steady state amounts of UDP-GlcNAc inside the Golgi (Dennis et al, 2009).
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and Mgat5 are limited by UDP-GlcNAc concentrations generated by the HBP. This implies that
initial branching by Mgat1 and 2 depends on the rate of protein synthesis, while N-glycan
branching by Mgat4 and 5 is determined mostly by UDP-GlcNAc availability. Indeed,
supplementation of extracellular GlcNAc has been shown to increase intracellular UDP-GlcNAc
levels, Mgat5-mediated N-glycan branching, and glycoprotein retention at the cells surface, with
increased sensitivity of cells to growth factors and cytokines (Johswich et al, 2014; Lau et al, 2007;
Mendelsohn et al, 2007).
1.1.3.2.2 Mgat Branching Enzymes
The Man5GlcNAc2 glycan is substrate for the first N-acetylglucosaminyltransferase, or
GlcNAc-transferase enzyme Mgat1 (GlcNAc-TI) (Schachter, 2010). Through transfer of GlcNAc
from UDP-GlcNAc in the medial Golgi, Mgat1 modifies the high-mannose structure to a hybrid
N-glycan. This is an essential step required for synthesis of either hybrid or complex-type glycans,
and cells deficient in Mgat1 can only generate high-mannose type structures (Schachter, 2010).
The product of Mgat1 can then be further stripped of remaining Man residues by α-mannosidase
II, rendering it a substrate for Mgat2 (GlcNAc-TII) (Schachter, 2010). The conversion of a mono-
antennary to a complex bi-antennary structure requires addition of GlcNAc by Mgat2. Specific
Mgat activity requires prior action of a distinct Mgat for catalysis to occur. One exception to this
is Mgat3, which catalyzes the transfer of GlcNAc residue to the core to form a bisected N-glycan.
However, due to the steric hindrance resulting from the presence of a bisecting GlcNAc, this
structure cannot be used as an acceptor by other Mgats, thus preventing further branching reactions
and formation of tri- and tetra-antennary N-glycans (Taniguchi & Korekane, 2011). Since Mgat3
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inhibits further N-glycan branch formation it has been suggested to play a regulatory role in
biosynthesis of complex and hybrid-type N-glycans (Taniguchi & Korekane, 2011).
In instances where Mgat3 is not involved, additional N-glycan structures can be generated
by Mgat5 (GlcNAc-V) and Mgat4a/b/c (GlcNAc-IVa/b/c) isoenzymes (Taniguchi & Korekane,
2011). Therefore, the number of antennas formed on an N-glycan depends on expression and
dynamic action of different GlcNAc-transferases, the concentration of common donor UDP-
GlcNAc, and acceptor glycoprotein with its immature N-glycan moving through the medial-Golgi.
The abundance of these factors and execution of these processes dictates the ultimate N-glycan
structure produced. Indeed, this varies among species, tissues, cells, glycoproteins, and even varies
with respect to different glycosylation sites on the same glycoprotein. For instance, when protein
synthesis slows down UDP-GlcNAc is spared, providing greater opportunity for the late branching
enzymes Mgat4 and Mgat5 to act and increase branching. Indeed, low glucose media conditions
increase surface β1,6-GlcNAc-branched N-glycans in mouse embryonic fibroblasts (Cheung et al.
2007).
N-glycan products of Mgat1, 2, 4 and 5 can be extended further through sequential addition
of Gal, and terminal capping with Fuc and Sia (Dennis et al, 2009). N-linked glycans represent the
most complex and functionally diverse covalent modification characterized (Ohtsubo & Marth,
2006). N-glycosylation is an inherently noisy and variable process, with potential N-glycan sites
not always being occupied with glycans, and even those that are occupied often varying in their
structure. The structural variability of N-glycans has been described in terms of
microheterogeneity and macroheterogeneity. Microheterogeneity refers to the site-specific
composition, chain-length, and branching pattern variability that occurs at a specific glycosite
amongst different molecules of the same glycoprotein (Schachter, 1986). On the other hand,
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macroheterogeneity results from variable Asn-X-Ser/Thr sequon usage, suggesting that local
three-dimensional conformation of the polypeptide, and the immediate microenvironment in the
vicinity of the potential glycosite, influences its accessibility to oligosaccharyltransferase
(Schachter, 1986).
1.1.3.2.2.1 Mgat1 in N-Glycan Branching
Although N-glycosylation is dispensable for survival of isolated cells in vitro, it is crucial
for proper functioning in vivo (Schachter, 2010). To elaborate on the N-glycan branching pathway
and impairments caused when its synthesis is perturbed at different points, I will review
phenotypes associated with mutations in genes coding for Mgat enzymes. Mgat1 is the first
branching enzyme to act in the Golgi N-glycan branching pathway, which then allows additional
branches to be added through action of other Mgats. The gene Mgat1 encodes for the enzyme
which starts the branching process by adding GlcNAc to the trimmed core producing the hybrid
N-glycan required for recognition by α-mannosidase II in the medial Golgi (Dennis et al, 2009).
Mgat1 activity is essential for the synthesis of complex-type N-glycans. Genetic ablation studies
in mice have proved informative concerning the structure-function relationship of Mgats.
Mgat1-dependent N-glycans are required for normal mammalian development, as
evidenced by systemic Mgat1 null mouse embryos dying in utero at around embryonic day 9.5,
and presenting underdevelopment including fewer somites, a tube-like heart, defective
vascularisation, and an open neural tube (Schachter, 2010). The phenotype would have been more
severe, and Mgat1 null mice would most likely die much earlier if it was not for maternally derived
Mgat1 gene transcripts, which rescue early embryos (Shi et al, 2004). This suggests that hybrid
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and/or complex N-glycans might be required for blastocyst formation or implantation (Shi et al,
2004). Lack of Mgat1 affects N-glycan structures at the cell surface, with all hybrid and complex
N-glycans being replaced by Man5GlcNAc2 (Schachter, 2010). Since Mgat1-dependent N-
glycosylated glycoproteins on the cell surface are required for normal cell to cell interaction, as
well as cell surface residency and activity of growth factor receptors and nutrient transporters,
combined disturbance in these fundamental biological processes are most likely responsible for
the lethality of Mgat1 null mice.
Mgat1 loss of function mutation in rice Oryza sativa causes severe developmental defect
with early lethality due to reduced sensitivity to the plant growth hormones cytokinins (Fanata et
al, 2013). Deletion of Mgat1 in Drosophila melanogaster results in viable flies exhibiting defects
in locomotion, brain abnormalities, and severely shortened lifespan (Sarkar et al, 2010). This
phenotype is rescued by neuronal Mgat1 expression, which also increases lifespan in wild-type
flies (Sarkar et al, 2010). These results imply that neuronal glycoproteins dependent on Mgat1
modification play a role in control of fly lifespan by affecting global metabolic changes (Sarkar et
al, 2010). Knockdown of Mgat1 in prostate cancer cells reduces tumor progression both in terms
of tumor size and metastasis (Beheshti Zavareh et al, 2012). This is interesting, as a link between
cancer and metabolism has been rediscovered in recent years (Vander Heiden et al, 2009).
1.1.3.2.2.2 Mgat2 in N-Glycan Branching
Mgat2 is required for synthesis of complex N-glycans, and is widely expressed in
mammalian cells and tissues (Wang et al, 2001). Mgat2 encodes the enzyme that transfers GlcNAc
onto the tri-mannosyl core. Homozygous deletion of Mgat2, which abolishes complex-type N-
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glycans but retains hybrid branched structures, results in prenatal and perinatal death with few null
mice surviving to adulthood (Wang et al, 2001). These mice displayed postnatal phenotype similar
to that observed in human patients with congenital disorders of glycosylation IIa (CDG-IIa),
including failure to thrive, dysmorphic facial features, and poor psychomotor development (Wang
et al, 2001). Furthermore, Mgat2 null mice were runted in comparison to wild-type littermates,
had decreased blood glucose, showed gastrointestinal abnormalities, and exhibited reduced body-
weight at all developmental stages (Wang et al, 2001).
1.1.3.2.2.3 Mgat4 in N-Glycan Branching
Bi-antennary N-glycans can be further modified through addition of GlcNAc onto the tri-
mannosyl core by Mgat4 isoenzymes (Taniguchi & Korekane, 2011). Interestingly, Mgat4b is
upregulated in the liver of fast-growing chickens, and is thought to promote fat deposition in this
context (Claire D'Andre et al, 2013). Mgat4a is expressed in most mouse and human tissues, but
its levels are much higher in pancreas and small intestine (Ohtsubo et al, 2011; Ohtsubo et al,
2005). In pancreatic β-cells, Mgat4a-dependent N-glycosylation is necessary for generating multi-
antennary N-glycans on the glucose transporter 2 (Glut2), which enables galectin-glycan binding
to maintain cell surface residency of Glut2 for proper glucose transport and sensing (Ohtsubo et
al, 2005). Indeed, Mgat4a null mice are hyperglycemic, displaying elevated free fatty acids and
triglycerides, with reduced insulin levels and impaired glucose-stimulated insulin secretion
(Ohtsubo et al, 2005). With aging, glucose intolerance in these mice progressed to metabolic
dysfunction, insulin resistance, and liver steatosis (Ohtsubo et al, 2005).
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Furthermore, a high-fat diet strongly attenuated Mgat4a expression in pancreatic β-cells of
wild-type mice. This resulted in reduced branching of Glut2 N-glycans required for proper cell
surface retention, which in turn lead to impairment of insulin secretion, and eventually type 2
diabetes and hepatic steatosis (Ohtsubo et al, 2005). In pancreatic β-cells, Mgat4a expression is
transcriptionally regulated by FoxA2 and Hnf1-α, whose intracellular distribution is regulated by
cellular redox balance that might be affected by high-fat diet induced oxidative stress (Ohtsubo et
al, 2011). Protection from high-fat diet-induced metabolic disease was conferred by ectopic
Mgat4a constitutive expression in β-cell of transgenic mice, in which Glut2 glycosylation and its
cell surface residence was maintained (Ohtsubo et al, 2011). Mgat4a was also reduced in
enterocytes from human obese subjects, who exhibited endosomal Glut2 accumulation, most likely
resulting from altered N-glycan branching on Glut2 (Ait-Omar et al, 2011).
1.1.3.2.2.4 Mgat5 and β1,6-linked GlcNAc Branching
Mgat5 encodes for a medial-Golgi N-glycan branching enzyme responsible for catalyzing
addition of β1,6-linked GlcNAc to Man residue, thereby forming complex-type tri- or tetra-
antennary N-glycan structures on glycoproteins (Dennis et al, 2009). The red kidney bean
(Phaseolus vulgaris) lectin L-phytohemagglutinin (L-PHA) exhibits specific and selective
reactivity toward β1-6GlcNAc-branched N-glycans (Grigorian et al, 2009). Since Mgat5 has a
high Km value for UDP-GlcNAc, in comparison to enzymes Mgat1 or Mgat2, the intracellular
concentration of this substrate determines the amount of complex tri- and tetra-antennary N-
glycans found on glycoproteins (Dennis et al, 2009; Lau et al, 2007). Mgat5 products are the
preferred substrate for elongation with N-acetyllactosamine and poly-N-acetyllactosamine, which
is comprised of repeating units of GlcNAc and Gal of variable length, allowing for further
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modification of the N-glycan chain by fucosylation and sialylation (Dennis et al, 2009; Grigorian
et al, 2009).
Mice deficient in Mgat5 lack tri- and tetra-antennary N-glycans. This results in altered
distribution or clustering of glycoproteins at the cell surface, due to reduced affinity for galectins,
and diminished signal transduction intensities from certain growth factor receptors and adhesion
proteins (Dennis et al, 2009; Grigorian et al, 2009). Mgat5 null mice are viable and appear similar
to wild-type littermates at birth. However, later on they display metabolic phenotypes such as
leaner body composition and smaller size, resistance to weight-gain on high-fat diet,
hypoglycemia, sensitivity to fasting in terms of exaggerated glycogen depletion and lipid
mobilization, as well as increased oxidative respiration by reliance on fatty acid oxidation (Cheung
et al, 2007). Glucose uptake is impaired in mouse embryonic fibroblasts derived from Mgat5 null
mice, especially under conditions of low-glucose or serum-free medium, suggesting that Mgat5-
modified N-glycans promote glucose uptake and anabolic metabolism (Cheung et al, 2007). Mgat5
null mice also display adult phenotypes that may be linked in part through metabolism, including
delayed oncogene-induced tumorigenesis, sensitivity to autoimmune disease, loss of adult stem
cells, and premature aging with accelerated loss of bone and muscle mass (Cheung et al, 2007;
Dennis et al, 2009; Grigorian et al, 2009).
The levels of β1,6-branch on N-linked glycans regulates the cell surface residency of
glycoproteins to affect their function (Dennis et al, 2009). Biological processes regulated by
Mgat5-dependent N-glycan modification include metabolic homeostasis, cell adhesion, motility
and migration, cytokine and growth factor signaling, and cellular proliferation and differentiation
(Dennis et al, 2009). Also, alterations in Mgat5 activity or function, leading to changes in N-glycan
branching pattern on glycoproteins, have been linked to cancer progression and invasive
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metastastic dissemination. In different types of cancer, Mgat5 transcript levels and enzymatic
activity are upregulated, which in turn leads to increased β1,6-branched N-linked glycan structures
and poly-N-acetyllactosamine content on membrane glycoproteins (Dennis et al, 2009). Mgat5
transcript levels are regulated by a Ras-Raf-Mek-Erk-Ets oncogenic signaling pathway, and
typically show a three to five fold increase when this pathway is activated through transformation
(Taniguchi & Korekane, 2011). The phenotypic effects of overexpression of Mgat5 are decreased
cell–cell and cell–matrix adhesion, and promotion of motility and invasiveness (Taniguchi &
Korekane, 2011). Transgenic mice expressing the polyoma virus middle T (PyMT) oncoprotein
under control of the murine mammary tumor virus (MMTV) long terminal repeat develop
multifocal mammary epithelium carcinomas that metastasize to lungs. Mgat5 null mice
intercrossed with transgenic mice expressing PyMT show a considerable delay in mammary tumor
development, and a reduction in tumor growth and lung metastasis, compared to those observed
in MMTV-PyMT;Mgat5+/- or MMTV-PyMT;Mgat5+/+ mice (Dennis et al, 2009). Mgat5-/- adult
mice also showed suppression of tumor progression in HER2/neu-induced mammary oncogenesis
model, and Pten+/- lymphoma and carcinoma tumor model (Cheung & Dennis, 2007; Granovsky
et al, 2000; Guo et al, 2010). In these models, the effects of Mgat5 deletion were traced to
alterations in PI3K and ERK signaling pathways.
1.1.3.2.2.5 Mgat6 in N-Glycan Branching
Mgat6 (GlcNAc-VI) catalyzes the formation of the most highly branched penta-antennary
complex-type N-glycan, the β1,4-linked branch (Sakamoto et al, 2000; Watanabe et al, 2006).
Prior action of Mgat5 is a prerequisite for Mgat6 activity (Brockhausen et al, 1989). Mgat6
enzymatic activity was found in avian tissues, such as in the hen oviduct, chicken liver, duck colon
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and turkey intestine (Brockhausen et al, 1989). This activity has also been seen in fish ovaries
(Brockhausen et al, 1989). The gene coding for Mgat6 is expressed in various chicken (Gallus
gallus) tissues (Sakamoto et al, 2000). However, Mgat6 gene and its enzymatic activity have not
been detected in mammalian tissues (Brockhausen et al, 1989; Sakamoto et al, 2000).
1.1.3.2.2.6 Functional Redundancy of N-Glycan Branches
Genetic studies with Mgat knockout mice revealed that loss of an Mgat enzyme is more
severe the earlier that enzyme acts in the N-glycan branching pathway. In later stages of the
branching pathway a degree of redundancy between N-glycan structures generated by Mgat4 and
Mgat5 may offer compensation for functional defects (Dennis & Brewer, 2013). This is contingent
on substrate availability and spatiotemporal expression of genes coding for each enzyme (Dennis
& Brewer, 2013). Experiments with Mgat4a and Mgat4b single and double knockout mice show
induced glycomic biosynthetic compensation (Dennis & Brewer, 2013). This occurs through
compensatory induction of other Mgats to generate similar N-glycan epitopes, as means of
maintaining overall expression of N-glycan ligands on glycoproteins for cross-linking galectins at
the cell surface (Dennis & Brewer, 2013). Furthermore, functional redundancy or compensation
between N-acetyllactosamine branches in structurally related N-glycans can be promoted by UDP-
GlcNAc, the rate-limiting substrate in their biosynthesis in the Golgi N-glycan branching pathway
(Dennis & Brewer, 2013; Lau et al, 2007). The N-acetyllactosamine units, although repositioned
within N-glycans with GlcNAc treatment, have been shown to substitute and compensate
functionally by rescuing affinity for galectins' association with N-glycan branches at the cell
surface (Dennis & Brewer, 2013; Lau et al, 2007). In fact, GlcNAc supplementation in Mgat5-/-
cells doubled the tri-antennary N-glycan levels, and rescued levels of EGF and TGF-β cell surface
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receptors and their signaling, suggesting that less-branched N-glycans in larger quantities are
sufficient to restore galectin binding (Dennis & Brewer, 2013; Lau et al, 2007).
1.1.4 HBP and UDP-GlcNAc Formation
The general route for incorporating monosaccharides into a glycan starts with their
conversion into activated sugar-nucleotides, which serve as donors for glycosylation reactions
(Ohtsubo & Marth, 2006). Activated sugar-nucleotides are synthesized by covalently linking a
monosaccharide to a nucleotide, typically nucleoside diphosphate, and in many cases one sugar-
nucleotide can be converted into another (Du et al, 2009). The de novo biosynthetic steps to
generate UDP-GlcNAc follow a variant of the Leloir pathway, elucidated biochemically in the
1950s (Marshall, 2006). However HBP, also called the hexosamine signaling pathway, was only
well delineated and formally described based on genetic and biochemical evidence in the early
1990s (Marshall, 2006). UDP-GlcNAc is the major product of the HBP, and is the primary
substrate in virtually all glycoprotein processing pathways, including Golgi N-glycan branching
pathway.
1.1.4.1 de novo UDP-GlcNAc Biosynthesis
HBP is one of several pathways that divert minor amounts of glucose from glycolysis or
thereby regulating its own synthesis through competitive feedback inhibition, and controlling the
amount of glucose entering HBP (Du et al, 2009; Love & Hanover, 2005). The next reaction in
UDP-GlcNAc synthesis involves transfer of an acetyl group from acetyl coenzyme A (acetyl-CoA)
by GlcN-6P acetyltransferase (GNPNAT1 or GNA1), to obtain GlcNAc-6P (Hardiville & Hart,
2014). Isomerization of GlcNAc-6P to GlcNAc-1P is catalyzed by GlcNAc phosphomutase
(PGM3 or AGM1), which is autophosphorylated and dephosphorylated during the reaction cycle.
Finally, activation of monosaccharides as sugar-nucleotides is an essential step in the biosynthetic
pathway. Hence, uridylation of GlcNAc-1P by UDP-GlcNAc pyrophosphorylase (UAP1 or
AGX1) results in formation of the end-product UDP-GlcNAc, which is then transported into the
rER and Golgi apparatus and used as a donor for N-glycosylation by a number of GlcNAc-
transferases (Dennis et al, 2009).
1.1.4.2 Fate of UDP-GlcNAc
In the secretory pathway, UDP-GlcNAc serves as a basic building block for synthesis of
glycans on secreted and membrane-bound glycoproteins. UDP-GlcNAc is also used to make
glycolipids, cell surface O-glycans, glycosaminoglycan (GAG) chains used in proteoglycans of
the extracellular matrix, and hyaluronic acid (2009). In addition, some UDP-GlcNAc is epimerized
to generate UDP-N-acetylgalactosamine (UDP-GalNAc), by the enzyme UDP-galactose-4-
epimerase (GALE), or cleaved into UDP and ManNAc by UDP-GlcNAc-2-epimerase/N-
acetylmannosamine kinase (GNE), the bi-functional and rate-limiting enzyme of sialic acid
biosynthesis (Galeano et al, 2007). The same pool of UDP-GlcNAc generated by HBP also
22
Figure 1.5 Hexosamine Pathway for Biosynthesis of UDP-GlcNAc
An offshoot of the glycolytic pathway, the HBP integrates the metabolism of carbohydrates (glucose), amino acids (glutamine), fats (acetyl-CoA), and nucleotides (uridine-diphosphate) in the synthesis of UDP-GlcNAc. The enzymes and metabolites involved in the synthesis of UDP-GlcNAc are presented, with different precursors color-coded to denote their contribution to UDP-GlcNAc synthesis.
23
functions as the obligatory substrate for intracellular O-GlcNAcylation (Love & Hanover, 2005).
O-GlcNAcylation is a dynamic and reversible posttranslational modification where a single
GlcNAc moiety is attached to the hydroxyl group of serine or threonine residue in cytoplasmic and
as glycogen, as well as glycolysis to generate pyruvate, which in liver and adipose tissue is then
converted to fatty acid and triglyceride (Postic et al, 2004). Glucose is also metabolized by the
pentose phosphate pathway to generate ribose 5-phosphate, a precursor in nucleotide biosynthesis,
and NADPH, which is required for biosynthesis of fatty acids (Postic et al, 2004).
1.2.2.1 Liver Glycogen
Once absorbed, carbohydrates are carried to the liver where they are converted to glucose.
Glycogen, a branched polymer of glucose consisting of up to 50,000 residues, is the primary form
of stored carbohydrate in animal tissue. Liver is the central organ involved in glycogen formation
(glycogenesis), storage and breakdown (glycogenolysis) (Lin & Accili, 2011). These processes are
35
coordinated to maintain normal glucose levels in the blood during glycemic fluctuations associated
with states of feeding and starving. Due to its hydrated state, glycogen requires ample storage
space, and as such liver can only store a limited amount of glucose in this form. Liver glycogen
represents a short-term reserve of glucose for peripheral organs and tissues. Indeed, glycogen-
metabolizing enzymes have the property that enables the liver to act as a sensor of blood glucose
and to store or mobilize glycogen according to peripheral needs. Glycogen synthase (GS) is the
rate-limiting enzyme for glycogen synthesis and deposition, responsible for transfer of glucose
from UDP-glucose, its nucleotide-sugar donor form (Postic et al, 2004). GS phosphorylation on
Ser641 leads to inactivation, while glycogenesis is stimulated by activating GS through
dephosphorylation by protein phosphatase 1, which is controlled by insulin (Postic et al, 2004).
1.2.3 Feeding and Fasting
All cells and organs in the body have a constant requirement for nutrients and metabolites,
which serve as energy substrates. Since nutrient supply in nature is irregular and variable in amount
and type, organs such as the liver and adipose tissue act as temporary reservoirs storing energy
bearing metabolites and nutrients that can be readily deployed. In metabolism, a distinction is often
made between the fed state, also known as the absorptive state, immediately following a meal, and
the fasting or starving state, which develops later if food is not consumed. The two phases operate
on different metabolic programs, which are dictated by plasma levels of various metabolites and
the hormonal signaling cascades they trigger (Schwartz et al, 2013). During the absorptive state,
the insulin to glucagon ratio increases and the availability of substrates trigger an anabolic phase,
whereby liver forms increased amounts of glycogen and fats, muscle synthesizes proteins from
amino acids and stores glycogen, and adipose tissue synthesizes triglycerides and stores them in
36
lipid droplets (Lin & Accili, 2011). Without food intake the post-absorptive state gradually evolves
into the fasting state (Figure 1.6).
During fasting, blood glucose and insulin levels decrease, and glycogen synthesis is
inhibited, as is fatty acid synthesis (Schwartz et al, 2013). In response to lowered blood glucose
concentration, pancreatic α-cells release increased amounts of glucagon, which act on the liver to
stimulate glucose release by glycogenolysis to maintain blood glucose homeostasis and provide
other tissues with an energy source (Lin & Accili, 2011). Glucagon also plays an important role in
initiating and maintaining hyperglycemic conditions in diabetes (Lin & Accili, 2011). In fact,
insulin resistance is manifested by hyperinsulinemia, increased hepatic glucose production, and
decreased glucose disposal. The reduced insulin to glucagon ratio results in a switch to different
fuel metabolism, as the body engages in using its energy reserves by breaking down glycogen, fats
and proteins, and distributing these energy supplying metabolites between organs (Lin & Accili,
2011). Increased level of glucagon relative to insulin also stimulates the mobilization of fatty acids
from adipose tissue. Glucagon release also activates hormone-sensitive lipases, which catalyze
triglyceride hydrolysis in adipocytes to release glycerol and free fatty acids into the circulation
(Lin & Accili, 2011).
Liver is a major site for fatty acid oxidation to acetyl-CoA and fat-derived ketone body
formation (Lin & Accili, 2011). Free fatty acids and ketone bodies released into the blood serve as
important energy sources during hunger. After prolonged glucose starvation, muscles and other
tissues begin to degrade protein to amino acids, which are then oxidized or secreted and used in
gluconeogenesis by the liver. Gluconeogenesis is the formation of glucose from non-carbohydrate
sources, primarily glycerol produced through degradation of fats, and lactate, alanine, glutamine
and other glucogenic amino acids derived from muscles (Lin & Accili, 2011). In animals
37
Figure 1.6 Glucose Production in Liver During Fasting
During fasting, when plasma glucose levels drop due to lack of new supplies or rapid use, the pancreatic islet cells increase glucagon secretion, and decrease insulin release. To maintain plasma glucose homeostasis, glucagon stimulates liver glycogenolysis and increases the enzymatic activity required for gluconeogenesis, utilizing glycerol, lactate and amino acids released by adipocytes and muscle cells into the bloodstream.
38
gluconeogenesis takes place primarily in the liver, and its main function is to generate sufficient
glucose supply for organs such as brain and muscles during fasting or starvation (Lin & Accili,
2011). Typically this system is activated once hepatic glycogen has been depleted. Most of the
reaction steps involved in gluconeogenesis represent a reversal of glycolysis reactions, catalyzed
by the same enzymes that are used in glycolysis (Postic et al, 2004). Other enzymes are specific to
gluconeogenesis and are only synthesized under the influence of glucagon when needed (Lin &
Accili, 2011).
1.2.4 Glycoprotein Receptors for Glucagon and Insulin, and Glucose Transporters
Transmembrane receptors for the hormones glucagon and insulin are N-glycosylated, as
are glucose transporters. All of these are specialized glycoproteins residing at the cell surface,
where they respond to the extracellular environment and trigger changes in function of the cell.
1.2.4.1 Glucagon Receptor (Gcgr)
The glucagon receptor (Gcgr) is a G-protein coupled receptor with four and five N-glycan
sequons in man and mice, respectively. Glucagon binding to Gcgr results in a conformational
change that activates adenylate cyclase, forming the secondary messenger cyclic AMP (cAMP),
which in turn activates protein kinase A (PKA) (Johswich et al, 2014). PKA then phosphorylates
and inactivates GS, thereby terminating the synthesis of glycogen (Lin & Accili, 2011). Recently,
the Dennis laboratory has shown in primary hepatocytes that Mgat5-branched N-glycans on Gcgr
increase receptor binding to galectin-9, which slows its membrane mobility and enhances
39
sensitivity to glucagon (Johswich et al, 2014). This study suggests that N-glycan branching on
Gcgr acts as a positive regulator of glucagon responsiveness.
1.2.4.2 Insulin Receptor (Insr)
The insulin receptor (Insr) belongs to a family of related tyrosine kinase receptors.
Interestingly, human and murine Insr both have eighteen N-glycan sequon motifs. A natural variant
of human Insr has a substitution of Lysine for Asparagine at a position immediately preceding the
first N-glycan sequon (Kadowaki et al, 1990). This was found to decrease the affinity of insulin
binding to the receptor (Kadowaki et al, 1990). Furthermore, site-directed mutagenesis of the first
and second Insr N-glycan sequons significantly reduced insulin binding to the receptor on the cell
surface (Caro et al, 1994). Insulin binding to Insr leads to tyrosine phosphorylation of several
insulin receptor substrates (IRSs), which then act as docking proteins for other signaling proteins
(Lin & Accili, 2011). Phosphorylation of IRSs proteins activates the phosphatidylinositol-3-kinase
(PI3K) and Akt signaling pathway, which is responsible for most of the metabolic actions of insulin
(Lin & Accili, 2011). One of these is to increase translocation of glucose transporters (Gluts) from
cytoplasmic vesicles to the cell surface, where they can mediate glucose import from the blood via
facilitated diffusion (Lin & Accili, 2011).
1.2.4.3 Glucose Transporters (Gluts)
Most Glut proteins, encoded by Slc2a genes, typically have a single N-glycan sequon.
Dependence of Gluts on N-glycan branching for increased residency and activity at the cell surface
has been shown for Glut1, Glut2 and Glut4 (Haga et al, 2011; Kitagawa et al, 1995; Lau et al,
40
2007; Ohtsubo et al, 2005; Zhu et al, 2012). In both mouse and human pancreatic β-cells, cell
surface expression of Glut2 depends on binding of its Mgat4-mediated N-glycan structure to
galectin-9, which slows mobility at the cell surface and loss to endocytosis, thereby enhancing
glucose transport (Ohtsubo et al, 2011; Ohtsubo et al, 2005). There is also pharmacological
evidence for N-glycan requirement on nutrient transporters at the cell surface. Indeed, membrane
transport of three major classes of nutrients, namely glucose, amino acids and uridine, was
inhibited by tunicamycin, an inhibitor of protein N-glycosylation in the ER (Olden et al, 1979). A
similar result was obtained using a different tool to perturb N-glycan function, suggesting that
interfering with N-glycans on glycoproteins alters plasma membrane transport systems to impair
nutrient uptake. In this case, wheat germ agglutinin, a lectin that binds GlcNAc within N-glycans
on cell surface glycoproteins, has been shown to have an inhibitory effect on transport of amino
acids through the plasma membrane (Li & Kronfeld, 1977).
1.2.5 Nutrient Sensing and Signaling Pathways
Cells have evolved several sensory systems and homeostatic regulatory mechanisms to
sense nutrient levels, cellular energy and metabolic status, as well as to respond to fluctuations by
adjusting flux through metabolic pathways. Pathways that detect differences in extracellular and
intracellular levels of nutrients and metabolites are often integrated and coordinated. The
mammalian target of rapamycin (mTOR) and the AMP-activated protein kinase (AMPK)
pathways are two well characterized nutrient sensing and signaling pathways.
1.2.5.1 mTOR
41
mTOR controls cell growth and metabolism in response to nutrients, growth factors and
cellular energy, by positively and negatively regulating several anabolic and catabolic processes,
respectively, to collectively determine mass accumulation (Cornu et al, 2013; Zoncu et al, 2011).
mTOR is a Ser/Thr protein kinase that interacts with several distinct proteins to form two
complexes named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Cornu et al,
2013). mTORC1 activates p70 S6 kinase, which in turn phosphorylates ribosomal protein S6, thus
activating translation and transcriptional up-regulation of anabolic pathways (Cornu et al, 2013).
The abundance of phosphorylated ribosomal protein S6 is directly related to mTORC1 activity,
and inversely related to level of cellular autophagy (Zoncu et al, 2011). Autophagy is involved in
regulating intracellular lipid metabolism in the liver through macrolipophagy (Singh et al, 2009).
Macrolipophagy promotes breakdown of intracellular lipids stored in lipid droplets to
supply fatty acids for β-oxidation or other uses (Singh et al, 2009). Hepatic mTORC1 activity
negatively regulates autophagy and production of ketone bodies for peripheral tissues to use as
energy source in response to fasting (Zoncu et al, 2011). Hence, fasting response typically
suppresses mTORC1 activity, however its chronic activation by overabundance of nutrients and/or
hyperinsulinemia can drive ectopic accumulation of lipids in the liver (Zoncu et al, 2011). Indeed,
mTORC1 activity is significantly elevated in liver, muscle, and adipose tissue of both genetically
induced and high-fat diet induced obese mice, suggesting its involvement in the pathogenesis of
obesity and obesity-associated metabolic disorders (Rui, 2007). In addition to its key role in lipid
metabolism within the liver, hepatic mTORC1 signaling also affects systemic glucose and insulin
homeostasis, most likely due to its effects on Akt and hepatic glucose uptake (Albert & Hall, 2014).
Hyper-activation of mTORC1 signaling upon fasting results in metabolic stress due to systemic
and hepatic glutamine depletion, and thereby an inability of glutaminolysis to sustain the TCA
42
cycle (Albert & Hall, 2014). This is consistent with the finding that mTORC1 inhibition by
rapamycin increases intracellular glutamine levels (Albert & Hall, 2014). In certain settings, such
as low-nutrient conditions, AMPK acts as a negative regulator of mTORC1 signaling (Hardie,
2014).
1.2.5.2 AMPK
AMPK is a master regulator of energy homeostasis, as well as cellular and organismal
metabolism (Hardie, 2014). It senses low ATP levels and restores cellular energy by inhibiting
energy consuming anabolic while stimulating catabolic pathways such as autophagy and fatty acid
oxidation (Hardie, 2014). To maintain energy homeostasis, a delicate balance between fat storage
and breakdown is crucial. The adipose tissue hormone adiponectin, whose actions are mediated by
AMPK, inhibits fatty acid synthesis, stimulates fatty acid uptake and oxidation, and sensitizes liver
and muscle tissues to insulin (Unger et al, 2010). AMPK activity can be measured through its
phosphorylation at Thr172, as well as by phosphorylation of its downstream target acetyl-CoA
carboxylase (ACC) at Ser79 (Hardie, 2014). ACC catalyzes the pivotal step in fatty acid synthesis,
the formation of malonyl-CoA, by attaching a bicarbonate ion to acetyl-CoA (Jump, 2011).
Malonyl-CoA inhibits transport of acyl-CoA into the mitochondrial matrix and its subsequent
breakdown (Jump, 2011). Thus, de novo fatty acid synthesis is in part negatively regulated by
AMPK. Dephosphorylated ACC is enzymatically active, while phosphorylation inactivates its
enzymatic activity and promotes fatty acid oxidation instead (Hardie, 2012). The inactivating
phosphorylation of ACC is catalyzed by AMPK, which in turn is regulated by an activating
phosphorylation by cAMP dependent PKA, while the reactivating dephosphorylation of ACC is
catalyzed by protein phosphatase 2A (Hardie, 2014).
43
1.2.6 Fatty Acid Synthesis
The rate of fatty acid synthesis can be influenced by diet. In well-fed state with an ample
supply of glucose, insulin is secreted, which causes a low degree of ACC phosphorylation, and
increased formation of malonyl-CoA (Jump, 2011). The biosynthesis of fatty acids, or de novo
lipogenesis, occurs in many tissues, however post-absorptively this occurs primarily in the liver
and fatty tissues. Fatty acid synthase (FASN) is a key biosynthetic multi-enzyme protein complex
responsible for de novo lipogenesis (Jump, 2011). By catalyzing the synthesis of long-chain fatty
acids from acetyl-CoA and malonyl-CoA, in the presence of NADPH as a reducing agent, FASN
produces long-chain saturated fatty acids for storage in the liver or export to other tissues (Jump,
2011). FASN uses acetyl-CoA as a starter molecule, and in a cyclic reaction, acetyl residue is
elongated by two carbon units at a time for seven cycles, until formation of sixteen carbon palmitic
acid as the major product (Jump, 2011). The liver is the most important site for synthesis of fatty
acids and triglycerides, most of which are subsequently released into the blood. The cytosolic
buildup of acetyl-CoA encourages de novo hepatic lipogenesis, which over time can lead to
triglyceride accumulation and even development of fatty liver disease, or hepatic steatosis (Jump,
2011). FASN is transcriptionally regulated by sterol regulatory element binding protein-1c
(SREBP-1c) in response to feeding and insulin (Jump, 2011). Long-term adaptive control of
lipogenesis occurs though changes in the rate of synthesis at the transcriptional level, and
degradation of the participating enzymes (Jump, 2011).
1.2.7 Lipid Storage and Breakdown
44
Due to the limited storage capacity of glycogen, all carbohydrates consumed beyond
energy needs are converted to and stored as fat. Fats are the most important energy reserves in an
animal. This reserve pool is practically almost unlimited. Triglycerides are neutral lipids, each
composed of three fatty acids in ester linkage with a single glycerol (Walther & Farese, 2012). In
animals, triglycerides are highly concentrated stores of metabolic fuel, and yield more energy per
weight upon oxidation than either proteins or carbohydrates (Speakman & O'Rahilly, 2012).
Although most tissues are capable of producing triglycerides, their primary site of accumulation
is the adipose tissue. In vertebrates, lipid droplets are the major cytoplasmic storage organelles for
triglycerides (Walther & Farese, 2012). They are characterized as ubiquitous components of
different cell types, even those that, unlike adipocytes, only have a limited capacity for triglyceride
storage (Walther & Farese, 2012). Lipid droplets play a crucial role in regulating cellular lipid
levels through hydrolysis and trafficking, and provide energy and substrates for synthesis and
repair of cell membranes (Walther & Farese, 2012).
During fasting or starvation the body’s strategy is to minimise the use of carbohydrate and
protein, and to obtain as much energy as possible from fat stores. Hence, lipogenesis is inhibited,
and fatty acids activated in a process requiring ATP to form acyl-CoA, which can then be
transported into the mitochondrial matrix for β-oxidation and acetyl-CoA generation (Walther &
Farese, 2012). Resulting acetyl residues can be oxidized to carbon-dioxide in the TCA cycle,
producing reduced coenzyme and ATP derived through oxidative phosphorylation (Vander Heiden
et al, 2009). Fatty acid breakdown in the liver is increased when levels of free fatty acids are
elevated, such as after consumption of high-fat foods, and increased lipolysis during fasting or
starvation (Jump, 2011). Short chain fatty acids are dissolved in plasma, while longer chain fatty
acids are bound to albumin. Fatty acids are transported across the cell membrane by membrane-
45
associated fatty acid-binding proteins, or fatty acid transporters, which not only facilitate but also
regulate cellular fatty acid uptake (Schwenk et al, 2010). A number of fatty acid transporters have
been identified, including CD36, which is extensively glycosylated. Indeed, mouse CD36 has eight
N-glycan sequons, while human CD36 has ten. These are situated in a region coding for the large
extracellular loop. Insulin treatment stimulates simultaneous recruitment of CD36 and glucose
transporter Glut4 from the recycling endosomal storage compartment to the cell membrane in order
to increase fatty acid and glucose uptake, respectively (Schwenk et al, 2010).
1.3 Body Weight and Obesity
Body weight is a simple and effective way to measure tissue mass and estimate body
composition of an animal. In humans, the body mass index (BMI) provides a more accurate
measurement, since it is independent of height and correlates fairly well with the total body fat
(Speakman & O'Rahilly, 2012). BMI is calculated from body weight in kilograms divided by the
square of height in meters. To specifically measure body composition in terms of fat and lean
muscle mass, various imaging modalities are available. Dual-energy X-ray absorptiometry
(DEXA) passes two photons of varied energy intensity through body tissues, and body fat
percentage is estimated based on the attenuation patterns of these photons. Alternatively, to obtain
even more accurate measures of fat mass, computed tomography (CT) or magnetic resonance
imaging (MRI) can be used. In humans, obesity is clinically defined as BMI of greater than 30
kg/m2, while a number of 25 up to 30 indicates that a person is overweight. Around the globe, in
both industrialized and developing countries, there has been an increase in adiposity among people,
often progressing to obesity. It is estimated that currently about ~2 billion people worldwide are
overweight and ~700 million are obese (Speakman & O'Rahilly, 2012). These are staggering
46
numbers compared to 50 years ago (Speakman & O'Rahilly, 2012). Obesity is a risk factor for
chronic diseases such insulin resistance, diabetes, metabolic syndrome, cardio-vascular disease,
fatty-liver disease, and even some forms of cancer (Speakman & O'Rahilly, 2012; Unger et al,
2010). The organs affected in these conditions, including the liver, pancreatic islets, skeletal
muscle, heart and kidney, display ectopic lipid accumulation (steatosis), which can ultimately lead
to lipotoxicity, apoptosis, and gradual organ failure (Unger et al, 2010). The increased prevalence
of obesity and these chronic diseases related to nutrition and metabolism have prompted
considerable efforts to understand their origin and to identify effective prevention strategies or
treatments.
1.3.1 Body Weight Regulation
The biology of body weight regulation is complex and operates through effects on energy
intake, metabolic activity, energy expenditure, and caloric partitioning into tissues such as fat and
muscle. Since there is a constant flux in the nutritional supply and energy needs of an organism,
strategies that maintain a steady state under varying nutritional conditions are crucial for healthy
functioning of an organism. Although body weights across a population vary considerably,
individuals show a remarkable weight stability, suggesting that body weight is physiologically
regulated (Flier & Maratos-Flier, 2007). Regulation of fat depots involves a coordinated interplay
between central regulators of feeding behavior in the hypothalamus, neuroendocrine signals, and
metabolic regulators of energy expenditure and fat storage (Flier & Maratos-Flier, 2007). Dramatic
alterations in body weight have been shown to result from mechanical or pharmacological lesions
of the hypothalamus (Flier & Maratos-Flier, 2007). Peripheral tissues such as the gut, white and
brown adipose, and skeletal muscles also regulate body weight and its composition (Flier &
47
Maratos-Flier, 2007). One of the most dramatic illustration of this comes from animals with a
mutation in myostatin, or animals treated with compounds that block or antagonize the activity of
myostatin such as follistatin, which results in significantly larger skeletal muscle mass and
increased muscle strength (Gangopadhyay, 2013).
1.3.1.1 Leptin
The adipose-derived hormone leptin regulates systemic energy homeostasis by linking an
individual’s fat stores with caloric intake and expenditure (Flier & Maratos-Flier, 2007). The
receptors for leptin are located in the hypothalamus, and have eighteen N-glycan sequons in
humans, and seventeen and sixteen in rat and mouse, respectively. Leptin functions as a satiety
signal released from adipose tissue in proportion to fat stores, and is part of a signaling pathway
that acts to maintain the size of body fat depot (Flier & Maratos-Flier, 2007). Leptin activity
impinges on the hypothalamic regulatory neuro-circuitry to increase energy expenditure and
inhibit feeding (Flier & Maratos-Flier, 2007). Both leptin and insulin have been characterized as
adiposity signals, since their plasma levels positively correlate with body weight and are in direct
proportion to the amount of energy stores in adipose tissue (Schwartz et al, 2013). Leptin also
stimulates fatty acid oxidation in non-adipose tissues, so as to minimize ectopic lipid accumulation
and protect against lipotoxicity (Unger et al, 2010). Humans with a rare congenital loss-of-function
mutations in the leptin gene are clinically obese, due to abnormalities in energy expenditure and
increased food intake (Speakman & O'Rahilly, 2012). These symptoms are reversed by
administration of recombinant human leptin (Speakman & O'Rahilly, 2012). Mice deficient in
leptin or in the leptin receptor exhibit overfeeding and develop obesity and diabetes (Flier &
Maratos-Flier, 2007).
48
1.3.2 Determinants of Body Weight and Obesity
Factors affecting weight regulation and obesity have been identified at a number of
different levels of analysis. These span the whole spectrum, from social to economic,
psychological, behavioral, developmental, physiological, biochemical, metabolic, genetic and
molecular, with interaction and feedback across and between factors. Adiposity is a highly
heritable trait, and genetics appears to explain around 65% of weight variation between individuals
(Speakman & O'Rahilly, 2012). Although many polymorphisms associate robustly with obesity,
much of the genetic variation that underpins differences in adiposity across the normal population
remains unexplained (Speakman & O'Rahilly, 2012). It appears that body weight and composition
is a phenotype at the interface of genes and environment, where genes most likely determine
susceptibility to environmental factors. Environmental factors such as gut microbiota, stress,
endocrine disruptors have also been linked to risk of developing obesity (Speakman & O'Rahilly,
2012).
The most widely accepted explanation for the rise in adiposity around the globe involves
increased availability and access to palatable calorie-dense foods, which leads to overeating and
excess calorie intake; combined with decreased energy expenditure through sedentary lifestyle
resulting from modernization and technological advances, which have greatly reduced labour
intensive activities (Speakman & O'Rahilly, 2012; Unger et al, 2010). The idea of a toxic-
environment and exposure to chemical obesogens, such as bisphenol-A and pesticides, has also
been proposed to account for the rise in obesity rates in the last few decades (Grun, 2010). This
theory argues that our environment has become contaminated due to pharmaceutical drugs,
chemical pollutants, as well as hormones and antibiotics used in farming, which disrupt the energy
49
balance, endocrine function, lipid metabolism and fat storage by a variety of mechanisms (Grun,
2010). However, the prevailing explanation for the observed increase in body weight and obesity
is that of energy imbalance, i.e. eating too many calories and not getting enough physical activity
(Speakman & O'Rahilly, 2012).
1.3.2.1 Energy Balance
The guiding principle of body regulation bioenergetics is that of energy balance or energy
homeostasis, which proclaims that to maintain a constant body weight, energy or calorie intake
from food consumed must equal energy or calories burned by the metabolism of the organism
(Flier & Maratos-Flier, 2007). Hence, body weight is maintained in a steady state by a balance
between caloric intake and energy expenditure. Any imbalance between energy intake and
expenditure, which includes total physical activity, metabolic rate and thermogenesis, is reflected
in a change in the amount of stored energy as fat (Flier & Maratos-Flier, 2007). Thus, when
nutritional intake chronically exceeds the energy needs of an organism, the excess energy is stored
in body fat, which in turn leads to weight gain. This dominant theory is firmly rooted in the first
law of thermodynamics, i.e. the law of conservation of energy, which states that energy can neither
be crated nor destroyed, but only converted from one form to another. However, the dogma of
energy imbalance between calories consumed and calories expended being the fundamental cause
of obesity is not universally accepted (Lustig et al, 2012; Taubes, 2013).
1.3.2.2 The Carbohydrate Hypothesis of Obesity
50
The theory of energy balance has been challenged with arguments that physiology is not
physics, and that not all calories are created equal, but rather that nutrient composition affects fat
accumulation (Taubes, 2012). In this theory, sugar has been singled out as the toxic culprit
responsible for increase in obesity, diabetes and the metabolic syndrome around the globe (Lustig
et al, 2012; Taubes, 2013). This alternative hypothesis suggests that adiposity is not caused by
excess calories alone, but rather by the quantity and quality of carbohydrates and starches
consumed. The logic behind this theory is that since insulin regulates carbohydrate metabolism
and stimulates the synthesis and storage of fats in the liver and fat depots, and blood levels of
insulin are effectively determined by carbohydrate intake, then consumption of sweet
carbohydrates with a high-glycemic index will result in more insulin release and more fat
accumulation (Taubes, 2013). With elevated insulin levels (hyperinsulinemia) in the bloodstream
for prolonged periods, adipocytes respond by accumulating more fat, thus promoting more weight
gain (Taubes, 2013).
A variant of this theory suggests that it is specifically excess consumption of fructose,
found in sucrose and high-fructose corn-syrup, that is responsible for the current epidemic of
obesity and chronic metabolic disease (Lustig, 2013). Indeed, fructose consumption has increased
worldwide, as it is often used as a sweetener during food processing, paralleling the increase in
obesity and chronic metabolic diseases over the last few decades (Lustig et al, 2012). Unlike
glucose, fructose is very sweet, and does not generate an insulin response (Lustig, 2013). Its
hepatic metabolism in the post-absorptive state impairs β-oxidation, and promotes de novo
lipogenesis, triglyceride formation and hepatic steatosis (Lustig, 2013). This is often followed by
hepatic insulin resistance, hyperglycemia, as well as formation of reactive oxygen species,
resulting in cellular toxicity and dysfunction (Lustig, 2013). Furthermore, fructose also has
51
dependence-producing properties, which promote changes in the brain’s reward system, leading
to excessive consumption (Lustig, 2013). Consequently, fructose has been deemed toxic and a
danger to individuals and society requiring prompt regulation, since it exerts its detrimental health
effects beyond its calories, and in ways that are similar to alcohol (Lustig et al, 2012).
However, it should also be noted that the idea that carbohydrates are mostly to blame for
making animals fat has many detractors, who instead point to fat as the real culprit. Most nutrients
consumed in excess of energy needs are converted to fat, however since carbohydrates must first
be metabolized for this purpose, their conversion is actually more ineffective than fats, which can
be directly integrated into triglycerides and lipid droplets. Moreover, there is plenty of evidence
from mouse experimental research to support the claim that calorie enriched fatty diet leads to
body weight increase (Cheung et al, 2007). The incompleteness in our understanding of body
weight regulation and what causes obesity is illustrated by the lack of a general consensus on the
issue, or a universally accepted explanation. Indeed, competing diet programs promising success
and results are often polar opposites of one another. Furthermore, the fact that dieting, weight-loss
and fad diets are a billion dollar industry, with most being ineffective, and those that do lose weight
initially not being able to maintain the weigh-loss they have obtained either through hard-work or
deprivation, is a testament to the fact that we do not fully understand the underlying biology of
body weight regulation, and hence lack actionable knowledge for impactful intervention in public
health advice.
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1.4 Rationale, Objectives and Summary
N-glycosylation and Golgi N-glycan branching are essential modifications of proteins
translated in the secretory pathway. Glycoproteins traffic through the Golgi apparatus, where
exposure to N-glycan branching enzymes is dependent on enzyme expression and UDP-GlcNAc
supply. Glucose transporters Glut1, Glut2 and Glut4 are dependent on N-glycan branching
pathway and galectin binding for optimal cell surface retention and glucose transport (Dennis et
al, 2009). Mutations in genes encoding branching enzymes Mgat4a and Mgat5 in the N-glycan
branching pathway disrupt glucose homeostasis in mice (Cheung et al, 2007; Ohtsubo et al, 2005).
When I began my research project, Mgat4a expression in the pancreas was reported to be regulated
at the level of gene expression, and both Mgat5 and Mgat4a mutant mice displayed aberrant
regulation of body-weight on high-fat diet (Cheung et al, 2007; Ohtsubo et al, 2005). Relatively
little consideration had been given to the contribution of metabolic feedback through the HBP or
GlcNAc salvage to UDP-GlcNAc, the common donor substrate used by Mgat enzymes, until a
publication from the Dennis laboratory reported that elevated UDP-GlcNAc supply to the N-
glycan branching pathway enhanced cell surface residency and activity of growth factor receptors
and Glut4 (Lau et al, 2007).
The main objective of my thesis was to determine whether metabolic input through the
HBP to UDP-GlcNAc contributed to control of N-glycan branching pathway in vivo, and whether
this could be seen to occur cell-autonomous in vitro. Another objective was to examine the effect
of increased expression of N-glycan branching enzymes and/or UDP-GlcNAc supply on cellular
metabolism using targeted mass-spectrometry based metabolomics, and to explore the potential
mechanisms by which HBP-mediated increase in N-glycan branching exerts its effect on
metabolism.
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Effects of overexpressing Mgat1, Mgat5 or Mgat6 on central metabolites were investigated
in cells growing under normal cell culture media conditions supplemented with GlcNAc. Induced
overexpression of Mgat enzymes increased respective N-glycan branches on cell surface
glycoproteins, and increased central metabolites in different metabolic pathways. Further increases
were observed by using GlcNAc to supplement the HBP and increase UDP-GlcNAc pool for N-
glycan branching enzymes. Importantly, GlcNAc supplementation and Mgat5 overexpression
displayed synergistic increase in Mgat5-mediated N-glycan branching. Moreover, my findings
suggest that N-glycan branching cooperates with the HBP to regulate nutrient import and
metabolism in a cell-autonomous manner.
I also examined whether UDP-GlcNAc levels are sensitive to dietary GlcNAc
supplementation in vivo, and might increase nutrient uptake and promote anabolic metabolism via
Gln is not an essential amino acid, but high growth rates in embryonic and cancer cells
depend on the import of Gln, and anaplerotic conversion to α-ketoglutarate which supports the
TCA cycle (DeBerardinis et al, 2008). SLC7A5/SLC3A2 is a bidirectional transporter that imports
branched-chain essential amino acids (BCAA) (Leu, Ile,Val) in exchange for Gln efflux (Nicklin
et al, 2009). The Gln transporter, SLC1A5 is widely expressed and required to support BCAA
uptake, although in some cells, Gln biosynthesis from α-ketoglutarate and glutamate can support
internal needs and SLC7A5/SLC3A2 activity (Hassanein et al, 2013). Tet-induced Mgat5 alone in
Hek293 cells was sufficient to increase uptake of Gln and EAA in low Gln/Glc culture conditions.
Gln is also a positive regulator of HBP (Abdel Rahman et al, 2013) and may drive reciprocal
positive feedback between N-glycan branching and metabolism (Figure 2.4D and 2.9). Mgat5-
deficient mice are hypoglycemic and display a reduced sensitivity to glucagon (Johswich et al,
2014), and here we present the first evidence of a cell autonomous regulation of metabolism by N-
glycan branching. Up-regulation of Mgat5 in Hek293 cells stimulated amino acid uptake and
increased metabolite levels under low Gln/Glc culture conditions. Further work is required to
identify the various nutrient transporters regulated by N-glycan branching and their contribution
to development, disease and environmental stress.
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Figure 2.1 Branching pathway and inducible expression of branching enzymes.
(A) N-glycan branching pathway modified from Essentials of Glycobiology 2nd edition, Chapter 8 Figure 5 (Varki et al, 2009). GlcNAcT-I (Mgat1), GlcNAcT-V (Mgat5), avian GlcNAcT-VI (Mgat6) are circled. (B-D) Expression of transgene proteins detected by Western blots probed with antibodies to Flag and tubulin. Flag-tagged GlcNAcT-I, GlcNAcT-V (upper band), and GlcNAcT-VI have expected molecular weights of 53kD, 86kD and 54kD, respectively. Flp-In-TREx HeLa clones were cultured in DMEM with 25 mM Glc, 4 mM Gln, 10% FBS (normal culture conditions), with or without 1 ug/ml of tetracycline (tet) for 24h. (E) Tet dose response in clone 8 Mgat5 Flp-In-TREx HeLa cells. (F-H) N-acetylglucosaminyltransferases activities measured in cell lysates from tet-inducible expression of Flag-tagged Mgat1, Mgat5 and Mgat6 in different Flp-In-TREx HeLa cell clones.
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Figure 2.2 N-glycan profiles of transgenic Flp-In-TREx HeLa cells.
(A) ConA and (B) L-PHA lectin binding to Mgat1, Mgat5 and Mgat6 in Flp-In-TREx HeLa clones, with and without 1 ug/ml tet for 24h, measured by fluorescence microscopy, *p<0.05 and **p<0.01 by student t-test. (C-E) LC-ESI MS chromatogram for N-glycans in Flp-In-TREx HeLa cells expressing (C) Mgat1 clone 3, (D) Mgat5 clone 8, and (E) Mgat6 clone 6. The red chromatogram is tet-induced and black is non-induced. Orange circles indicate increases, and blue circles indicate decreases. Cells were grown in DMEM, 25 mM Glc and 4 mM Gln +10% FBS (standard conditions) with and without tet for 24h.
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Figure 2.3 N-glycan profiles of transgenic Flp-In-TREx Hek293 cells by LC-ESI MS.
(A) Expression of transgene proteins detected by western blot probed with anti-Flag and tubulin antibodies in three different Flp-In-TREx Flag-tagged Mgat5 Hek293 clones, and Mgat5 enzyme activity measured in cell lysates from tet-inducible expression of Flag-tagged Mgat5 in clone 4. (B) Tet-induced Mgat5 increases complex-type N-glycan branching in Hek293 Mgat5 clones 4 and 8, quantified by Alexa-488 conjugated L-PHA fluorescence imaging. For quantification, mean ±SD, one-way ANOVA with Dunnett's multiple comparison test. (C) L-PHA binding of tri- and tetra-antennary N-glycans displays a synergistic effect for GlcNAc supplementation with tet-induced Mgat5 in clone 4. *p<0.05 and **p<0.01 by student t-test compared to -tet control. (D-F) LC-ESI MS chromatogram of N-glycans in Flp-In-TREx Hek293 cells expressing (D) Mgat1, (E) clone 4 Mgat5, and (F) Mgat6. Experimental conditions as described in Figure 2.
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Figure 2.4 Metabolite levels are sensitive to tet-induced branching and HBP stimulation.
(A) Mgat1, Mgat5 and Mgat6 Flp-In-TREx HeLa and Hek293 cells were grown in standard culture conditions with and without tet (T) and 0, 15 or 30 mM GlcNAc supplementation for 24h. Each row represents a biological replicate (n=4-5). Metabolites in cell lysates were measured by targeted LC-MS/MS and normalized to cell number. Data for 129 metabolites was analyzed by unsupervised clustering and presented as heat maps. (B) Lactate and (C) GSH / GSSG ratios in Flp-In-TREx Hek293 cells with and without tet-induced Mgat1, Mgat5 and Mgat6 induction for 24h. Additive effects of tet and GlcNAc, *p<0.05 by student t-test. (D) Scheme for increased nutrient uptake and flow to catabolism with tet-induced increased N-glycan branching and GlcNAc supplementation. Green and yellow arrows indicate putative positive feedback from central metabolism through de novo HBP to N-glycan branching, which in turns promotes cell surface residency of transporters and more nutrient uptake.
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Figure 2.5 Growth of Mgat5 Flp-In-TREx Hek293 cells in defined Glc and Gln conditions.
Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured with and without tet in medium modified for Glc/Gln content as indicated + 10% FCS for 48h. (A) Cell growth as a function of Glc and Gln concentrations, with and without tet-induced Mgat5 (cell count ± SD, n=3). (B) Growth conditions (grey bars) corresponding to (C) metabolite profiling by LC-MS/MS, and analyzed by principal component analysis (PCA).
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Figure 2.6 Amino acid levels increase with Mgat5 expression under Gln-deprived conditions.
(A) Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured in the four Gln/Glc conditions indicated, and data was normalized to 2.5/10 Gln/Glc no-tet (second bar from the right, red line) and plotted as fold change (mean ±SD, n=5, *p<0.05). The axis labels are shown for Ala at the top. The pathway scheme is from Chapter 20 (Amino Acid Degradation and Synthesis) Lippincott's Illustrated Reviews: Biochemistry. (B) Heat map of amino acid levels in cells and medium showing data for each of 4-5 replicates. In limiting conditions, tet-induced Mgat5 increased cellular amino acids content, and the depleted growth medium of the same, indicating increased amino acids uptake. Red is high, green is low. White boxed area highlights contrasting effect of +tet on cells and medium.
Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured in the four Gln/Glc conditions indicated. The data was normalized to 2.5/10 mM Gln/Glc no-tet conditions (second bar from the right, red line). *p<0.05 by student t-test for tet-induced change at 0/2.5 mM Gln/Glc condition.
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Figure 2.8 Tet-induced Mgat5 increases HBP and glycolysis metabolites under Gln/Glc limiting conditions.
Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured in the four Gln/Glc conditions, and normalized data graphed as fold change. The data was normalized to 2.5/10 mM Gln/Glc no-tet conditions (second bar from the right, red line). *p<0.05 by student t-test for tet-induced change at 0/2.5 mM Gln/Glc condition.
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Figure 2.9 Summary of metabolite changes with tet-induced Mgat5 in Hek293 cells under low Gln/Glc conditions.
Metabolites in bold were measured by LC-MS/MS, and the EAA are marked in blue. * indicates 2 fold or more decrease as a result of growth in low Gln/Glc 0/2.5 mM conditions. Green arrows mark metabolites increased by tet-induced Mgat5.
Flp-In-TREx Hek293 (A) Mgat5 clone 4 and (B) Mgat1 clone 7 cells, were grown with and without tet and 15 mM GlcNAc in standard culture conditions. Gln was removed for 16 h and cells were pulsed with 1 mM 15N15N Gln for the indicated times. 15N15N Gln and 15N Gln levels in cell lysates were measured by LC-MS/MS. *p<0.05 and **p<0.01 by student t-test comparing nil to tet or GlcNAc to GlcNAc+tet, with n=5-6 samples. The bar graphs on the left compare branching by L-PHA staining of Mgat1 and Mgat5, with and without tet-induction.
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Chapter 3
Metabolic Reprogramming by the Hexosamine Biosynthetic Pathway and
Golgi N-Glycan Branching
A version of this chapter is in revision at the Journal of Biological Chemistry
Michael C. Ryczko, Judy Pawling, Rui Chen, Anas M. Abdel Rahman, Kevin Yau,
Daniel Figeys, and James W. Dennis
Attributions:
Mouse-work and targeted metabolomics mass-spectrometry experiments in Figures 3.1A-F, 3.2A-
G, 3.3A,D-F,I, 3.5E-G, 3.7A,G,I, 3.8A-C,E, and 3.9A-D,G-I were performed together with Judy
Pawling.
Site-specific characterization of N-glycan structures on liver glycoproteins summarized in Table
3.2 and displayed in Figure 3.6 was performed by Rui Chen using LC-MS/MS.
All other experiments were designed, performed and analyzed by Michael Ryczko.
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3.1 Summary
UDP-GlcNAc is an essential substrate for protein N-glycosylation and Golgi remodeling
of N-glycans, a crucial modification to cell surface receptors and nutrient transporters. de novo
synthesis requires glucose, glutamine, acetyl-CoA and uridine-triphosphate, and cellular levels of
UDP-GlcNAc are sensitive to the supply of these central metabolites. GlcNAc is salvaged from
dietary and glycoconjugate turnover into the HBP to generate UDP-GlcNAc. Herein I examined
the effects of GlcNAc supplementation and salvage on metabolism in C57BL/6 mice. Fat and
body-weight increased without affecting calorie-intake, activity, or energy-expenditure. Chronic
oral GlcNAc increased hepatic UDP-GlcNAc, and GlcNAc content in N-glycans on hepatic
Table 3.1 Phenotypic differences in serum biochemistry between GlcNAc treated and untreated mice on 9% fat diet, under fasted and fed conditions.
At sacrifice mice were 10 months old (n=4-5 per group), and have been on 0.5 mg/ml oral GlcNAc supplementation for 30 weeks, or 7 months. Data are mean ±SEM. *p<0.05 versus fasted or fed ad libitum control mice on 9% fat diet (2-tailed, unpaired Student's t-test). ALT alanine aminotransferase, HOMA2-IR homeostatic model assessment-insulin resistance.
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Diet
GlcNAc content (GlcNAc treated/control)
Glycopeptides with N-glycans
p-values
4% fat 1.54 361 <0.0001
4% fat, fasted 1.99 394 <0.0001
9% fat 1.52 292 <0.0001
9% fat, fasted 1.54 218 0.2785
Table 3.2 Global analysis of relative GlcNAc content in liver N-glycans.
N-glycans from liver glycopeptides, from control and GlcNAc treated mice, were identified by differential labelling with stable light and heavy isotope dimethyl labelling followed by LC-MS/MS. Liver was isolated from mice on 4% and 9% fat diets, in fasted and fed conditions. Sign test with probability of 0.5 and two-tail p-value was performed.
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Figure 3.1 Oral GlcNAc is rapidly absorbed by gut to enter bloodstream and be taken up by tissue from circulation.
(A) Change in body-weight for wild-type C57BL/6 male mice on diets containing different percentages of fat. Data shown are mean ± SEM, n=8, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test compared with 4% fat diet, with significant differences indicated as #p<0.05 and ##p<0.01 versus 9% fat diet, and *p<0.05 and **p<0.01 versus 22% fat diet. (B) Respiratory exchange ratio (RER) of mice fed different percentage fat diets for 50 weeks, (C) quantification of data in (B) for night and day. Data shown are mean ±SEM, analyzed by one-way ANOVA followed by Tukey's multiple comparison test, with significant differences indicated as *p<0.05, **p<0.01, and ***p<0.001. (D) Time-course of relative abundance of 13C6-GlcNAc in serum of mice orally gavaged with a bolus administration of 13C6-GlcNAc at 20 µg/g of mouse. (E) Time-course of relative abundance of Glc-d7 in serum of mice orally gavaged with a bolus administration of Glc-d7 at 50 µg/g of mouse. (F) At 180 minutes following oral gavage with 20 µg/g 13C6-GlcNAc, UDP-13C6-GlcNAc was detected as a strong peak in different mouse tissues.
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Figure 3.2 Oral GlcNAc increases UDP-GlcNAc level and promotes weight-gain in mice.
(A) Analysis of change in body-weight of wild-type C57BL/6 male mice on low fat diet over 90 days supplemented with oral GlcNAc. (B) Relative abundance of liver metabolites in the distal portion of HBP measured by LC-MS/MS in 90 day GlcNAc treated mice on low fat diet. (C) Principle component analysis of all measured liver metabolites in mice on low fat diet and GlcNAc supplied in drinking water at 0.5, 5.0 and 15 mg/ml. Steady-state liver metabolites in the glycolysis and gluconeogenesis pathways (D), tricarboxylic acid (TCA) cycle (E), amino acids (F), and oxidized and reduced forms and ratios of glutathione and nicotinamide adenine dinucleotides (G). All metabolites measured by LC-MS/MS and expressed as fold change in 90 day GlcNAc treated mice on low fat diet. Relative levels of specific metabolites were normalized to liver weight. Metabolomic data shown are mean ±SEM, n=10, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test compared with vehicle control of 0 mg/ml GlcNAc, with significant differences represented as *p<0.05, **p<0.01, and ***p<0.001.
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Figure 3.3 Oral GlcNAc promotes weight-gain and lipid accumulation.
(A) Change in body-weight for wild-type C57BL/6 male mice on 4% and 9% fat diet over weeks of oral GlcNAc or GlcN supplied in drinking water at 0.5 mg/ml. Data shown are mean ± SEM, n=10, analyzed by 2-tailed unpaired Student's t-test, with significant differences indicated as #p<0.05 for 4% fat versus GlcNAc 4% fat, and *p<0.05 for 9% fat versus GlcNAc 9% fat. (B) Body-weight and (C) calorie intake per body-weight per day at 34 weeks of age, or following 20 weeks of GlcNAc treatment. Terminal body-weight (D), tissue composition (E), and epidydymal-fat and liver weight normalized to body-weight (F) at sacrifice. Error bars represent ±SEM, n=10, *p<0.05 or **p<0.01 GlcNAc treated versus control, on either 4% or 9% fat diet (2-tailed, unpaired Student's t-test). Serum free fatty acids (FFA) (G), triglycerides (TG) (H), and significant steady-state metabolite changes (I) in mice on 9% fat diet and supplemented with 0.5 mg/ml oral GlcNAc for 90 days. Error bars represent ±SEM, n=5, *p<0.05, or **p<0.01 versus control (2-tailed, unpaired Student's t-test). (J) Western blot analysis of metabolic signaling pathways in liver lysates from mice maintained on 9% fat diet supplemented with GlcN or GlcNAc, probed for phosphorylated AMP-Activated Protein Kinase (AMPK-α), Acetyl-CoA Carboxylates Kinase (ACC) and ribosomal protein S6 (S6). Tubulin was used as a loading control.
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Figure 3.4 Oral GlcNAc promotes fatty acid oxidation without affecting activity or energy expenditure.
(A) Analysis of total activity, (B) oxygen consumption rate, (C) carbon dioxide emission rate, and (D) Respiratory Exchange Ratio (RER=VCO2/VO2) over 20 hour period, (E) quantification of data in (D) for night and day, (F) energy expenditure, and (G) food and (H) water intake. Liver free fatty acids (FFA) (I) and triglycerides (TG) (J) in mice on 9% fat diet and orally supplemented with 0.5 mg/ml GlcNAc. Error bars represent ±SEM, n=5, *p<0.05, or ** p<0.01 versus control (2-tailed, unpaired Student's t-test). (K) Representative images of liver histology sections stained with oil red O to detect TG and neutral lipids in mice on 9% fat diet, fed ad libitum or fasted for 18 h, and supplemented with 0.5 mg/ml GlcNAc in drinking water for 30 weeks.
(A) Representative images of liver histology sections stained with periodic acid-Schiff, with glycogen deposits detected as purple-magenta areas, obtained from mice on 9% fat diet mice, fed ad libitum or fasted for 18 h, and supplemented with 0.5 mg/ml GlcNAc in drinking water for 30 weeks. (B) Liver glycogen content. Error bars represent ±SEM, n=5, *p<0.05 versus control (2-tailed, unpaired Student's t-test). (C) Immunoblot analysis of metabolic signaling pathways with FASN, and phosphorylated versions of GS, Akt kinase, ribosomal protein S6, AMPK-α, and ACC in liver lysates of fasted mice maintained on 9% fat diet and treated with GlcN or GlcNAc. (D) Quantification of immunoblots using tubulin as loading control. (E) Steady-state relative abundance of metabolites in blood serum from mice maintained on 9% fat diet and GlcNAc for 30 weeks. (F) Intraperitoneal glucagon tolerance test, and (G) intraperitoneal glucose tolerance test, with Area Under the Curve (AUC) quantification in mice treated with 0.5 mg/ml oral GlcNAc for 23 weeks. Error bars represent ±SEM, n=10, analyzed with 2-tailed unpaired Student's t-test *p<0.05 versus control on the same diet.
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Figure 3.6 Oral GlcNAc increases tri-antennary N-glycan structures on glycosite Asn89 of CEACAM1 hepatic transmembrane glycoprotein.
(A) By matching the Y1 ion (peptide + GlcNAc) from the MS/MS spectrum to the list of deglycosylated peptides identified by Mascot database search with accurate molecular weight and retention time, the peptide sequence was identified as Asn89 of CEACAM1 transmembrane glycoprotein. (B) The extracted ion chromatogram peak area for control (light) and GlcNAc (heavy) labelled precursor from deglycosylated CEACAM1 peptides. With a ratio of GlcNAc to control of 1.68, no significant difference was found, which demonstrates that the increase in abundance of intact glycopeptides with tri-antennary complex structure was due to increased branching of complex N-glycans. (C) Annotated MS/MS spectrum of heavy-labelled intact glycopeptide identified with complex tri-antennary N-glycan structure. Terminal sialylation could be verified by the existence of oxonium ion with m/z 292 and 274. (D) The extracted ion chromatogram (XIC) of control (light) and GlcNAc (heavy) labelled peptide precursor from full MS scan indicates the abundance of tri-antennary glycopeptide being much higher in liver lysates from GlcNAc treated mice, with a ratio of 21 in GlcNAc to control. (E) Hybrid bi-antennary N-glycan structure with unsubstituted terminal mannose residues. (F) XIC from control and GlcNAc bi-antennary hybrid N-glycan with mannose, with a peak area ratio of GlcNAc to control of 1.21. (G) Complex bi-antennary N-glycan structure. (H) XIC from control and GlcNAc complex bi-antennary N-glycan, with a peak area ratio of GlcNAc to control of 1.17.
(A) Fold changes of distal HBP metabolites upon GlcNAc treatment for 20 h. (B) Analysis of Mgat5-mediated β1,6-GlcNAc branched complex-type N-glycans on cell surface glycoproteins quantified with fluorophore conjugated lectin L-PHA. (C) Analysis of oligomannose-type and hybrid-type N-glycans on cell surface glycoproteins with fluorophore conjugated lectin ConA. (D) Immunoblot analysis of O-GlcNAcylation detected with monoclonal-antibody CTD110.6. (E) Protein expression level by western blot analysis of FASN enzyme and loading control tubulin, used for relative quantification of immunoblot. (F) Intracellular lipid droplets, quantified microscopically with lipophilic fluorescent probe BODIPY 493/503. (G) Fold change in specific metabolites involved in fat metabolism, normalized to cell number. (H) Glucose uptake in cells treated with GlcNAc for 20h, grown in the presence of fluorescent glucose analog 2-NBD-Glc for 1h, and quantified using flow cytometry as mean fluorescent intensity (MFI). (I) Analysis of heavy-isotope labelled 15N2-Gln uptake in cells treated with GlcNAc for 20 h, pulsed with 15N2-Gln for designated times, and quantified using mass spectrometry. (J) Tri- and tetra-antennary Mgat5-modified N-glycans, and (K) intracellular lipid accumulation in the absence and presence of GlcNAc and/or Swainsonine (SW), quantified microscopically with fluorophore-conjugated lectin L-PHA or BODIPY 493/503. Data shown are mean ±SEM, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test compared with vehicle control (A-C, E-G, J-L), with significant differences represented as *p<0.05, **p<0.01, and ***p<0.001.
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Figure 3.8 HBP and N-glycan dependent regulation of cellular metabolism.
(A) Analysis of 15N2-Gln uptake and its downstream metabolites (B) glutamate (15N-Glu) and (C) 15N-Gln in HeLa cells treated with GlcNAc for 20 h, pulsed with 15N2-Gln, and quantified using LC-MS/MS. (D) Analysis of intracellular lipid in HeLa cells. (E) Fold change (FC) in L-PHA and UDP-GlcNAc as a function of GlcNAc treatment in HeLa cells. (F) Kinetic reading monitoring uptake of non-esterified long-chain-fatty-acid-analog (BODIPY-FA) quantified as Area Under the Curve (AUC) for Relative Fluorescence Units (RFU). (G) Analysis of intracellular lipid and (H) complex-type N-glycans in 3T3-L1 adipocytes. Data are mean ±SEM, analyzed by t-test or one-way ANOVA followed by Dunnett’s test, significant differences represented as *p<0.05, **p<0.01, and ***p<0.001. (I) GlcNAc salvaged by HBP increases UDP-GlcNAc, the substrate for N-acetylglucosaminyltransferase enzymes (Mgat1, 2, 4, 5) acting on glycoprotein acceptors trafficking through Golgi en route to the cell surface. Km values for UDP-GlcNAc decline from Mgat1, Mgat2, Mgat4 to Mgat5, making biosynthesis of tri- and tetra-antennary N-glycans sensitive to UDP-GlcNAc levels. N-glycan branching increases the affinity of glycoproteins for galectins, which cross-link and oppose loss of receptors and transporters to endocytosis. This increases cell surface residency of glucose (Glc), glutamine (Gln), and fatty-acid (FA) transporters (Glut, SLC and CD36 respectively), and other transmembrane glycoproteins such as CEACAM1. The improved cell surface retention allows more nutrients to enter the cell and contribute to increased lipid accumulation via FASN. A positive-feedback loop is formed by increasing uptake and flux of Glc, Gln and Ac-CoA through de novo HBP to UDP-GlcNAc and N-glycan branching on receptors and transporters.
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Figure 3.9 Oral GlcNAc promotes lipid storage in male and female Mgat5 wild-type and null mice.
(A) Change in body-weight for Mgat5 wild-type (wt) and null male mice on 9% fat diet supplemented with 0.5 mg/ml oral GlcNAc in drinking water for 30 weeks. (B) Fat and lean tissue mass, as measured by magnetic resonance imaging. (C) Diurnal and nocturnal respiratory exchange ratio (RER). (D) Relative abundance of liver GlcNAc-P and UDP-GlcNAc determined using LC-MS/MS and expressed as fold change. Error bars represent ±SEM, n=4-5, with statistical significance indicated as *p<0.05 and **p<0.01 versus control for the same genotype (2-tailed, unpaired Student's t-test). (E) Analysis of intracellular lipid accumulation in lipid droplets as a function of overnight exogenous GlcNAc supplementation in primary hepatocytes obtained from Mgat5 wt and null mice, quantified microscopically using the lipophilic fluorescent probe BODIPY 493/503. Data shown are mean ±SEM, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test compared to 0 mM GlcNAc control of respective genotype, with significant differences indicated as *p<0.05, **p<0.01, and ***p<0.001. (F) Histological analysis of liver tissue sections stained with oil red O, identifying lipid deposits as red-stained areas. (G) Change in body-weight of Mgat5 wt and null female mice on 9% fat diet supplemented with 0.5 mg/ml oral GlcNAc in drinking water for 21 weeks. (H) Fat-tissue to body-weight (g/g) ratio, and (I) lean-tissue to body-weight (g/g) ratio, as measured by DEXA. (J) Serum concentration of leptin. Error bars represent ±SEM, with 4 to 6 mice per group, *p<0.05 versus control for the same genotype (2-tailed, unpaired Student's t-test).
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Chapter 4
Discussion and Future Directions
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4.1 GlcNAc and N-Glycan Branching in Mgat5 Null Mice
Mammalian glycans are involved in molecular and cellular mechanisms that control health
and disease, and changes in glycosylation have been observed in both genetic and acquired disease
states (Dennis et al, 2009; Freeze et al, 2015; Ohtsubo & Marth, 2006). The HBP and Golgi N-
glycan branching pathway are linked to metabolic homeostasis (Dennis et al, 2009; McClain,
2002). Null mutations in genes encoding N-glycan branching enzymes Mgat4a and Mgat5, which
are sensitive to UDP-GlcNAc levels from the HBP, disrupt mouse glucose homeostasis and result
in abnormal body-weight (Dennis et al, 2009). Mgat5-/- mice are leaner, smaller in size, and exhibit
reduced fat pad depots despite maintaining daily calorie intake and physical activity similar to
wild-type mice, who show large abdominal fat deposits on the same diet (Cheung et al, 2007).
In my studies, oral GlcNAc supplementation partially restored fat accumulation in Mgat5-
/- mice and primary hepatocytes. This result is consistent with functional redundancy of N-glycan
branches, where increased levels of UDP-GlcNAc drive compensating increases in N-glycan
branching by other Mgat enzymes (Dennis & Brewer, 2013). Indeed, even though Mgat5 enzyme
is absent and hence no β1,6-linked GlcNAc can be added, there is evidence of functional
redundancy or compensation in the remaining structurally related N-glycan branches to maintain
branch complexity on glycoproteins for cross-linking endogenous galectins at the cell surface
(Dennis & Brewer, 2013; Johswich et al, 2014). This can occur provided there is an abundant
supply of the common substrate UDP-GlcNAc, since the Golgi N-glycan branching pathway is
sensitive to its levels, especially for generating tri- and tetra-antennary N-glycans (Dennis et al,
2009). In fact, GlcNAc supplementation in Mgat5-/- cells rescued levels of EGF and TGF-ß cell
surface receptors and their signaling by restoring the glycan-galectin lattice (Lau et al, 2007).
Therefore it was reasonable to assume that additional metabolic supply of UDP-GlcNAc, through
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GlcNAc supplementation to HBP, may rescue fat accumulation defects observed in Mgat5-/- mice,
as it partially did, supporting the idea of functional redundancy between N-glycans. Also, GlcNAc
supplementation rescued glucagon receptor sensitivity and signaling in Mgat5-/- primary
hepatocytes and in vivo in Mgat5-/- mice in the glucagon tolerance test (Johswich et al, 2014). In
my study, we further show that GlcNAc enhanced glucagon sensitivity in wild-type mice in the
glucagon tolerance test, which might be indicative of enhanced glucagon receptor activity through
increased N-glycan branching.
4.2 HBP and N-Glycan Branching Reprogram Metabolism to Promote Fat Accumulation
Experimental evidence suggests that UDP-GlcNAc concentration is at least 20-fold higher
in the Golgi than in the cytoplasm (Waldman & Rudnick, 1990). This concentration gradient is
established by the Golgi UDP-GlcNAc antiporters, which exchange uridine monophosphate
(UMP) for UDP-GlcNAc, thus forming a direct proportionality between the steady-state amounts
of UDP-GlcNAc inside the Golgi and the cytosol (Lau et al, 2007). The Vmax of UDP-GlcNAc
transport is on the order of ~0.2 mM/sec, which corresponds to mM concentration of UDP-GlcNAc
in the Golgi (Lau et al, 2007). Previous calculations used to establish a computational model of
Golgi N-glycan branching ultrasensitivity estimate the basal physiological Golgi concentration of
UDP-GlcNAc at ~1.5 mM (Lau et al, 2007). Moreover, a recent publication reported that the UDP-
GlcNAc antiporter SLC35A3 forms a complex with Mgat5 enzyme in the Golgi membrane, and
thus augments its catalytic activity by proximity (Maszczak-Seneczko et al, 2015). SLC35A3
ensures a localized subcellular supply of substrate for the late Mgat enzymes to regulate N-glycan
branching pathway. Indeed, cells deficient in SLC35A3 activity displayed reduced amount of
145
highly branched tri- and tetra-antennary N-glycans (Maszczak-Seneczko et al, 2013). Thus, UDP-
GlcNAc distribution in the Golgi does not appear to be homogenous, but is rather highly localized.
A relatively restricted localization of UDP-GlcNAc transporters within the Golgi suggests that this
common donor substrate may be preferentially supplied to specific Golgi compartments where
Mgat branching enzymes reside. GlcNAc supplementation and Mgat5 overexpression displayed
synergistic increase in branching, consistent with Mgat5’s high Km value and pathway
ultrasensitivity to UDP-GlcNAc (Abdel Rahman et al, 2015; Lau et al, 2007).
Numerous lines of evidence indicate that metabolic flux through the HBP and availability
of intracellular UDP-GlcNAc regulate activities of Golgi Mgat enzymes, and control the N-glycan
branching pathway and cell surface retention of transmembrane glycoproteins (Dennis et al, 2009;
Johswich et al, 2014). In cultured cells, GlcNAc directly enters the HBP to increase total cellular
UDP-GlcNAc pool, enhance N-glycan branching, and improve the association of glycoprotein
receptors and transporters with galectins, thereby increasing their cell surface retention and
sensitivity to extracellular factors (Abdel Rahman et al, 2015; Dennis et al, 2009; Johswich et al,
2014). I hypothesized that GlcNAc supplementation to the HBP could increase the surface level
of nutrient transporters to facilitate efficient nutrient uptake, i.e. transport more fatty acids, glucose
and/or glutamine, and synthesize and store more lipids, via an Mgat5-mediated N-glycan
dependent mechanism (Abdel Rahman et al, 2015).
I examined the role of increasing UDP-GlcNAc pool through GlcNAc supply in order to
elucidate its effects in vitro and in vivo at the molecular, cellular and physiological levels of
analysis. GlcNAc supplemented to mice, delivered by ad libitum drinking water, resulted in
increased hepatic UDP-GlcNAc pool, indicating that the salvage pathway has the capacity to
elevate UDP-GlcNAc levels in vivo. I show that extended GlcNAc supplementation in mice
146
increased their body-weight without affecting calorie-intake, activity, or energy expenditure.
These results show that GlcNAc treated mice are not indolent or lethargic, but rather that chronic
et al, 2013). This demonstrates that multiple genes encoding components of the hexosamine
biosynthetic and N-linked glycosylation pathways have the potential to harbor mutations that cause
this syndrome, and suggests a general framework that might be operational in other diseases and
disorders.
In a GWAS carried out in individuals from diverse European populations a SNP located
downstream of the Mgat1 gene was shown to be significantly associated with body-weight
(Jacobsson et al, 2012; Johansson et al, 2010). Furthermore, two other SNPs in the Mgat1 gene
157
were found to be nominally associated to body-weight (Johansson et al, 2010). The molecular
mechanisms by which these SNPs may contribute to the observed associations are unclear. Genetic
variants can result in changes in expression, or SNPs in the HBP and Mgat genes can affect the
functional efficacy of the corresponding enzymes in the HBP and Golgi N-glycan branching
pathway. For instance, missense mutations that dramatically increased the apparent Km value of
Mgat1 for both UDP-GlcNAc and the glycoprotein acceptor were identified in CHO mutant cells
(Chen et al, 2001). Cells harboring a genetic variant of this kind synthesized hybrid and complex
type N-glycans on cell surface glycoproteins, albeit in reduced amounts compared to parental CHO
cells (Chen et al, 2001). This alteration might in turn translate into functional consequences for
growth factor signaling and nutrient transport.
Human multiple sclerosis patients frequently show partial deficiencies in Mgat5 modified N-
glycans, which lowers their T-cell sensitivity to autoimmune activation (Grigorian et al, 2009).
Disease associated human Mgat1 SNPs showed a gain-of-function mutation that increased Mgat1
mRNA and protein levels, which in turn increased Mgat1 activity and decreased Mgat5-mediated
β1,6-GlcNAc-branched N-glycans (Mkhikian et al, 2011). When metabolism limited substrate
availability in the form of UDP-GlcNAc, the Mgat1 gain-of-function haplotype lowered N-glycan
branching by limiting UDP-GlcNAc availability to downstream Mgat branching enzymes
(Mkhikian et al, 2011). In contrast, when UDP-GlcNAc supply increased, as occurs in presence of
high glucose or supplementation with GlcNAc and uridine, the Mgat1 haplotype had the opposite
effect on late N-glycan branching and glycoprotein surface residency (Mkhikian et al, 2011). These
studies demonstrate that small changes in few genes and/or metabolites affecting a common
biochemical pathway can have drastic effects on N-glycosylation, and provide a framework for
understanding how genetic and environmental effects converge and interact at the molecular level.
158
Mgat5-deficient mice display lower thresholds to T-cell receptor clustering, T-cell
activation and autoimmune disease (Grigorian et al, 2009). However these phenotypes of Mgat5-/-
mice appear more penetrant on certain mouse genetic backgrounds than others, suggesting the
existence of strain-dependent modifier genes or strain-specific SNPs in the HBP and/or N-glycan
branching pathway for these phenotypes (Dennis et al, 2009; Grigorian et al, 2009). The PL/J strain
of mice, which show reduced Mgat activities, is hypersensitive to spontaneous demyelinating
autoimmune disease, a model of multiple sclerosis (Grigorian et al, 2009). Multiple sclerosis
increases in frequency and onset in PL/J Mgat5-/- mice, while the 129/sv Mgat5-/- mice do not display
the clinical or histopathological symptoms of this autoimmune disease at any age (Grigorian et al,
2009). Importantly, GlcNAc supply to the HBP and N-glycan branching regulates PL/J T-cell
hypersensitivity and susceptibility to autoimmune disease in vitro and in vivo, suggesting that some
of these polymorphic differences are amenable to environmental influences through metabolism
(Grigorian et al, 2009; Mkhikian et al, 2011). This implies that genetic variation and conditional
regulation via environmental factors that influence metabolic pathways interact through the HBP and
Golgi N-glycan branching pathway to regulate cellular homeostasis (Dennis et al, 2009; Mkhikian et
al, 2011).
It would be of interest to determine if variant alleles in the HBP and/or Golgi N-glycan
enzymes could be linked with body-weight regulation, obesity, or other metabolic diseases. The
C57BL/6 strain of mice used in the GlcNAc supplementation experiments is sensitive to
development of diet-induced obesity and insulin resistance. It would be interesting to find out if
GlcNAc supplementation in a different strain of mice, such as PL/J or 129/sv, would generate a
similar phenotype to that observed in C57BL/6 mice. Future studies could also include direct
sequence comparison of genes in the HBP and N-glycan branching pathway to identify genetic
159
polymorphisms between strains of mice exhibiting different body-weight and/or composition, or
in human populations displaying obesity and severe body-weight phenotypes.
Variants of the gene glucosamine-6-phosphate deaminase 2 (GNPDA2), which codes for
an enzyme participating in the HBP, is strongly linked to BMI and obesity in children and adults
from different human populations (Renstrom et al, 2009; Willer et al, 2009; Wu et al, 2010; Zhao
et al, 2009). GNPDA2 transcript levels were down-regulated in the hypothalamus of high-fat diet
fed rats (Gutierrez-Aguilar et al, 2012). GNPDA2 catalyzes the reversible reaction converting
GlcN-6P back to Fru-6P and ammonium, thus opposing the function of GFAT which catalyzes the
conversion of glutamine and Fru-6P to GlcN-6P and glutamate at the start of the HBP. The function
of GNPDA2 has not been explored but it is reasonable to assume that a deletion or deleterious
mutation in this gene would result in enhanced flux through the HBP and increased UDP-GlcNAc
formation. Such an outcome would support my findings of increased body-weight observed with
GlcNAc supplementation experiments, and relate it to the epidemiological findings linking SNPs
in GNPDA2 with obesity. The caveat however is that there is also a GNPDA1, which could be
redundant in function, and that both enzymes GNPDA and GFAT are allosterically controlled by
GlcNAc-6P, in a positive and negative manner respectively (Broschat et al, 2002; Lara-Lemus &
Calcagno, 1998).
4.10 GlcNAc Supplementation and O-GlcNAcylation
Most studies where the HBP is supplemented to increase UDP-GlcNAc levels are very
selective in terms of which downstream pathway utilizing UDP-GlcNAc is analyzed, either
pursuing O-GlcNAcylation, N-glycosylation, or hyaluronan synthesis. However the fact remains
160
that any of these could be impacted by increased activity in the HBP. In theory any glycoconjugate,
be it a glycoprotein, glycolipid, or glycosaminoglycan, that utilizes GlcNAc from UDP-GlcNAc
could potentially be affected. The complex nature of the effect of increasing flux in the HBP, with
few potential targets involved, makes it difficult to unambiguously dissect its role. I focused on N-
glycosylation, for numerous valid reasons described above and Dennis laboratory’s long-term
commitment to N-glycan branching, but given that the role of O-GlcNAcylation is firmly
established in metabolism and energy homeostasis it is reasonable to assume that it could also have
been a contributing factor to the metabolic phenotype observed with GlcNAc supplementation
(Hardiville & Hart, 2014). For instance, O-GlcNAcylation is involved in short-term fasting
response. In response to glucagon and cAMP, CRTC2 is dephosphorylated and O-GlcNAcylated
at the same site, which promotes its translocation into the nucleus, binding to CREB, and induction
of gluconeogenic gene expression (Dentin et al, 2008). In this study no major changes were
detected in O-GlcNAcylation at the level of western-blot analysis probed with the O-β-GlcNAc-
specific monoclonal antibody CTD 110.6, a reagent heavily used in O-GlcNAcylation research
(Hardiville & Hart, 2014). However, it is reasonable to argue that one limitation of this approach
was the coarse level of resolution employed in my analysis of O-GlcNAcylation. Thus, I cannot
exclude the possibility that more subtle changes in O-GlcNAcylation, such as that occurring on
CRTC2, did take place on specific signaling proteins, transcription factors, metabolic enzymes, or
even histones, which would have eluded the analysis performed. Increasing however, increasing
O-GlcNAcylation globally by using a selective inhibitor of O-GlcNAcase for a few months did
not affect body-weight, or glucose or lipid metabolism in rodents (Macauley et al, 2010a;
Macauley et al, 2010b).
161
4.11 GlcNAc Supplementation and Gut Microbiota
It has been shown before, and confirmed and extended in my studies herein, that GlcNAc
supplementation in cells in culture contributes to UDP-GlcNAc pool and increases β1,6-GlcNAc
branching (Abdel Rahman et al, 2015; Abdel Rahman et al, 2013; Dennis et al, 2009; Johswich et
al, 2014; Wellen et al, 2010). My results show that GlcNAc supplementation affects metabolism
in a cell-autonomous manner in cell culture (Abdel Rahman et al, 2015). In mammalian cells in
culture, salvaged GlcNAc does not appear to re-enter glycolysis to be used as a fuel source for
energy production (Abdel Rahman et al, 2013; Chertov et al, 2011; Wellen et al, 2010). However
in vivo the scenario could be much different, with the gut microbiota acting as a confounding
variable. The microbiome is a diverse ecosystem containing trillions of microbes such as bacteria,
fungi, archaea, protozoa, and viruses, living in the host’s digestive system (Greiner & Backhed,
2011). There is a complex relationship and an intimate connection between the host and the
microbiome, primarily though diet, and more and more studies are being published describing how
dietary interventions affect the host metabolism and physiology in health and disease via their
impact on the microbiome (Greiner & Backhed, 2011). Since nutrients affect the composition of
the gut microbiota, such changes are possible to occur over the course of dietary GlcNAc
supplementation.
The gut microbiota contributes to host metabolism by several mechanisms including
increased inflammatory tone, increased energy harvest from the diet, modulation of lipid
metabolism, and altered endocrine function (Greiner & Backhed, 2011). Recent data have revealed
that the gut microbiota has a strong effect on energy homeostasis, and thus could be considered an
environmental factor that promotes adiposity, and contributes to obesity and other metabolic
diseases (Fang & Evans, 2013; Greiner & Backhed, 2011). Studies in rodents and humans have
162
shown that the microbiome of obese has lower bacterial diversity and a significantly greater ratio
of the phylum Firmicutes to Bacteriodetes (Fang & Evans, 2013; Greiner & Backhed, 2011). It is
possible that GlcNAc supplementation in mice promoted selective advantage to certain microbial
communities, thereby altering the composition of the gut microbiome to modulate the host
metabolism, physiology and homeostatic regulation. Experiments are currently underway to
examine the effect of GlcNAc supplementation on gut microbiota in mice. A recent study reported
that supplementation with specific human breast milk oligosaccharides influenced the selective
intestinal bacterial colonization pattern in mice, as indicated by changes in the abundance of
specific strains (Weiss et al, 2014). The sources and amounts of GlcNAc available to the host
and/or the gut microbiome have not been studied. However GlcNAc residues are common
constituents of glycoprotein N-glycans, mucus glycans, glycosaminoglycan polysaccharides of
extracellular matrix, milk oligosaccharides, fungal and exoskeleton chitin, and bacterial
peptidoglycan (Konopka, 2012; Koropatkin et al, 2012). Thus, in theory, GlcNAc can be obtained
from the host glycocalyx, dietary sources, and the microbiome itself. The microbiome has a variety
of carbohydrate-active enzymes, such as the glycosidases, that may release GlcNAc from all of
these sources during metabolic breakdown in the gut, and perhaps even share it with the host
(Koropatkin et al, 2012). Indeed, the microbiome associated with obesity might have more
glycosidase activity, and hence may liberate more GlcNAc to be used by the host.
In many microorganisms GlcNAc has a significant role in cell signaling (Konopka, 2012).
For example, GlcNAc stimulates the human fungal pathogen Candida albicans to undergo changes
in morphogenesis and expression of virulence genes (Konopka, 2012). GlcNAc also regulates
virulence properties of pathogenic bacteria such as E. coli, and stimulates soil bacteria to sporulate
and produce antibiotics (Konopka, 2012). Bacteroidetes, Firmicutes and a variety of other bacterial
163
taxa actively grow in the presence of GlcNAc (Tada & Grossart, 2014). Therefore, unlike
mammalian cells, bacterial cells are well adapted at utilizing GlcNAc as a carbon and energy
source in glycolysis (Brigham & Malamy, 2005; Rigali et al, 2006; Tannock, 1977). Bacteria
fermenting GlcNAc as a carbon source remove the acetyl group and excrete acetate and propionate
(Kotarski & Salyers, 1981). In this study the serum level of propionate was actually lower in
GlcNAc supplemented mice, suggesting that perhaps GlcNAc utilization by the gut bacteria was
not a significant factor.
Future in depth experiments would be necessary to determine what effects, if any, GlcNAc
supplementation has on gut microbiota, and its consequences on the host metabolism and
physiology. It should be noted that many of these bacteria reside in the cecum, the beginning of
the large intestine, while monosaccharides and disaccharides are primarily absorbed much earlier
in the small intestine. Hence, the majority of orally ingested GlcNAc could have already been
absorbed by the host, with little spared for the gut bacteria. To address this experimentally, the
effect of GlcNAc supplementation on the production of bacterial metabolites such as the short
chain fatty acids acetate, propionate, and butyrate should be measured in the colon fluid. Changes
in the production of these metabolites following GlcNAc supplementation would support a
contribution of intestinal microbes in the regulation of nutrient uptake and energy homeostasis. In
the future, in order to bypass the possible issue of GlcNAc and microbiota interaction altogether,
germ-free mice might be used to perform the same experiment. Alternatively, instead of oral
supplementation, GlcNAc might be delivered parenterally, i.e. administered via a route other than
through the digestive tract, i.e. intraperitoneally, intramuscularly or subcutaneously. Such
approach would also provide precise knowledge of the dose delivered to the animal.
164
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