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Glucosamine-Induced Insulin Resistance in
Primary Rat Hepatocytes and the Role of Selenium
as an Insulin Mimetic
Sandhya N. Adiyodi Veetil
Western Michigan University
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Insulin Mimetic" (2011). Dissertations. Paper 475.
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GLUCOSAMINE INDUCED INSULIN RESISTANCE IN PRIMARY
RAT HEPATOCYTES AND THE ROLE OF SELENIUM
AS AN INSULIN MIMETIC
by
Sandhya N Adiyodi Veetil
A Dissertation
Submitted to the
Faculty of The Graduate College
in partial fulfillment of the
requirements for the
Degree of Doctor of Philosophy
Department of Chemistry
Advisor: Susan R. Stapleton, Ph.D.
Western Michigan University
Kalamazoo, Michigan
June 2011
GLUCOSAMINE INDUCED INSULIN RESISTANCE IN PRIMARY
RAT HEPATOCYTES AND THE ROLE OF SELENIUM AS AN
INSULIN MIMETIC
Sandhya N Adiyodi Veetil, Ph.D.
Western Michigan University, 2011
Type 2 diabetes is mediated by insulin resistance, the inability of insulin to elicit a
normal biological response in insulin responsive tissues. Several cellular models have
been utilized to determine the mechanism of induction of insulin resistance but questions
remain unanswered. One model, implicates the products of the Hexosamine Biosynthetic
Pathway (HBP) in the induction of insulin resistance under hyperglycemia. The major
end product of HBP, UDP-GlcNAc, is the substrate for O-GlcNAc transferase, an
enzyme that catalyzes the O-GlcNAcylation of numerous proteins. This modification
may play a role in induction of insulin resistance and thus needs to be evaluated in
different cell types. Therefore, we set out to test whether or not insulin resistance could
be established in primary rat hepatocytes. To accomplish this we used a HBP precursor,
glucosamine and first assessed whether or not insulin resistance was established by
evaluating insulin's effect on a key signaling protein, Akt.
Results indicate that the insulin induced phosphorylation of Akt was decreased in
the presence of glucosamine when compared to the control, suggesting an insulin
resistant state. Signal protein activation is very important for the insulin regulation of
gene expression. If the activation of signal proteins is altered, then the effect on gene
expression should also be altered. Results show that under glucosamine treatment, insulin
was no longer able to control the gene expression of a number of key enzymes in major
metabolic pathways. Additionally an increase in O-GlcNAc modified proteins under
glucosamine compared to the control was observed and a number of these proteins were
identified through LC-MS.
Lastly, the effect of selenium, an insulin mimetic agent, was examined in this
model system. The effects of selenium on the phosphorylation of the insulin signaling
protein, Akt and on the expression of the key enzymes of the major metabolic pathways
both under normal and insulin resistant conditions were examined. Results show that
selenium is a potent insulin mimetic not only under the normal/control condition, but also
under the insulin resistant condition as well.
UMI Number: 3467829
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Copyright by
Sandhya N Adiyodi Veetil
2011
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my advisor, Dr. Susan R.
Stapleton. I am grateful for her guidance, understanding, patience and continuous support
throughout my graduate training.
I would also like to thank my committee members Drs Michael Barcelona, David
Reinhold and Pamela Hoppe for their patience, encouragement and guidance.
I would also like to thank present and previous members of the Stapleton's lab. You are
all such amazing people and I have been fortunate to have the privilege to work with you.
Finally, and most importantly I would like to give my deepest thanks to my
husband Raghu for his support, patience and understanding. I could not have achieved
my degree without you. Finally, I would like to thank my sister and my father for their
support and encouragement to whom I dedicate this thesis.
Sandhya N Adiyodi Veetil
11
TABLE OF CONTENTS
ACKNOWLEDGMENTS
ii
LIST OF TABLES
vii
LIST OF FIGURES
viii
CHAPTER
1. INTRODUCTION
1
Glucose homeostasis
1
Glucose metabolic pathways of the liver
5
Insulin signaling pathway
10
PI 3-kinase -Akt pathway
11
Insulin resistance and type 2 diabetes
14
Prevalence of diabetes
15
Molecular mechanism of insulin resistance
18
Decreased expression of insulin signaling components
18
Increase in the ratio of regulatory subunit/catalytic subunit
ofPB-kinase
19
Serine phosphorylation of IRS
20
Suppresors of cytokine signaling (SOCS)
21
Phosphatase
22
O-GlcNAcylation of serine/threonine residues
23
Hexosamine biosynthetic pathway (HBP)
26
Role of hexosamine biosynthetic pathway in insulin resistance
27
iii
Table of Contents- Continued
CHAPTER
Selenium-insulin mimetic agent
31
Significance of study
34
Objectives of study
35
2. MATERIALS AND METHODS
37
Primary rat hepatocytes isolation and maintenance
37
Cell treatment and processing
38
Protein isolation and western blot analysis
41
Protein isolation
41
Determination of protein content
42
Immunoprecipitation
42
Western blot analysis
43
LDH Assay
44
RNA Isolation
44
Determination of RNA content and quality assessment
45
Reverse transcriptase polymerase chain reaction (RT PCR)
46
Isolation and purification of O-GlcNAcylated proteins
47
Wheat germ agglutinin lectin affinity chromatography
47
SDS-PAGE (sodium dodecyl polyacrylamide gel
electrophoresis)
48
Identification of O-GlcNAc proteins by LC-MS analysis
48
iv
Table of Contents- Continued
CHAPTER
Statistical Analysis
48
3. RESULTS
49
Effect of glucosamine on insulin induced Akt phosphorylation
50
Effect of glucosamine on insulin induced G6PDH
gene expression
51
Effect of glucosamine on insulin induced fatty acid synthase
(FAS)
55
Effect of glucosamine on insulin inhibition of PEPCK gene
expression
56
Effect of glucosamine on insulin regulation of Foxa2
59
Effect of selenium on Akt phosphorylation
61
Effect of selenium on G6PDH gene expression
63
Effect of selenium on FAS gene expression
64
Effect of selenium on PEPCK gene expression
65
Effect of selenium on Foxa2 gene expression
68
Lactate dehydrogenase assay
70
Effect of glucosamine on O-GlcNAc modification
71
Identification of O-GlcNAc modified proteins by LC-MS
74
Functions of the identified O-GlcNAc modified proteins
86
v
Table of Contents- Continued
CHAPTER
4. DISCUSSION
89
APPENDIX
112
IACUC approval form for animal studies
BIBLIOGRAPHY
112
113
VI
LIST OF TABLES
1. Treatment conditions to establish an insulin resistance model
in primary rat hepatocytes
40
2. Treatment conditions to study the effect of selenium, an
insulin mimetic agent
41
3. Primers for RT-PCR
47
4. O-GlcNAc modified proteins found uniquely in the glucosamine
treatment as compared to control (no addition)
78
5. O-GlcNAc modified proteins found uniquely in glucosamine
plus insulin treatment compared to insulin
82
6. O-GlcNAc modified proteins found in the glucosamine plus selenium
treatment compared to selenium
83
7. O-GlcNAc modified proteins found in both glucosamine and glucosamine
plus insulin treatment
85
8. O-GlcNAc modified proteins found uniquely in glucosamine plus insulin
treatment but not present in glucosamine plus selenium treatment
85
vn
LIST OF FIGURES
1. Summary of insulin actions in different tissues
4
2. Estimates of the global prevalence of diabetes in the year 2000
and projections for 2030
17
3. Hexosamine Biosynthetic Pathway
27
4. Effect of glucosamine on insulin induced Akt phosphorylation
53
5. Effect of glucosamine on G6PDH gene expression
54
6. Effect of glucosamine on FAS gene expression
56
7. Effect of glucosamine on PEPCK gene expression
58
8. Effect of glucosamine on Foxa2 gene expression
60
9. Effect of glucosamine on selenium induced Akt Phosphorylation
62
10. Effect of selenium on G6PDH gene expression
64
11. Effect of selenium on FAS gene expression
65
12. Effect of selenium on PEPCK gene transcription
67
13. Effect of selenium on Foxa2 gene transcription
69
14. Effect of glucosamine on lactate dehydrogenase release into the media
71
15. Effect of glucosamine on O-GlcNAc modification
73
16. SDS-PAGE of O-GlcNAcylated proteins
75
viii
CHAPTER 1
INTRODUCTION
Glucose homeostasis
Glucose is an essential fuel for the body and is a regulated metabolite. The
concentration of glucose in the peripheral circulation is highly regulated such that over a
24-h period, the concentration is 5.5-6 mmol/L with a maximum of approximate 9
mmol/L postprandially (after meal) and a minimum of approximate 3 mmol/L in a state
of fast (Gerich 1993). In the fasting state, circulating glucose is derived from two
processes: 1) glycogenolysis which is the release of glucose from stored glycogen, and 2)
gluconeogenesis which is the formation of glucose from metabolites in the body such as
amino acids, glycerol, lactate, pyruvate and intermediate metabolites of the tricarboxylic
acid (TCA) cycle. In the fed state, circulating glucose is derived from dietary glucose and
from absorbed gluconeogenic substrates, i.e. amino acids, fructose and galactose, as well
as from endogenous gluconeogenic substrates.
Blood glucose level is controlled by a system, which involves several body organs
and tissues. The maintenance of glucose in a low range is important for two major
reasons. The first is the requirement of the brain for glucose as a fuel. In a fed state, the
brain uses glucose almost exclusively, and the rate of utilization is estimated to be ~ 117-
1
142g/day (Felig, Marliss et al. 1969; Gottstein 1979). The second reason for regulating
glucose concentration is that glucose is toxic.
The amount of glucose in the bloodstream is tightly regulated by actions of
insulin and glucagon during fed and fasting states (Flakoll PJ 2000). Insulin, a hormone
secreted by the P- cells of the islets of Langerhans in the pancreas, plays an important
role in maintaining glucose homeostasis and is secreted in response to elevated blood
glucose. Glucagon, a hormone secreted by the a- cells of the pancreas is another major
player in glucose homeostasis and is secreted in response to a fall in blood glucose.
During the fed state the increase in circulating insulin level promotes glucose
disposal mainly by skeletal muscle and adipose tissues, while inhibiting glycogenolysis
and gluconeogenesis in the liver (Flakoll PJ 2000). However, a drop in insulin levels
during the fasting state results in increased gluconeogenesis, glycogenolysis, and lipolysis
in order to maintain the plasma glucose concentration. Besides glucose homeostasis,
insulin also is involved in the anabolism of protein through a number of signaling
cascades that promote ion and amino acid transport and protein synthesis in skeletal
muscle, liver, and adipose tissue (Flakoll PJ 2000). In addition, insulin promotes de
novo synthesis of fatty acids in liver and adipose tissues (Flakoll PJ 2000). A summary
of the variety of insulin actions in different tissues is presented in Figure 1.
Hepatic glucose production is one of the factors responsible for glucose
homeostasis. It is a major determinant of fasting glucose level (Weyer, Bogardus et al.
1999). Insulin inhibits glucose production by both direct (activation of insulin signaling
in hepatocytes) and indirect actions (activation of insulin receptors in extra hepatic sites).
2
The direct action takes place in the liver itself, by responding to sinusoidal insulin levels.
In contrast, the indirect insulin action occurs in peripheral tissues such as the pancreas,
skeletal muscle, adipose tissue, and brain, which accordingly influence the direct actions
of insulin in the liver. Examples of indirect insulin action include the inhibition of
glucagon secretion from the pancreas that consequently decreases hepatic glucose
production (Ishihara, Maechler et al. 2003) caused by a decrease in the flow of free fatty
acids from adipose tissue (Sindelar, Chu et al. 1997) and gluconeogenesis precursors
from skeletal muscles (Sindelar, Balcom et al. 1996) in response to insulin induced
inhibition of lipolysis and proteolysis as well as a decrease in hepatic gluconeogenesis.
Furthermore, it has been shown that insulin signaling in a discreet area of the
hypothalamus plays a role in regulation of insulin action in liver (Obici, Feng et al. 2002),
showing the importance of the brain in regulating hepatic glucose output. In this study, a
transient defect in hypothalamic insulin signaling was generated by infusing an antisense
oligodeoxynucleotide designed to blunt the expression of the insulin receptor (IR) protein
in rat hypothalmic area. This selective decrease in hypothalamic insulin receptors resulted
in increased hepatic glucose production even in the presence of equal plasma insulin
concentrations.
In skeletal muscle, insulin increases glucose utilization and enhances the
perfusion of muscle by increasing vasodilation in skeletal muscle under euglycemia
(Baron 1994). After glucose is taken up into skeletal muscle, it is largely oxidized and the
rest is stored in the form of glycogen. In addition, the utilization rate of glucose in
skeletal muscle can be extremely high during hyperglycemia (Mandarino, Consoli et al.
3
1993). Glucose uptake in skeletal muscle determines the postprandial glucose level
(DeFronzo, Jacot et al. 1981). If the actions of insulin are impaired, the earliest
manifestation of this will be a reduction of glucose uptake in skeletal muscle (Zierath and
Wallberg-Henriksson 2002).
tijfc,*
Muscle
Suppression of gluconeogenesis
-Elevation of glycogen and fat synthesis -Elevation of glucose uptake
-Elevation of glycogen and protein synthesis
-Inhibition of lipolysis
-Elevation of glucose uptake
-Elevation of fat synthesis
Glucose homeostasis
Figure 1: Summary of insulin actions in different tissues.
In adipose tissue, the actions of insulin are responsible for promotion of glucose
uptake and suppression of lipolysis. In fact, adipose tissue serves as a depository of an
alternative source of energy during glucose deprivation. When glucose availability is
limited and the insulin level is low (Avruch (2001), breakdown of fat in the form of
triglyceride will result and serve as an alternative source of energy. In obesity,
suppression of lipolysis by insulin becomes less sensitive compared to normal
individuals. Oversupply of free fatty acids to the liver eventually will cause hepatic
insulin resistance, elevation of glucose production and hypertriglyceridemia (Dimitriadis
GD 2000).
In summary, insulin regulates glucose homeostasis by integration of its function
in liver, skeletal muscle, and adipose tissues. Insulin controls hepatic glucose production
by direct effects of the hormone. Insulin is also responsible for the glucose uptake in
skeletal muscle as well as adipose tissue. In addition, insulin exerts an indirect effect by
inhibiting lipolysis in adipose tissues to further control glucose metabolism.
Glucose metabolic pathways of the liver
Glucose first enters the liver via the glucose transporter, GLUT2, which allows
for passive movement of glucose across the cell membrane. GLUT2 transporters are
facilitative glucose transporters with a high km for glucose (17-20mM) (Gould and
Holman 1993). They have high transport capacity which increases as a direct function of
extracellular glucose concentration and are not saturated under most physiological
conditions. Thus, glucose transport is never rate limiting for the entry of glucose into the
liver (Rencurel and Girard 1998). GLUT2 transporters are constitutively present on the
cell membrane and are not regulated by insulin (Gould and Holmanl993). Once glucose
has entered the cell, it is phosphorylated by glucokinase (GK) to glucose-6- phosphate
(G6P). The expression level of glucokinase is regulated by insulin (Weinhouse 1976;
5
Iynedjian, Gjinovci et al. 1988).The resulting G6P can enter glycolysis or the pentose
phosphate pathway, resulting in various metabolic intermediates, reducing equivalents.
Glycogen can be synthesized via the glycogenesis pathway or oxidized to provide
energy or substrate for macromolecule synthesis via the glycolysis pathway. Glycogen
synthesis predominates in the fed state. The formation of glycogen in the liver can occur
via a direct pathway, in which G6P is produced through the phosphorylation of glucose
obtained from the circulation, or an indirect pathway, in which G6P is formed from
gluconeogenic precursors (Kuwajima, Golden et al. 1986). Once G6P is formed through
either pathway, it is converted to glucose 1-phosphate by phosphoglucomutase and to
UDP-glucose by UDP-glucose phosphorylase. UDP-glucose serves as the substrates for
glycogen synthase, which catalyzes the addition of glucose residues to the glycogen
particle via the formation of a-1, 4-glycosidic bonds. A branching enzyme then links
blocks of glucose residues through a-l,6-glycosidic linkages to form the highly branched
structure of mature glycogen (Kuwajima, Golden et al. 1986). The rise in insulin in the
fed state stimulates phosphorylation of glycogen synthase kinase 3 (GSK-3) via a branch
of the insulin signaling pathway that includes PI3-Kinase and Akt-1 (protein kinase B).
Phosphorylation of GSK-3 reduces its kinase activity, thereby resulting in reduced
phosphorylation of glycogen synthase and an increase in its enzymatic activity (Frame,
Cohen etal. 2001).
In glycolysis, one molecule of glucose is converted into two molecules of
pyruvate via a series of 10 enzymatic reactions, yielding energy in the form of ATP.
Seven of the ten steps in this pathway are catalyzed by enzymes with equilibrium
6
constants that allow the forward (glycolytic) or the reverse (gluconeogenic) direction of
the reaction to proceed depending on physiologic changes in the relative concentrations
of substrates and products. The three other enzymatic steps of the pathway are catalyzed
by glucokinase (GK) (glucose phosphorylation step), phosphofructoinase (PFK)
(conversion of fructose-6-phosphate to fructose-1, 6-bisphosphate), and pyruvate kinase
(conversion of phosphoenolpyruvate to pyruvate), and are considered to be irresversible
because of the large release of free energy associated with these reactions. Modulation of
the concentrations and activities of GK, PFK and pyruvate kinase are the primary
mechanisms for control of glycolytic rate.
Hepatic glucose output is mainly from breakdown of glycogen (glycogenolysis)
or de novo synthesis from lactate, pyruvate, or amino acids through the gluconeogenesis
pathway (Pilkis and Granner 1992; Moore, Hsieh et al. 1999; Nordlie, Foster et al. 1999).
Glycogenolysis is the process by which the liver produces glucose from its own glycogen
reserves. This process provides a glucose supply during short periods of fasting. In
prolonged fasting, hepatic glycogen stores are reduced and glucose is formed from noncarbohydrate precursors such as lactate, pyruvate, glycerol and a-ketoacids. This process
is called gluconeogenesis. Gluconeogenesis is basically a reversal of the glycolytic
pathway except for three irreversible steps in glycolysis that are bypassed by enzymes
unique to gluconeogenic pathway. The first of these bypasses involves a series of
cytosolic and mitochondrial reactions that catalyze the conversion of pyruvate to
phosphoenolpyruvate. In the mitochondria, excess acetyl coenzyme A (CoA) in the
presence of adequate energy activates pyruvate carboxylase and then converts pyruvate to
7
oxaloacetate. Oxaloacetate is reduced to malate for entry into the cytosol, malate is
reoxidized to oxaloacetate and oxaloacetate is then decarboxylated by cytosolic
phosphoenolpyruvate carboxylase (PEPCK) to form phosphoenolpyruvate (Rognstad
1979).
The gluconeogenic and glycolytic pathway are reciprocally regulated in order to
prevent pointless cycling of substrates that are common to both pathways. Enzymes in the
gluconeogenic and glycolytic pathways can be regulated in the short term by allosteric
and covalent modifications and in the long term by changes in gene expression. A unique
characteristic of the PEPCK enzyme is that it is exclusively regulated at the level of gene
transcription, rather than by allosteric or covalent modifications, which is unlike the
majority of the other enzymes involved in these pathways (Hanson and Reshef 1997).
Insulin decreases gluconeogenesis directly by inhibiting PEPCK gene
transcription. Insulin also decreases PEPCK gene expression indirectly by inhibiting
glucagon gene expression and by increasing cAMP degradation, thus accelerating the
degradation of PEPCK mRNA. When blood glucose levels decrease, insulin levels drop
and glucagon levels rise. This activates a series of reactions that are mediated through the
intracellular second messenger, cAMP, with a net effect being an increase in hepatic
gluconeogenesis and glycogenolysis.
As mentioned before, following conversion into glucose-6-phosphate, glucose can
be channeled to the pentose phosphate pathway rather than glycolysis. The pentose
phosphate pathway (PPP) is a pathway that generates NADPH and synthesizes pentose
(5-carbon sugars) (Au, Gover et al. 2000). There are two major phases in the PPP, the
8
oxidative phase and the non-oxidative phase. During the oxidative phase reducing
equivalents are generated in the form of nicotinamide adenine dinucleotide phosphate
(NADPH) for reductive biosynthesis reactions within the cell. The synthesis of 5-carbon
sugars, which are essential for DNA and RNA synthesis, occur during the non-oxidative
phase. Glucose-6-phosphate dehydrogenase (G6PDH) is the first and the key enzyme of
the PPP. It catalyzes the oxidation of G6P into 5-glucono-l,5-lactone-6-phosphate, while
reducing one NADP+ molecule to NADPH by transferring an electron from one molecule
to another. Being the key rate determining step of the PPP, G6PD is responsible for
maintaining adequate levels of NADPH within the cell (Au, Gover et al. 2000).
The liver has a role not only in controlling glucose homeostasis (described
above), but also in lipid homeostasis. In addition to being able to store ingested
carbohydrates as glycogen, the liver can use these carbohydrates to synthesize lipids de
novo through the lipogenesis pathway. Glucose flux through glycolysis is crucial for
providing carbons from glucose for lipid synthesis.
Lipid metabolism involves the synthesis of fatty acids, which is a condensation
reaction involving the 2 carbon units of acetyl-CoA as the subunit of a fatty acid chain.
Key enzymes in this process are acetyl-CoA carboxylase (ACC), which catalyzes the
formation of malonyl-CoA from acetyl-CoA, and fatty acid synthase (FAS), which
catalyzes successive rounds of the condensation reaction. Lipogenesis is regulated
according to the nutritional state.
9
Insulin signaling pathway
The biological effects of insulin are initiated by activation of its receptor
on the surface of the cell. Insulin receptors (IR) are present in all the vertebrate tissues,
although the concentration varies from as few as 40 to more than 200,000 receptors per
cell (White 1997). This receptor consists of 2a and 2p subunits, which are held together
by disulphide bonds and noncovalent interactions (Cheatham and Kahn 1995;
Ottensmeyer, Beniac et al. 2000). Binding of insulin to the a -subunit of the insulin
receptor induces a conformational change in the p-subunits. Subsequent
autophosphorylation of specific tyrosine residues leads to increased intrinsic tyrosine
kinase activity of the receptor. This results in subsequent recruitment of docking proteins
such as insulin receptor substrates IRSl and IRS2, which are tyrosine phosphorylated by
the insulin receptor and coupled to several important signaling pathways. Stimulation of
the insulin receptor results in the activation of three major pathways: 1) the mitogenactivated protein (MAP) kinase cascade, which mediates the mitogenic, growth and cell
differentiation effects; 2) the phosphatidylinositol 3-kinase (PBK)-Akt pathway, which
is mainly involved in the control of metabolic actions by insulin (glucose, lipid, protein
metabolism) and has been extensively studied (Saltiel and Kahn 2001; Lizcano and
Alessi 2002); and 3) the CAP/Cbl/TclO pathway, which controls the membrane
translocation of the glucose transporter (GLUT4).
The protein of interest is Akt, it will be discussed in detail in the PI 3-Kinase-Akt
pathway, and the other two pathways are beyond the scope of the study.
10
PI 3-kinase -Akt pathway
Activation of insulin receptors leads to the recruitment of insulin receptor
substrate 1(IRS1) and its tyrosine phosphorylation. Some of the tyrosine phosphorylation
sites are recognized by the src homology-2 domains (SH2) of p85, the regulatory subunit
of PI 3kinase. P85 contains a src homology -3 domain (SH3) and two SH2 domains;
between the SH2 domains lies a binding site for pi 10, the PI3-kinase catalytic subunit.
Recruitment of this p85/pl 10 activates the enzyme and brings it to the membrane, where
its substrate phosphatidylinositol 4 phosphate (PI3 4P) lies and subsequently generates PI
3, 4-bisphosphate (PI 3,4P2) and PI 3, 4, 5-triphosphate (PI 3, 4 P3) in the cell
membrane. The pleckstrin homology domain (PH domain) of Akt is highly specific to PI
3, 4-bisphosphate (PI 3, 4P2) (Klippel, Kavanaugh et al. 1997). The binding of PH
domain to PI 3, 4P2 recruits Akt to the plasma membrane where it encounters the
phospholipid -associated protein kinases, PDK1 (Cohen, Alessi et al. 1997). PDK1
contains a PH domain that binds to phospholipids products of the PI3 kinase, which
mediates its association with membranes where it catalyzes phosphorylation of Thr 309
of Akt (Meier and Hemmings 1999). The phosphorylation of Thr 309 partially activates
Akt, but full activation occurs after the phosphorylation of Ser 474 by a second enzyme
called PDK-2 (Meier and Hemmings 1999).
Akt, a serine threonine kinase is a 57 kDa protein, also known as protein kinase B
(PKB) or RAC -PK (protein kinase related to protein kinase A and C). It is known to
exist in three isoforms a, P and y (Akt-1, Akt-2 and Akt-3) (Vanhaesebroeck and Alessi
2000). Each isoform possesses an amino terminal pleckstrin homology (PH) domain , a
11
kinase domain and a carboxy-terminal regulatory domain (Coffer, Jin et al. 1998). The
pleckstrin homology (PH) domain consists of the first 100 amino acids at the amino
terminal and is responsible for binding phospholipids. The regulatory domain consists of
the last 70 amino acids at the carboxy terminal tail. The kinase domain consists of 258
amino acids. The phosphorylation of Akt occurs at two specific regulatory sites, one
localized in the kinase domain (Thr 309) and the other in the C-terminal regulatory
domain (Ser 474).
All three Akt isoforms are ubiquitously expressed in mammals, although the
level of expression varies among tissues (Jones, Jakubowicz et al. 1991; Altomare, Guo
et al. 1995; Brodbeck, Cron et al. 1999). Aktl is the predominant isoform in most tissues.
The highest expression of Akt2 is observed in the insulin responsive tissues like skeletal
muscle, heart, liver and kidney, suggesting that this isoform is important for insulin
signaling (Altomare, Guo et al. 1995; Kim, Peroni et al. 2000; Vuguin, Raab et al. 2004).
Higher levels of Akt 3 were detected in testis and brain and low levels in the adult
pancreas, heart and kidney (Brodbeck, Cron et al. 1999; Nakatani, Sakaue et al. 1999).
Akt mediates many metabolic actions. Akt is an important mediator of the
biological functions of insulin. The initial evidence for the involvement of Akt in insulin
signaling came from the work carried out by Cross et al (1995), which demonstrated that
Akt is responsible for the inactivation of glycogen synthase kinase 3 (GSK-3) and thus
may regulate glycogen synthesis in insulin-sensitive tissues. Since then, Akt has been
linked to a number of insulin and insulin-like growth factor-1 (IGF-l)-induced responses,
including glucose uptake (Kohn, Summers et al. 1996), protein synthesis (Hajduch,
12
Alessi et al. 1998; Ueki, Yamamoto-Honda et al. 1998), preadipose cell differentiation
(Magun, Burgering et al. 1996), the regulation of gene expression (Cichy, Uddin et al.
1998; Liao, Barthel et al. 1998; Wang and Sul 1998), and cell survival (Dudek, Datta et
al. 1997; Kulik, Klippel et al. 1997; Chen, Su et al. 1998).
In response to growth factors, Akt signaling regulates nutrient uptake and
metabolism through a variety of downstream targets. One of the most important
physiological roles of Akt is to stimulate glucose uptake in response to insulin. Akt2 has
been found to associate with glucose transporter 4 (Glut 4)- containing vesicles upon
insulin stimulation of adipocytes (Calera,_Martinez et al. 1998) and Akt activation leads
to Glut 4 translocation to the plasma membrane. The exact molecular mechanisms by
which Akt stimulates Glut 4 translocation to the plasma membrane are still being
investigated. It has been found that Rab-GAP AS 160 (also known as TBC1 domain
family member 4; TBC1D4) is a direct target of Akt involved in this process (Sano, Kane
et al. 2003; Eguez, Lee et al. 2005).
Akt activation can also alter glucose and lipid metabolism. Upon entry into the
cell, glucose is converted into glucose-6-phosphate by hexokinases. Akt has been shown
to stimulate the association of hexokinase isoforms with the mitochondria (Racek,
Holecek et al.), where they phosphorylate glucose, but the direct target of Akt responsible
is currently unknown. Glucose -6-phosphate can be stored by conversion to glycogen or
catabolized to produce energy through glycolysis, and Akt signaling can regulate both
these processes. In muscle and liver, Akt phosphorylates and inhibits GSK-3 which inturn
13
prevents GSK3 from phosphorylating and inhibiting its substrate glycogen synthase,
thereby stimulating glycogen synthesis. Akt activation also increases the rate of
glycolysis (Elstrom, Bauer et al. 2004). Akt's ability to enhance the rate of glycolysis is
due, at least in part, to its ability to promote the expression of glycolytic enzymes through
HIFa (Semenza, Roth et al. 1994; Majumder, Febbo et al. 2004; Lum, Bui et al. 2007)
Akt-mediated phosphorylation and inhibition of FOXOl also contribute to glucose
homeostasis, as FOXOl promotes hepatic glucose production and regulates the
differentiation of cells involved in metabolic control (Accili 2004). In hepatocytes, Akt
can also inhibit gluconeogenesis and fatty acid oxidation through direct phosphorylation
of serine 570 on PGC-la (Li et. al. 2007), which is a coactivator that can regulate genes
with FOXOl and other transcription factors. Akt signaling regulates lipid metabolism
through phosphorylation and inhibition of GSK3. GSK3 has been shown to promote
degradation of the sterol regulatory element-binding proteins (SREBPs), which are
transcription factors that turn on the expression of genes involved in cholesterol and fatty
acid biosynthesis (Sundqvist, Bengoechea-Alonso et al. 2005). Therefore, Akt-mediated
inhibition of GSK3 promotes SREBP stability and enhances lipid production.
Insulin resistance and type 2 diabetes
Insulin resistance is the failure to respond to normal concentrations of circulating
insulin. Clinically, insulin resistance, accordihg to the National Institute of Diabetes and
Digestive and Kidney Disease (NIDDK 2005), is indicated by a plasma glucose
concentration between 100 and 125 mg/dL (milligrams of glucose per deciliter of blood)
following a 12-hour fast. In the 1930s, Himsworth first differentiated patients with
14
diabetes mellitus into "insulin sensitive" and "insulin insensitive" based on the ability of
subcutaneous insulin administration to dispose of an oral glucose load. The former is now
classified a type 1 diabetes mellitus and the latter "insulin insensitive" classified as type 2
diabetes mellitus. With the development of the radioimmunoassay technique in 1960,
Yalow and Berson demonstrated that patients with adult-onset diabetes (Type 2 diabetes)
had higher circulating insulin levels than non-diabetic subjects. It was thus concluded that
"the tissues of the maturity onset diabetic do not respond to insulin as well as the tissues
of the nondiabetic subjects respond to insulin"
Impaired insulin action occurs when target tissues are unable to respond to the
normal circulating concentration of insulin. In response, P- cells in the pancreas need to
secrete increased amount of insulin to maintain normal blood glucose level. However,
overtime, functional defects in insulin secretion prevent the P- cells from maintaining
high rates of insulin secretion, and hyperglycemia and eventually type 2 diabetes develop
(DeFronzo, Bonadonna et al. 1992). Insulin resistance affects glucose disposal in muscle
and fat and has an effect on insulin suppression of hepatic glucose output (DeFronzo
1988; DeFronzo, Bonadonna et al. 1992; Reaven 1995).
Prevalence of diabetes
It is estimated that diabetes affects more than 220 million people worldwide
according to world health organization (WHO). Recent WHO calculations indicate that
worldwide almost 3 million deaths per year are attributed to diabetes. The number of
people with diabetes is increasing due to population growth, aging, urbanization, and
increasing prevalence of obesity and physical inactivity. Estimates of the global
15
prevalence of diabetes in the year 2000 (as used in the World Health Organization
[WHO] Global Burden of Disease Study) and projections for 2030 are shown in Figure 2.
Type 1 diabetes is referred to as insulin- dependent diabetes mellitus (IDDM) or
juvenile- onset diabetes. It develops when the body's immune system destroys pancreatic
beta cells, which are responsible for making the hormone insulin that regulates blood
glucose. Children and young adults are usually affected by this form of diabetes. In
adults, type 1 diabetes accounts for 5% to 10% of all diagnosed cases of diabetes. Risk
factors for type 1 diabetes may be autoimmune, genetic, or environmental.
Type 2 diabetes is referred to as non-insulin-dependent diabetes mellitus
(NIDDM) or adult onset diabetes. In adults, type 2 diabetes accounts for about 90% to
95% of all diagnosed cases of diabetes. As discussed before, it usually begins as insulin
resistance, a disorder in which the cells do not use insulin properly. As the need for
insulin rises, the pancreas gradually loses its ability to produce it. Type 2 diabetes is
associated with older age, obesity, a family history of diabetes, history of gestational
diabetes, impaired glucose metabolism, physical inactivity, and race/ethnicity. According
to the Center for Disease Control and Prevention (CDC), African Americans,
Hispanic/Latino Americans, American Indians, and some Asian Americans and Native
Hawaiians or other Pacific Islanders are at particularly high risk for type 2 diabetes and
its complications. Type 2 diabetes in children and adolescents, although still rare, is being
diagnosed more frequently among American Indians, African Americans,
Hispanic/Latino Americans, and Asians/Pacific Islanders.
16
Prevalence of diabetes
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ure 2: Estimates of the global prevalence of diabetes in the year 2000 and projections
for 2030. (http://www0whoint/diabetes/actionnow/en/mapdiabpreVopdf)
17
Molecular mechanism of insulin resistance
The molecular mechanisms that cause insulin resistance are not yet entirely clear
and several factors have been proposed to contribute to the pathogenesis of this metabolic
effect. Multiple mechanisms of how insulin resistance occurs have been documented. A
single factor can cause insulin resistance by several mechanisms. On the other hand,
multiple factors may share the same molecular target. Understanding the molecular
mechanisms of insulin resistance precisely will explain and help predict the pathological
consequences. Some of the molecular mechanisms of insulin resistance are:
Decreased expression of insulin signaling components
Any abnormality in the insulin receptor or its signaling cascade may lead to
insulin resistance. In normal physiology, insulin binds to its tyrosine kinase receptor,
initiates a cascade of intracellular signal pathways, and eventually facilitates glucose
transport, glycogen and lipid synthesis, and appropriate gene expression (Le Roith and
Zick 2001). Mutations in the insulin receptor (IR) gene could result in impaired receptor
biosynthesis, decreased affinity of insulin binding (Kolterman, Gray et al. 1981; Sinha,
Pories et al. 1987) or impaired tyrosine kinase activity (Caro, Sinha et al. 1987;
Maegawa, Shigeta et al. 1991). Decreased expressions of IR and IRS have been reported
in a number of studies of insulin resistant animal models (Biddinger, Miyazaki et al.
2006). For example, the expression of IR and IRS-1 was decreased significantly by 32
and 60%, respectively, in the genetically obese Spontaneously Hypertensive Koletsky rat
(SHROB) (Friedman, Ishizuka et al. 1997). This effect seems to be reversible, because an
antihyperglycemic agent restored the expression of IR and IRS-1 in this model (Haxhiu,
18
Dreshaj et al. 1999). In addition, the ob/ob mice (a model of insulin resistance of obesity
and non-insulin-dependent diabetes mellitus) exhibit a significant decrease in IR, IRS-1,
and IRS-2 expression in skeletal muscle and in IRS-1 and IRS-2 expression in liver
(Kerouz, Horsch et al. 1997).This diminished receptor function will impact downstream
events. The majority of insulin resistance is still believed to be associated with postreceptor mechanism. Several lines of evidences from human and animal studies suggest
defects in insulin signaling pathways in target tissues. Reduced expression and/or
diminished phosphorylation of early insulin signaling molecules have been observed in
insulin target tissues of obese and Type 2 diabetic patients. For example, Goodyear et al
reported that impaired insulin stimulated glucose uptake in human skeletal muscle of
obese individuals was associated with decreased IRS-1 phosphorylation and decreased
IRS-1 associated PI3-kinase activity. (Goodyear, Giorgino et al. 1995; Bjornholm,
Kawano et al. 1997; Meshkani, Taghikhani et al. 2006).
Increase in the ratio of the regulatory subunit/catalytic subunit of PI3-kinase
As mentioned before, PI3-kinase has two subunits: p85a (regulatory subunit) and
pi 10 (catalytic subunit). Elevation of the p85a regulatory subunit of PI3-kinase has been
reported in some insulin resistant states. For example, a significant increase in expression
of the p85a monomer leads to insulin resistance in the mouse model of insulin resistance
during pregnancy (Barbour, Shao et al. 2004). p85a competes with the p85/pll0
heterodimer for binding to the IRS-1 protein. This competition impairs binding between
IRS-1 and PI3-kinase, subsequently decreasing activation of PI3-kinase (Barbour, Shao et
al. 2004). Moreover, increased p85/pl 10 expression correlates with decreased insulin
19
sensitivity in skeletal muscle of insulin resistant obese and type 2 diabetic subjects (Dey,
Mukherjee et al. 2005). In another study carried out in obese Zucker rats (OZR), the
regulatory subunit of PI3-kinase (p85a) was increased significantly (Anai et al. 1998) and
some of the alternatively spliced isoforms of the regulatory subunits of PI3-kinase also
were elevated in liver of ob/ob mice compared to lean controls (Kerouz, Horsch et al.
1997). Targeted disruption of the gene encoding the murine p85 a subunit (Pik3rl-/-)
results in hypoglycemia and enhanced insulin sensitivity, which is due to increased
glucose transport in skeletal muscle and adipocytes. Insulin stimulated PI3-kinase activity
associated with IRSs was mediated through full-length p85 alpha in wild-type mice, but
through the p50 alpha alternative splice form of the same gene in Pik3rl-/-mice. This
isoform was associated with an increase in insulin-induced generation of
phosphatidylinositol (3, 4, 5) triphosphate (Ptdlns (3, 4, 5) P3) in Pik3rl-/- adipocytes
and facilitates Glut4 translocation to the plasma membrane (Terauchi, Tsuji et al. 1999).
Serine phosphorylation of IRS
Accumulating data have highlighted the critical role of serine phosphorylation of
IRS in insulin resistance. Yet, a mechanism of how serine phosphorylation could
decrease IRS-1 function has been difficult to formulate, in part because of a large number
of potential serine phosphorylation sites on IRS-1 (Aguirre, Werner et al. 2002). One
model is that serine phosphorylation induces a conformational change in the functional
domain of IRS-1. For instance, serine 307 of IRS-1 (serine 312 in human IRS-1) is
located at the end of the phosphotyrosine-binding (PTB) domain, which is involved in the
binding of IRS-1 to the IR. Serine 307 is phosphorylated by several different kinases,
20
depending upon the cellular conditions; this modification reduces the interaction between
IRS-1 and IR and could be one important mechanism for regulating the early stages of
insulin signaling. In addition, several studies have shown that phosphorylation of serine
residues could induce IRS-1 breakdown through the proteasome degradation pathway
(Pederson, Kramer et al. 2001). Many serine/threonine kinases have been proposed to
phosphorylate various serine residues of IRS-1, such as extracellular signal-regulated
kinase (ERK) (Bouzakri, Roques et al. 2003), c-Jun NH2-terminal kinase (JNK)
(Aguirre, Uchida et al. 2000), inhibitor KB kinase (IKK) (Gao, Hwang et al. 2002) and
the mammalian target of rapamycin (mTOR) (Carlson, White et al. 2004). For example,
full body reduction of JNKl resulted in a decrease in weight gain on a high fat diet and in
a reduction of insulin resistance with a decrease in IRSl serine phosphorylation
(Hirosumi, Tuncman et al. 2002). Hepatic reduction of JNKl activity improves insulin
sensitivity and hepatic steatosis. On the other hand muscle JNKl does not contribute to
insulin resistance (Singh, Wang et al. 2009). High levels of JNKl activity in adipose
tissue may promote insulin resistance through the deregulation of adipocytokines (cell
signaling proteins released by adipocytes) (Sabio, Das et al. 2008). Similarly, ERK1
deficient mice are protected against diet induced obesity and insulin resistance because of
decreased adipogenesis (Bost, Aouadi et al. 2005).
Suppresors of cytokine signaling (SOCS)
Another mechanism that may be involved in defective insulin signaling pathway
could be induction of inhibitory factors such as suppressors of cytokine signaling (SOCS1, 3). SOCS proteins block insulin signaling via competition with IRS-1 for association
21
with the insulin receptor and by augmentation of proteosomal degradation of IRS-1 (Kile,
Schulman et al. 2002). The expression of SOCS3 is enhanced by various inflammatory
cytokines and hormones, including IL-6 and leptin (Emanuelli, Peraldi et al. 2001;
Rieusset, Bouzakri et al. 2004; Shi, Tzameli et al. 2004). The levels of SOCS 1 and
SOCS6 have also been shown to be enhanced during cytokine-mediated inhibition of
insulin signaling (Mooney, Senn et al. 2001; Rui, Yuan et al. 2002). Overexpression of
SOCS1 or SOCS3 inhibited insulin-induced glycogen synthesis in myotubes and glucose
uptake in adipocytes (Ueki, Kondo et al. 2004), while hepatocyte-specific SOCS3
deletion improved insulin sensitivity in the liver (Torisu, Sato et al. 2007). SOCS proteins
inhibit insulin-induced signaling either by binding to the IR or reducing the receptor's
ability to phosphorylate IRS on tyrosine residues for activation, or by targeting IRS
proteins for proteosomal degradation (Rui, Yuan et al. 2002).
Phosphatase
The insulin signaling pathway is initiated by the tyrosyl phosphorylation of the
insulin receptor (IR), which is reversible. The IR dephosphorylation takes place rapidly in
intact cells even with the continued presence of insulin (Kole, Kole et al. 1998). Because
a critical regulatory step in insulin signal transduction is the dephosphorylation of
signaling molecules by protein tyrosine phosphatase (PTPs), it is plausible that enhanced
activity of one or more protein tyrosine phosphatase could lead to insulin resistance.
Support for the role of PTPs in the regulation of insulin action comes from transgenic and
gene knockout studies. The Khan group and Elchebly group generated PTP-1 null mice
and found that these mice have increased insulin sensitivity, manifested by enhanced
22
insulin stimulated phosphorylation of IR and IRS-1 in muscle and liver (Elchebly,
Payette et al. 1999; Klaman, Boss et al. 2000). In addition, insulin-stimulated whole body
glucose disposal is enhanced in protein tyrosine phosphatase-IB (PTPIB) deficient mice
(Klaman, Boss et al. 2000). Surprisingly, this effect is tissue-specific: insulin-stimulated
glucose uptake is elevated in skeletal muscle but not in adipose tissue. These data suggest
that overexpression of PTPIB in insulin-target tissues in vivo may contribute to insulin
resistance. Consistent with this hypothesis, Zabolotny et al. demonstrated that selective
overexpression of PTPIB in skeletal muscle impairs insulin-stimulated PI3K activity and
causes mild insulin resistance in vivo (Zabolotny, Haj et al. 2004). Recent studies have
indicated that liver-specific deletion of PTPIB improves insulin resistance and attenuates
diet-induced endoplasmic reticulum stress (Delibegovic, Zimmer et al. 2009). Similarly,
Haj et al. showed that liver-specific re-expression of PTPIB in PTPIB deficient mice
leads to marked attenuation of their enhanced insulin sensitivity (Arias-Salgado, Haj et al.
2005).
O-GlcNAcylation of serine/threonine residues
Traditionally protein glycosylation refers to the covalent attachment of complex
oligosaccharides to proteins that are in intralumenal compartments or cellular
membranes, or destined for secretion. A novel form of glycosylation surfaced when
proteins in the nucleus and cytosol were found to be modified with a single P-Nacetylglucosamine monosaccharide moiety through an O-P glycosidic attachment (OGlcNAc) (Torres and Hart 1984) to serine and threonine side chains of the polypeptide
backbone (Holt and Hart 1986; Holt, Snow et al. 1987). The addition of O-GlcNAc to
23
proteins is catalyzed by uridine diphospho-N-acetylglucosamine:polypeptide p-Nacetylglucosaminyltransferase O-GlcNAc transferase (OGT), which uses uridine
diphosphate N-acetylglucosamine (UDP-GlcNAc) as the direct sugar donor
(Haltiwanger, Blomberg et al. 1992). The removal of O-GlcNAc from proteins is
catalyzed by P-N-aetylglucosaminidase (O-GlcNAcase) (Dong and Hart 1994; Gao,
Wells et al. 2001). Both are found in the cytosol and nucleus. The O-GlcNAc is attached
and removed multiple times in the life of a polypeptide, at different rates and at different
sites in response to cellular signals. While there has been no consensus motif identified
for O-GlcNAc attachment, many sites appear to be identical to those used by
serine/threonine kinases. A reciprocal relationship as well as adjacent occupancy between
phosphorylation and O-GlcNAcylation has been observed for some proteins, and both
mechanisms are touted as the way O-GlcNAcylation can interfere with phosphorylation.
Interplay between O-GlcNAcylation and phosphorylation has been postulated to occur
through several different mechanisms: [1] direct competition for site occupancy at a
single hydroxyl group, [2] competition via steric hindrance by reciprocal modification at
proximal sites in a polypeptide, and [3] regulation of O-GlcNAc enzymes by
phosphorylation and conversely regulation of kinases or phosphatases by OGlcNAcylation. Studies of several proteins are consistent with all of these mechanisms.
For example, Thr-58 of c-Myc (Chou, Hart et al. 1995; Kamemura, Hayes et al. 2002)
and Ser-16 of estrogen receptor-P (Cheng and Hart 2001) reciprocally carry either the OGlcNAc or the O-phosphate modification at the same hydroxyl moiety. Furthermore, as
in the case of the tumor suppressor protein p53, an O-GlcNAc modification adjacent to a
24
known regulatory phosphorylation site can affect phosphorylation of that residue and
subsequent protein function (Shaw, Freeman et al. 1996).
A significant amount of research has been directed towards understanding the role
that O-GlcNAc plays in the development of type II diabetes (Buse 2006). However, the
exact role that this modification may play in this multifaceted, complex physiological
disease is still unclear. O-GlcNAc has been shown to be present on or added to
transcription factors involved in insulin regulation (Gao, Miyazaki et al. 2003; Andrali,
Qian et al. 2007; Vanderford, Andrali et al. 2007). For example, in Min6 cells, elevated
glucose concentration leads to an increase in O-GlcNAc modification of the transcription
factor, pancreatic/duodenal homeobox-1 protein (PDX-1), and this hyperglycosylation
correlates with an increase in DNA binding activity of PDX-1 (Gao, Miyazaki et al.
2003). Other studies, such as Andrali et al, presented data that showed increased OGlcNAcylation leading to relocalization of the P-cell specific transcription factor
NeuroDl (Andrali, Qian et al. 2007). Additionally, proteins that are normally activated
downstream of the insulin signaling pathway have been shown to be modified by OGlcNAc, such as IRS-1 and Akt (Park, Ryu et al. 2005; Ball, Berkaw et al. 2006). So, OGlcNAc modifications may directly affect the ability of a cell to respond to insulin as an
extracellular stimulus. Lastly, proteins known to be involved in membrane fusion events
that allow for the relocalization of the Glucose Transporter Type 4 (GLUT4) following
insulin stimulation have been modified by O-GlcNAc (Chen, Liu et al. 2003).
25
Hexosamine biosynthetic pathway (HBP)
Glucose upon entering the cell fluxes through various metabolic pathways. A
small portion of incoming glucose can enter a nutrient-sensing pathway called
hexosamine biosynthetic pathway (HBP). Amino sugars are physiologically synthesized
through the hexosamine biosynthetic pathway. This pathway results in the synthesis of
UDP-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor for all amino sugars used for
synthesis of proteoglycans, glycoproteins and glycolipids. This pathway involves the
transamidation and N-acetylation of glucose followed by the activation of the Nacetylated amino sugar. The activated acetylated sugar is then used in the synthesis of
glycoproteins, proteoglycans and glycolipids. Glucose is converted into glucosesphosphate by hexokinase. Phosphoglucose isomerase then catalyzes the reversible
isomerization of glucose-6-phosphate to fructose-6-phosphate (Fru-6-P).
The HBP (Figure 3) begins with conversion of fructose-6-phosphate to form
glucosamine-6-phosphate (GlucN-6-P). Glutamine serves as donor of the aminogroup.
The reaction is catalyzed by the rate-limiting enzyme glutamine: fructose-6-phosphateamidotransferase (GFAT). Acetyltransferase catalyzes the N-acetylation of glucosamine6-phosphate into N-acetyl-D-glucosamine-6-phosphate. N-acetyl-D-glucosamine-6phosphate isomerizes into N-acetyl-D-glucosamine 1-phosphate, which upon addition of
Uridine triphosphate (UTP) is converted into UDP-N-acetyl-D-glucosamine (UDPGlcNAc). UDP-GlcNAc, the end point of the HBP is the donor for a common form of
intracellular o-glycosylation of nuclear and cytoplasmic proteins, O-linked P- Nacetylglucosamine (O-GlcNAc).
26
Glucose
Glucosamine
*
Glc-6-P
(Glucose-6-phosphate)
GFAT
^ Fruc-6-P
1
^GIcN-i
6-P
(Fructose-6-phosphate)
^UDP-GlcNAc
(UDP-N-AcetylGlucosamine)
i
i
i
Glycolysis
O-GlcNAc-modified
cytosolic and nuclear proteins
TCA Cycle
GFAT-Glutamine Fructose-6-phosphate Amidotransferase
Figure 3: Hexosamine Biosynthetic Pathway
Role of the hexosamine biosynthetic pathway in insulin resistance
The adverse effect of glucose that is mediated through the HBP was first
demonstrated by Marshall et al (1991) who showed that the induction of insulin
resistance requires three components: insulin, glucose, and glutamine. When any one of
these components is omitted, little or no densensitization is observed. This was explained
by the fact that formation of hexosamine products requires a supply of both glucose (in
the form of F-6-P) and glutamine (as a cofactor for GFAT in the transfer of an amide
group to fructose-6-P). The primary role of insulin in this proposal is to facilitate the
uptake of glucose by increasing the number of cell surface glucose transporters. They
27
used two approaches to test their hypothesis that enhanced flux through the HBP
culminates in insulin resistance. They first demonstrated that glucose-induced
desensitization could be prevented by glutamine analogs that irreversibly inactivate
glutamine-requiring enzymes such as GFAT. Second, they showed that glucosamine, in
addition to glucose, could induce cellular insulin resistance. Glucosamine was shown to
enter adipocytes through the glucose transport system and directly enter the hexosamine
pathway distal to GFAT (through the formation of GlcN-6-P).
In 1993, Robinson et al. reported that glucosamine induces insulin resistance in
isolated rat skeletal muscles, revealing that this glucose sensing pathway is operative in
other tissues (Robinson, Sens et al. 1993). Further evidence of the involvement of HBP in
insulin resistance came from work carried out by Rossetti et al (1995), Baron et al
(1995.1998), and Giarccari et al (1995), who found that infusion of glucosamine for 4-6
hours under euglycemic conditions resulted in marked insulin resistance. Additional
studies by Baron et al showed that glucosamine induces insulin resistance in vivo by
causing a defect in insulin-responsive glucose transporter (GLUT 4) translocation from
the cell interior to the cell surface.
Further work carried out by McClain et al (1996) confirmed the involvement of
HBP in insulin resistance. He studied transgenic mice overexpressing GFAT in the
adipocytes. In response to excess nutrient flux as indicated by an increase in hexosamine
flux, the adipocytes became more insulin resistant while increasing GLUT4 expression,
glucose uptake, and fat synthesis, resulting in increased adiposity, and adipocytes size.
Subsequent transgenic studies carried out by McClain et al extended the glucose sensing
28
and regulatory role of the HBP to the liver and P-cells of the pancreas. In liver
overexpression of the rate limiting enzymes GFAT led to glucose intolerance with a
significant decrease in glucose disposal rates (Veerababu, Tang et al. 2000). Similarly,
overexpression of GFAT in pancreatic P-cell resulted in hyperinsulinemia and eventual
insulin resistance as the transgenic mice aged (Veerababu, Tang et al. 2000).
Later work carried out in both in vitro and in vivo supports the model where
increased UDP-GlcNAc, a results of increased flux through the HBP, raises O-GlcNAc
levels, and this result in insulin resistance (Buse, Robinson et al. 2002; McClain 2002;
Vosseller, Wells et al. 2002). Blocking the removal of O-GlcNAc by inhibiting OGlcNAcase, using 0-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-Nphenylcarbamate (PUGNAc), results in decreased glucose uptake in response to insulin in
3T3-L1 adipocytes (Vosseller, Wells et al. 2002). Treatment of rat primary adipocytes
with PUGNAc also reduced insulin-stimulated glucose uptake and glucose transporter 4
(GLUT4) translocation to the plasma membrane (Park, Ryu et al. 2005). In this model
system, reduced phosphorylation and increased O-GlcNAc modification of Akt were
thought to be the possible mechanisms by which O-GlcNAc altered insulin signaling in
the adipocytes (Park, Ryu et al. 2005).
In skeletal muscle, infusion of glucosamine in Wistar rats induces insulin
resistance of skeletal muscle through decreased phosphorylation of insulin receptor
substrate-1 (IRS-1) and its association with phosphatidylinositol 3-kinase (PI3-K) (Patti,
Virkamaki et al. 1999) and in turn results in impaired translocation of GLUT4 to the
plasma membrane (Baron, Zhu et al. 1995). Similarly, exposure of glucosamine also
29
inhibits insulin-stimulated glucose transport and glycogen synthesis in isolated rat
skeletal muscles (Robinson, Sens et al. 1993). Similar to what was observed in
adipocytes, treating isolated skeletal muscle with PUGNAc-impaired insulin stimulated
glucose transport (Arias, Kim et al. 2004). However, contrary to the adipocytes studies,
Akt phosphorylation in the skeletal muscle was not significantly altered (Arias, Kim et al.
2004). Other studies have also shown that glucosamine infusion in rats can also impair
insulin-stimulated glucose disposal through decreased IRS-1 association with PI3K
without affecting Akt activity (Kim, Nikoulina et al. 1999).
The effect of HBP on insulin signaling in liver was first demonstrated with the
overexpression of the rate-limiting enzyme GFAT under the control of the PEPCK
promoter. Those transgenic mice gained weight with age and developed glucose
intolerance with a significant decrease in glucose disposal rates (Veerababu, Tang et al.
2000). It was recently demonstrated that adeno viral-mediated overexpression of OGT in
mouse liver attenuated the ability of insulin to suppress hepatic glucose production. The
mechanism of this phenomenon involves decreased Akt phosphorylation and Akt
activity, which in increased expression of gluconeogenic enzymes phosphoenolpyruvate
carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). It was proposed that this
regulation was facilitated by OGT itself binding to phosphoinositide (3, 4, 5) phosphate
(PIP3), resulting in OGT translocation to the plasma membrane where it colocalized with
Akt (Yang, Ongusaha et al. 2008).
Taken together, these data suggest crucial and diverse roles for HBP in
mediating the adverse effects of hyperglycemia.
30
Selenium- an insulin mimetic agent
Insulin regulates many biological processes and pathways important to potently
understand but also find viable ways to either mimic effects or overcome with lack of or
no response. Thus, various agents have been studied that have been shown to stimulate
metabolic functions of insulin. Several investigators are studying trace elements that have
insulin- like properties. Selenium is one of the trace elements that has been shown to have
antidiabetic effects. Selenium is an integral component of several enzymes, such as
glutathione peroxidase, iodothyronine deiodinase, thioredoxin reductase, selenoprotein P
and W. In addition to its role as an antidiabetic agent, it is also involved in prevention of
cancer (Combs, Clark et al. 2001) and cardiovascular disease (Rayman 2002).
Osamu Ezaki first showed the effect of selenate as a potent insulin-like agent. In
this study, incubation of rat adipocytes with selenium stimulated glucose transport
activity in a dose dependent manner, and this increase in glucose transport activity was
due to the translocation of two types of glucose transporters (GLUT-1 and GLUT-2) to
the membrane surface (Ezaki 1990). Maximal transport activity (lOOuM for 30 min,
assessed from the rate of 3-0-14Cmethyl-D-glucose uptake) was equivalent to InM insulin
for 30 min incubation. Stimulation of glucose transport was observed 2 min after addition
of selenite and reached a steady state within 10 min (Ezaki 1990). Further studies carried
out on rat soleus muscle by Furnsinn C et al. also showed marked a increase in glucose
uptake when muscle was exposed to selenate (Furnsinn, Englisch et al. 1996). Expanding
on the work carried out by Ezaki, McNeill et al. showed that selenium acted as an insulinmimetic in vivo. Treating streptozotocin-induced diabetic animals with selenium
31
improved both food and water intake level to both control levels. Weight gain of the
animals improved, and within 2 weeks of treatment plasma glucose levels had improved
significantly and remained low throughout a seven week experiment (McNeill, Delgatty
et al. 1991). Similar studies carried out by Berg et al. in rats and Ghosh et al. in mice also
showed a blood glucose lowering effect of selenium (Ghosh, Mukherjee et al. 1994;
Berg, Wu et al. 1995) in diabetic animals.
In addition to influencing glucose uptake, selenium was shown to regulate the
activity and expression of various enzymes involved in the processes of glycolysis,
gluconeogenesis, fatty acid synthesis and the pentose phosphate pathway. Oral
administration of selenate to diabetic animals partly reversed abnormal liver expression
of both glycogenic and gluconeogenic enzymes (Becker, Reul et al. 1996). Regulation by
insulin-mimetics of the expression of lipogenic enzymes was found to be similar to that
of insulin. Treatment of the diabetic animals or rat hepatocytes in culture with selenate
restored the expression of both FAS and G6PDH, demonstrating that selenate was
capable of stimulating lipogenesis in the liver (Ghosh, Mukherjee et al. 1994; Berg, Wu
etal. 1995).
One of the main causes of mortality in diabetes is myocardial disease. A
depressed level of overall lipid metabolism in diabetics is believed to be a contributing
factor to a higher risk of heart disease and stroke. Selenate was shown to lower plasma
lipid levels. Studies carried out by Battell, M.L et al. in streptozotocin induced diabetic
rats treated with selenate, found that the plasma lipid levels, triglycerides, cholesterol and
free fatty acid levels were improved compared to untreated diabetic rats (Battell, Delgatty
32
et al. 1998). Further studies, showed that sodium selenate has cardioprotective effects. In
those studies, the effects of intra peritoneal administration of sodium selenite for 4 week
on the heart structure in streptozotocin-induced diabetic rats were evaluated. Examination
by electron and light microscopy revealed that selenite administration corrected the
changes in the diabetic heart. The structure of selenium-treated diabetic hearts appeared
very similar to the structure of control rat hearts (Ayaz, Can et al. 2002).
The enhanced phosphorylation of diverse cellular proteins is believed to be
responsible for an elevated translocation of glucose transporters, an increased glucose
uptake and modified expression of metabolic enzymes. Like insulin, selenium treatment
triggers a variety of phosphorylation events targeting proteins of the insulin signaling
pathway. For example, treatment of NIH3T3 HIR 3.5 cells with selenate stimulated
phosphorylation of the insulin receptor (Pillay and Makgoba 1992). In a later study,
incubation of either primary rat hepatocytes or 3T3-LI adipocytes in culture with selenate
caused not only a concentration and time dependent increase in phosphorylation of the P
subunit of the insulin receptor, but also increased phosphorylation of IRS-1 as determined
through immunoprecipitation studies (Stapleton, Garlock et al. 1997). This increase in
phosphorylation of IRS-1 was observed within lhr of selenium treatment, whereas only
few minutes of incubation is required for increased phosphorylation by insulin,
suggesting that more time is required for selenate to enter the cell and mediate insulinregulated processes through a post-insulin receptor kinase mechanism. These results are
supported by the fact that although selenate is capable of increasing phosphorylation of
33
the insulin receptor, this does not appear to directly increase insulin receptor tyrosine
kinase activity (Ezaki 1990) (McNeill, Delgatty et al. 1991).
Further downstream proteins of signal transduction pathway, triggered by
selenium have been identified over the years. For example, our laboratory has shown that
selenate increases PI 3-kinase activity in rat hepatocytes in culture. One protein that has
been identified to lie downstream of PI3-kinase is p70 S6 kinase. S6 kinase can play a
critical role in initiation of protein synthesis. Selenate has been shown to stimulate not
only S6 kinase phosphorylation but also kinase activation. In addition to the IRS family
and the PI 3-kinase pathway, selenate have been shown to activate mitogen activated
protein kinase (MAPK) (Stapleton, Garlock et al. 1997; Hei, Farahbakhshian et al. 1998).
In both hepatocytes in culture and 3T3-L1 adipocytes, selenate caused a marked increase
in not only the phosphorylation of MAPK in a both time and concentration dependent
manner but also its activity as measured by an 'in-gel' kinase assay (Stapleton, Garlock et
al. 1997).
Significance of study
The molecular mechanisms that cause insulin resistance are not yet entirely clear.
There is evidence that suggests that high glucose induced insulin resistance may be
mediated by products of the HBP. But, most of the research is focused on two insulin
sensitive tissues i.e. adipocytes and skeletal muscle and less attention has focused on the
liver. The liver plays a key role in coordinating the whole body metabolism including
carbohydrate, lipid and protein metabolism. It is important to understand the mechanism
34
of insulin resistance as mediated by products of the HBP in all the insulin sensitive
tissues.
Over the years, there is growing evidence that have shown that selenium, an
insulin mimetic agent, can mediate a number of insulin-like actions both in vivo and in
vitro models of type 1 diabetes. However, fewer studies have established its effectiveness
on models of type 2 diabetes or insulin resistance. Understanding the mechanism of
action of selenium, as insulin mimetic in insulin resistant models will aid in using it as a
therapeutic agent in management of type 2 diabetes.
Objectives of study
The goal of this project was to investigate the effect of glucosamine on a key
insulin signaling protein and key metabolic enzymes of the pentose phosphate pathway,
fatty acid biosynthesis pathway and gluconeogenesis. Here we hypothesize that increased
flux through the hexosamine biosynthetic pathway can generate insulin resistance
through increased glycosylation and that selenium, an insulin mimetic, can overcome the
insulin resistance state.
The goals of this study were to:
1) To assess the effect of glucosamine on an insulin induced key signaling
protein.
2) To assess the effect of glucosamine on the insulin induced gene expression of
key metabolic enzymes.
35
3) To test the effect of selenium on the activation of key insulin signaling
pathway in glucosamine induced insulin resistance.
4) To test the effect of selenium induced gene expression of key metabolic
enzymes in glucosamine induced insulin resistance.
5) To examine whether the treatment of glucosamine increases O-glycosylation
of proteins.
6) To identify O-GlcNAc modified proteins.
36
CHAPTER 2
MATERIALS AND METHODS
Primary rat hepatocytes isolation and maintenance
Male Sprague-Dawley rats (Charles River, Kalamazoo) weighing 180-200g were
housed in a facility approved by the WMU Institutional Animal Care and Use Committee
(IACUC). Rats were maintained on Lab diet 5001 standard rodent chow and drinking
water ad libitum. Animals were fasted for approximately 48 hrs prior to hepatocyte
isolation and anesthetized with an intra-peritoneal injection of pentobarbital (4550mg/Kg). Primary hepatocytes were isolated using collagenase-hyalurodinase perfusion
and digestion method (Stapleton, Stevens et al. 1993). At first the liver was perfused via
the portal vein with a perfusion solution containing 0.148M NaCl, 0.01M HEPES,
0.017M fructose, 0.049mM EGTA, 0.5% phenol red, and 6unit/ml heparin with pH-7.2.
Then a digestion solution containing lOOug/ml collagenase D, 93unit/ml hyaluronidase,
160unit/ml trypsin inhibitor, and 0.2% BSA was passed through the portal vein. The liver
was then excised and forced through three layers of gauze over a beaker using the
digestion solution, and then centrifiiged in 50ml tubes at 4°C for 3 minutes at 50xg.The
supernatant was aspirated and the cell pellet was suspended, washed and centrifiiged
twice with cold Waymouth's MB 752/1 (Sigma -Aldrich Corporation, St., MO) medium
containing 0.5% Bovine Serum Albumin (BSA). The cell pellet obtained after the final
37
washing was gently resuspended in the medium and an aliquot was used to determine cell
concentration and cell viability using trypan blue exclusion method with a
hemocytometer. Cells with greater than 85% viability were plated to 90% confluency on
sterilized 60mm collagen (rat tail) coated plates (Falcon-30002). The cells were incubated
in 4ml Waymouth's MB 752/1 medium containing 5% BSA (Sigma, St Louis, MO) and
supplemented with gentamicin (10|ag/mL) (Sigma) under a humidified atmosphere of 5%
CO2 and 95%) air at 37?C for 4 hrs. After a 4hr attachment period, the media was aspirated
and cells were washed once with 1ml BSA free Waymouth's medium and then incubated
with 4ml of fresh medium without BSA for an overnight incubation.
Cell treatment and processing
To assess the effect of glucosamine on an insulin induced key signal
protein and to investigate the effect of glucosamine on O-GlcNAc modification by
western blot, primary hepatocytes were incubated in DMEM low glucose media (5mM)
(Gibco, Grand Island, NY) with or without ImM glucosamine (Sigma) for 18hrs
followed by media change with low glucose media and then treated with 80nM (Sigma)
insulin for 1 hr (Table 1). To test the effect of selenium induced activation of a key
insulin signaling protein in glucosamine induced insulin resistance, primary hepatocytes
were incubated in DMEM low glucose media (5mM) with or without ImM glucosamine
for 18hrs followed by media change with low glucose media and then treated with
selenium (500|LIM) for 3hr (Table2). To investigate the effect of glucosamine on the
38
insulin or selenium induction of the gene expression of key metabolic enzymes, cells
were incubated in DMEM low glucose media with or without glucosamine for 18hrs
along with or without insulin at 44nM concentration at various time periods (1 or 18hr).
To test the effect of selenium, the cells were incubated in DMEM low glucose media with
or without glucosamine for 18hrs along with or without 500|uM selenium at various time
periods (1, 3 or 6 hrs). To study PEPCK and Foxa2 gene expression, the cells were
treated with 500|nM cAMP (Sigma) along with 44nM insulin for lhr or 500|uM selenium
for 1, 3 or 6hrs.
After the cells were treated using the various conditions, they were
processed by first removing the media and then washing twice with 1ml of cold
phosphate buffered saline (PBS) before isolating either protein or mRNA.
39
Table 1: Treatment conditions to establish an insulin resistance model in primary rat
hepatocytes
Assay
Signaling protein
Treatment
Glucosamine Insulin
Western blot
Akt*
18hr
lhr
NA
Number of
animals (n)
4
qRT-PCR
Metabolic
enzymes**
Glucose 6
phosphate
dehydrogenase
Fatty acid synthase
Phosphoenolpyruvat
e carboxylase
Foxa2
18hr
18hr
NA
4
18hr
18hr
18hr
lhr
NA
lhr
5
8
18hr
18hr
lhr
lhr
lhr
5
4
Mass
Spectrometry
*= Insulin treatment after 18hr glucosamine treatment
**= Insulin treatment along with 18hr glucosamine treatment
40
cAMP
Table 2: Treatment conditions to study the effect of selenium, an insulin mimetic agent
Assay
Signaling protein
Treatment
Glucosamine
Western blot
Akt*
18hr
3hr
qRT-PCR
Metabolic
enzymes**
Glucose 6 phosphate
dehydrogenase
Fatty acid synthase
18hr
1,3 and
6hrs
1,3 and
6hrs
1,3 and
6hrs
1,3 and
6hrs
3hr
Phosphoenolpyruvat
e carboxylase
Forkhead box A2
(Foxa2)
Mass
Spectrometry
18hr
18hr
18hr
18hr
Selenium
cAMP
NA
Number of
animals (n)
4
NA
4
NA
4
lhr
5
lhr
5
lhr
4
*= Selenium treatment after 18hr glucosamine treatment
**= Selenium treatment along with 18hr glucosamine treatment
Protein isolation and western blot analysis
Protein isolation
For isolating proteins from cells, a IX reporter lysis buffer (Promega-cat# E3971)
(500(il for each 60mm dish) was added and incubated at room temperature for 10
minutes. After the incubation, the cells were then scraped from the plates and the dish
containing the cell suspension was tipped at an angle and allowed to sit for an additional
10 minutes. The cell lysate was then collected in eppendorf tubes and centrifiiged at
10,000 rpm for 5 minutes at 4°C. The supernatants were then transferred to a new
41
eppendorf tube for protein assay, western blot protein analysis or affinity column
chromatography.
Determination of protein content
Total protein content of the sample was measured using the Micro BCA Protein
Assay Kit (Pierce). For the standard, a bovine serum albumin (Sigma) stock was prepared
at lmg/ml by dissolving BSA in water. In a micro titer plate, standard solution of BSA
from 2-20|il (2-20|^g) was used and the final volume of the standard was adjusted and
diluted to 110|nl with H2O. For the wells containing sample, lOjal of the sample was
diluted to 110|il with H2O and each sample was run in duplicate. Working reagent (WR)
was prepared freshly by mixing BCA reagent A (MA), 50 parts and BCA reagent B, 1
part. Each sample had
150JLX1
of WR added to it, and then the plate was incubated at 37°C
for 30 minutes. Samples were measured spectrometrically at 562nm.
Immunoprecipitation
A total of 300|ig of protein was used for immunoprecipitation. To investigate the
phosphorylation of Akt, two microliters of the Phospho-AKT (Ser 473) antibody (Cell
Signaling) was added and to investigate the O-GlcNAc modified proteins, two microliters
of the anti-O-P-GlcNAc specific monoclonal antibody (CTD 110.6) (Pierce) was added
and incubated overnight at 4°C on a shaker. In the morning a 50% agarose beads
(Upstate) slurry was prepared by washing it 3 times with PBS and collecting the pellet
after each wash. An equal amount of PBS was added to the pellet and the 50% slurry was
prepared. For each sample, 30(il of slurry was added and then incubated for 4hrs at 4°c on
a shaker. Samples were then centrifiiged at 5,000-6,000 rpm for 30 seconds and the pellet
42
was collected. The pellet was then washed an additional 3 times with PBS. To each of the
samples, 25|ul of 3X sodium dodecyl sulfate (SDS) sample buffer (Cell Signaling) and
ImM DTT (final concentration) was added. The samples were again centrifiiged at
14,000rpm for 2 minutes to break agarose beads. The supernatant was collected and
heated for 5minutes at 90°C. After this sample were cooled on ice and stored at -20°C for
further use.
Western blot analysis
Twenty microliters of immunoprecipitated sample was subjected to
electrophoresis in 10% Tris- Glycine gels at lOOv for 90 minutes with a IX running
buffer (25mM Trizma base, 192mM glycine, 0.1 % SDS). Five microliters of protein
marker (Cell Signaling) was used and 2ul of sample buffer was loaded in the empty lanes.
Proteins were then transferred to a PVDF membrane (Immobilin, Millipore, and CA).
After blocking with 5% non-fat dry milk, the blots were incubated overnight at 4°C with
diluted antibodies (1:1000) against Phospho-AKT (cell signaling) and (1:1000) against
O-P-GlcNAc. Blots were washed three times with 0.1% Tween-20 in Tris-buffered saline
(0.02M Tris base and 0.14 M NaCl in water, pH -7.6) and then blots were incubated with
diluted (1:2000) horseradish peroxidase-conjugated anti rabbit IgG, and diluted (1:1000)
anti-biotin antibody, for 1 hr at room temperature. The membranes were washes again as
above, and the bands were detected by chemiluminescence (HRP-Western Detection Kit,
Cell Signaling Technology, Inc). The membrane was then exposed to Kodak X-OMAT
autoradiography film and exposure time was varied depending on the intensity. Blots
were quantified using scanning densitometry (NIH Image).
43
LDH assay
Lactate dehydrogenase, an intracellular enzyme released into the extracellular
environment when the cell membrane is compromised (Rae 1977) was assayed using a
spectrophotometric method (Wroblewski 1955). After treatment with glucosamine for
18hrs, media was removed from the control as well as treated plates and assayed for
lactate dehydrogenase leakage. For the assay, a stock solution of 0.1 M phosphate buffer,
0.02mM pyruvate and 14mM NADH was prepared. The final concentration in a 1ml
assay was 83mM phosphate buffer, 0.67mM pyruvate and 0.23mM NADH. Two hundred
microliters of the media from each plate was used for the assay. The reaction velocity
was determined in a spectrometer by measuring a decrease in absorbance at 340nm
resulting from the oxidation of NADH. Protein concentration in the media was measured
using the BCA Protein Assay kit and units of enzyme activity were then calculated.
RNA isolation
Total RNA was isolated using TRIzol reagent (TRIzol reagent, Invitrogen). Cells
were lysed using 1ml of TRIzol/60mm dish. The cell suspension was incubated at room
temperature for 5minutes to dissociate nucleoprotein complexes. Chloroform (0.2ml per
lml TRIzol) was then added to the cell suspension and vigorously shaken for 15 seconds
which was then followed by incubation for 3 minutes at room temperature and then
centrifiiged for 15 minutes (13400 xg @ 4°C). Following centrifugation, the mixture
separates into lower red phenol chloroform phase, an interphase, and a colorless upper
aqueous phase that contains the RNA. The upper aqueous phase was transferred to a fresh
44
eppendorf tube. The volume of aqueous phase is about 60%> of the volume of TRIzol used
for cell processing. RNA is then precipitated from the aqueous phase with isopropyl
alcohol (0.5ml per 1.0 ml TRIzol reagent used). After incubation at room temperature for
10 minutes, samples were centrifiiged for 10 minutes (13400 xg @4°C). The RNA
precipitate forms a gel like pellet on the side and bottom of eppendorf tube. After
removing the supernatant, RNA pellet was then washed with 75% ethanol (1ml per 1ml
TRIzol/60mm dish used).The RNA pellet was again centrifiiged (3300 xg @ 4°C) for 5
minutes. The gel like RNA pellet at the bottom was dissolved in diethyl pyrocarbonate
(DEPC) treated water. RNA samples were then analyzed for both concentration and
quality using spectrophotometer.
Determination of RNA content and quality assessment
For quantification of the RNA isolated, spectrophotometer readings at 260 nm and
280 nm were taken. The readings at 260 nm give the concentration of nucleic acid in the
sample. The readings at 280 nm give the amount of protein in the sample. The isolated
RNA is of high quality has an OD 260/OD 280 value between 1.8 to 2.0. For
quantification of sample, 2ul of sample was diluted with 998ul DEPC water. The RNA
concentration read was then calculated by using the following formula:
OD 260* 40ng/ul * dilution factor.
1 O.D (optical density) at 260nm for RNA = 40ng/|nl of RNA
45
Reverse transcriptase polymerase chain reaction (RT PCR)
Two step real time quantitative PCR (qRT PCR) was carried out to evaluate the
changes in gene expression. All the reagents, kits and target gene probes were purchased
from Applied Biosystem (ABI). First 150ng total RNA was reverse transcribed into
cDNA on a thermocycler (Eppendorf Master Cycler gradient) using a Taqman Reverse
Transcription Reagent kit containing Taqman buffer, magnesium chloride, random
hexamers, dNTPs, RNase inhibitor and reverse transcriptase (product # N8080234, ABI).
The quality of cDNA was checked by agarose gel electrophoresis before the second step
of PCR amplification. The second step was performed on the Step One Plus Real time
PCR system (Applied Biosystems). Real time PCR reaction was carried out in a 96-well
plate containing 12.5ul of 2X Taqman Universal PCR Master Mix, 1.25ul of 20X
Taqman Gene expression Assays-containing specific PCR primers and FAM Tm dyelabeled probes, cDNA obtained from the first strand synthesis reaction and water to a
final reaction volume of 25|il. After an initial incubation step for 2 minutes at 50°C and
denaturation for 10 minutes at 95°C, qRT-PCR was carried out using 40 cycles of PCR
(95°C for 15s. 60°C for 60s), p actin was used as reference gene.
Primer/probe set for the genes studied was commercially obtained from Applied
Biosystems (Table 3).
46
Table 3: Primers for RT-PCR
Assay ID
Gene
Glucose 6 phosphate dehydrogenase
Fatty acid synthase
Rn00569117_ml
Phosphoenolpyruvate carboxy kinase
Rn01529009_gl
Forkhead box A2 (Foxa2)
Rn00562517_ml
Isolation and purification of O-GlcNAcvlated proteins
Wheat germ agglutinin lectin affinity chromatography
To Isolate and purify O-GlcNAcylated proteins from whole cell lysate, we
used glycoprotein isolation kit (Pierce). The sample containing 1 to 1.5 mg of total
protein was diluted (4:1) with 5x Binding/wash buffer solution. Then Wheat germ
agglutinin (McGarry, Kuwajima et al.) column was prepared by transferring
200JLL1
of
50% resin slurry to the column provided. The column was centrifiiged for 1 minutes at
1000 x g, to discard the storage buffer and to pack the column tightly. The column was
washed twice with 200JLX1 IX Binding/Wash buffer by centrifuging the column for 1
minute at 1000 x g. This step was performed to equilibrate the column with binding
buffer. Then samples were added and the column was incubated for 10 minutes at room
temperature with end -over-end mixing using a rotator. After incubation, the column was
washed again thrice with 400jil IX Binding/Wash Buffer, to remove any unbounded
47
proteins. Then 200|LI1 of elution buffer was added to the column and incubated for 20
minutes at room temperature with end-over-end mixing using a rotator. After
centrifugation for 1 minute at 1000 x g, the eluate was collected in the collection tubes
and stored on ice for immediate use or kept at -80°C for later use.
SDS-PAGE (sodium dodecyl polyacrylamide gel electrophoresis)
A total of 25|ng of eluted proteins from the wheat germ affinity
chromatography was subjected to electrophoresis in 10% Tris-Glycine gels at lOOv for 90
minutes with a IX running buffer (Trizma base, glycine, SDS). Before subjecting the
samples to electrophoresis, 3X SDS sample buffer (cell signaling) was added and the
samples were heated for 5minutes at 90°C. Five microliters of prestained protein marker
(Cell Signaling) was used as a standard. After the run was complete, proteins were
visualized by silver staining.
Identification of O-GlcNAc proteins by LC-MS analysis
After purification, protein samples were submitted to the Mass Spectrometry and
Proteomics Lab at the Van Andel Research Institute, directed by Greg Cavey.
Statistical Analysis
The results were expressed as the mean ±SEM of N number of animals used for
each experiment. The comparisons within groups were performed with one way analysis
of variance (ANOVA) followed by Student-Newman-Keuls test. Statistical significance
was tested at p<0.05.
48
CHAPTER 3
RESULTS
The first direct evidence supporting a link between flux through the hexosamine
biosynthetic pathway (HBP) and the development of insulin resistance came from in vitro
studies done in rat adipocytes (Marshall, Bacote et al. 1991). Since then, multiple in vitro
and in vivo studies have shown that chronic elevated flux through the HBP may represent
one mechanism by which hyperglycemia can lead to insulin resistance. There are three
routinely used experimental approaches to evaluate the roles of hexosamine flux in the
development of insulin resistance: 1) exposure to chronic hyperglycemia 2) exposure to
glucosamine and 3) perturbing GFAT activity. We have used glucosamine to induce
insulin resistance in primary rat hepatocytes.
One of the molecular mechanisms responsible for the insulin resistant condition in
type 2 diabetes is caused by alterations at one or several levels of the insulin-signaling
cascade in the insulin sensitive tissues, skeletal muscles, adipose and liver. Previous
studies in various other cell models have shown the involvement of the PI3-kinase
pathway in insulin resistance is mediated by the increased flux in HBP (Kim, Nikoulina
et al. 1999; Patti, Virkamaki et al. 1999). In adipocytes and pancreatic P cells, insulin
resistance is mediated by the impaired activation of Akt caused by reduced insulin
49
stimulated phosphorylation of Akt (Vosseller, Wells et al. 2002; Parker, Lund et al. 2003;
DAlessandris, Andreozzi et al. 2004; Park, Ryu et al. 2005). However, contrary to the
adipocytes and pancreatic P cells studies, Akt phosphorylation in the skeletal muscle was
not significantly altered (Arias, Kim et al. 2004). These contradictory results in these
insulin sensitive tissues led us to further investigate the phosphorylation of Akt in
primary rat hepatocytes. Hence, this was the first target for the investigation.
Effect of glucosamine on insulin induced Akt phosphorylation
To study the effect of glucosamine on insulin induced Akt phosphorylation,
primary rat hepatocytes were incubated in DMEM low glucose media with or without
ImM glucosamine for 18hrs, followed by media change with low glucose media and then
treatment with 80nM insulin for 1 hr. The conditions of ImM glucosamine and 18hrs of
incubation were established after treating the cells with varying concentrations over
different lengths of time. After treatment, cells were lysed and the phosphorylated Akt
was immunoprecipitated using phospho-Akt (Ser 473) antibodies and western analysis
performed. The film was quantified with densitometry using NIH software. The results
show insulin treatment increased the phosphorylation of Akt 7 fold as compared with the
basal level in control, whereas the insulin stimulated phosphorylation of Akt was
drastically decreased 4 fold in the presence of glucosamine when compared to insulin
treated phosphorylation of Akt in the control (Figure 4). Glucosamine by itself did not
induce phosphorylation of Akt. These results indicate that one of the mechanisms through
50
which glucosamine interferes with insulin signaling is through inhibiting insulin
stimulated Akt phosphorylation at Ser-307 in primary rat hepatocytes.
As mentioned before, insulin, after binding to its receptor, regulates many cellular
processes and the expression of several genes through the activation of various signaling
proteins and pathways. If the activation of the signaling proteins is affected, then gene
expression should also be altered. Insulin regulates the transcription of the genes for
several metabolic enzymes: Glycogen synthase (Sutherland, Leighton et al. 1993),
phosphoenolpyruvate carboxylase (PEPCK) (Forest, O'Brien et al. 1990), fatty acid
synthase (FAS) (Moustaid, Beyer et al. 1994) and glucose 6 phosphate dehydrogenase
(G6PDH) are rate limiting enzymes in glycogen synthesis, gluconeogenesis, fatty acid
biosynthesis and pentose phosphate pathway respectively, and have been the target of
studies with regard to action of insulin. Therefore, our next objective was to examine the
effect of glucosamine on the insulin regulation of the expression of key metabolic
enzymes.
Effect of glucosamine on insulin induced G6PDH gene expression
G6PDH is a lipogenic enzyme that catalyses the first reaction in the pentose
phosphate pathway, a metabolic pathway that supplies reducing energy to cells by
maintaining the level of the co-enzyme nicotinaminde adenine dinucleotide phosphate
(NADPH), which is required by fatty acid synthase for de novo lipogenesis. Early studies
in our lab using diabetic animals showed that G6PDH expression was regulated by
insulin (Berg, Wu et al. 1995). To understand the mechanism by which insulin and/or
carbohydrate regulates the expression of this gene, the DNA sequence was cloned for the
51
promoter region of the gene (Rank, Harris et al. 1994). Using this promoter sequence
cloned into a reporter gene, as well as by measuring endogenous mRNA levels, we
showed that G6PDH expression was regulated transcriptionally by insulin (Wagle, Jivraj
et al. 1998). To investigate the effect of glucosamine on the insulin induced expression of
G6PDH, primary rat hepatocytes were incubated in DMEM low glucose media with or
without glucosamine for 18hrs along with or without 44nM insulin for 18hrs. The cells
were then processed and the G6PDH mRNA was measured using qRT PCR. These
results show that insulin significantly increased the gene expression of G6PDH by 2 fold
when compared to control and these results are comparable to our previous reports using
northern analysis (Figure 5). Insulin stimulated expression of G6PDH under glucosamine
treatment decreased significantly compared to insulin stimulated G6PDH gene expression
in the control. In fact the levels measured were comparable to basal control (Figure 5),
suggesting that insulin was no longer able to regulate G6PDH expression. This decrease
in the insulin induced gene expression of G6PDH under glucosamine treatment is
significant metabolically since the pentose phosphate pathway is responsible for most of
the NADPH production and NADPH is needed not only for fatty acid synthesis but other
reductive biosynthetic processes such as nucleic acid synthesis.
52
NA
B
Ins
9.00
8.00
Gin +lns
Gin
^r
-
~~
< 7,00
+
.
~
~
3 6.00
£ 5.00
|
4
A
^0
&
£ 3.00
A
J 2 00
5
2 ioo
0.00
+
! I
§
NA
ins
!
i
•
'—
Gin
Gin* Ins
Figure 4: Effect of glucosamine on insulin induced Akt phosphorylation. A.
Representative western blot showing phospho-Akt (Ser-473). B. Quantitation
of Phospho-Akt blots. Primary rat hepatocytes were incubated in DMEM low
glucose media with or without ImM glucosamine for 18hrs followed by
media change with low glucose media and then treated with insulin at 80nM
for 1 hr. The cells were lysed and processed and immunoprecipitated using
Phospho-AKT (ser 473) antibodies. Western blot was run with samples and
blots were quantified. Data represent the mean +/- S.E.M (n=4), "n" is the
number of animals used. Statistical analysis was performed by one-way
analysis of variance (ANOVA) followed by Student -Newman Keuls test
(PO.05). tUf = Significant increase compared to NA. /V= Significant
decrease compared to insulin induced Akt phosphorylation in control. NANo addition, Ins- Insulin, Gin- Glucosamine, Gln+Ins- Glucosamine
+Insulin.
53
2.50
•
T
1
2.00
1.50
<U
JU.
A3
Q.
E
o
i
T>
1.00
u
<u
QuO
C
A
T
I
cz
JO
0.50
^
0.00
I
I
NA
Ins
-
i
Gin
Gln+lns
Figure 5: Effect of glucosamine on G6PDH gene expression. Primary rat hepatocytes
were incubated in DMEM low glucose media with or without ImM
glucosamine for 18hrs along with or without insulin at 44nM for 18hrs. The
cells were then processed and the G6PDH mRNA was measured using qRT
PCR. Data represent the mean +/- S.E.M (n=4), "n" is the number of
animals used. Amount of G6PDH mRNA was normalized to control probe,
P-actin. Statistical analysis was performed by one-way analysis of variance
(ANOVA) followed by Student -Newman Keuls test (PO.05).). All the
data in the figures are presented as fold changes compared to the control
(NA).*= Significant increase compared to NA. A = Significant decrease
compared to insulin induced G6PDH. NA- No addition, Ins- Insulin, GlnGlucosamine, Gln+lns- Glucosamine +Insulin.
54
Effect of glucosamine on insulin induced fatty acid synthase (FAS)
FAS synthesizes long-chain fatty acids by using acetyl coenzyme A (CoA) as a
primer, malonyl-CoA as a 2-carbon donor, and NADPH as a reducing equivalent. Insulin
is considered as a positive regulator of fatty acid synthesis by increasing fatty acid
synthase (FAS) mRNA transcription (Moustaid, Beyer et al. 1994). The effect of insulin
on FAS transcription is mediated by the activation of the phosphoinositide (PI) 3kinase/Akt pathway (Wang and Sul 1998). These results show that insulin significantly
increased the expression of FAS by 1.6 fold (Figure 6) when compared to control, which
is comparable to the published reports (Fukuda, Katsurada et al. 1992). On the other
hand, insulin stimulated gene expression of FAS under glucosamine treatment decreased
significantly compared to insulin stimulated FAS gene expression in control. This
indicates that glucosamine inhibits insulin stimulated FAS gene expression.
55
24
<
2:
2.1
o
TJ
<X)
u.
re
Q.
b
o
u
0)
w>
c
u
TJ
O
u_
1.8
1
*
T
1
1.5
i
*
.. _L.
1.2
±
0.9
0.6
0.3
1
0
NA
1
"
Ins
1
»
Gin
1
1
Gln+lns
Figure 6: Effect of glucosamine on FAS gene expression. Primary rat hepatocytes were
incubated in DMEM low glucose media with or without ImM glucosamine
for 18hrs along with or without 44nM insulin for 18hrs. The cells were then
processed and the FAS mRNA was measured using qRT PCR. Data
represent the mean +/- S.E.M (n=5), "n" is the number of animals used.
Statistical analysis was performed by one-way analysis of variance
(ANOVA) followed by Student -Newman Keuls test (PO.05). All the data
in the figure are presented as fold changes compared to the control (NA).
*k= Significant increase compared to NA. A = Significant decrease compared
to insulin induced FAS gene expression. NA- No addition, Ins- Insulin, GlnGlucosamine, Gln+lns- Glucosamine +Insulin.
Effect of glucosamine on insulin inhibition of PEPCK gene expression
Phosphoenolpyruvate carboxylase catalyzes the conversion of oxaloacetate to
phosphoenolpyruvate, which is the rate limiting step in gluconeogenesis.
Gluconeogenesis plays a key role in diabetes as it is the process by which the liver makes
56
glucose. The hepatic PEPCK gene is regulated at the transcriptional level by a variety of
hormones (Forest, O'Brien et al. 1990; Hall, Scott et al. 1992) including glucocorticoids
(GC), retinoic acid (RA), thyroid hormone, and glucagon (acting through cyclic AMP)
which increase the rate of synthesis of the enzyme (Sasaki, Cripe et al. 1984; Lucas,
O'Brien et al. 1991; Park, Jerden et al. 1995; Scott, Stromstedt et al. 1998). Insulin exerts
an opposite, dominant negative effect (O'Brien, Lucas et al. 1990) primarily through
inhibiting the initiation of hepatic PEPCK gene transcription but also reducing the rate of
transcript elongation. In H4IIE cells, the inhibitory effects of insulin are dominant, since
it prevents cAMP and glucocorticoid mediated increases to PEPCK gene transcription. In
this experiment, cAMP was used to induce the gene expression of PEPCK. It was found
that the addition of 500uM of cAMP for 1 hr in the control group caused an increase in
PEPCK gene expression by 27 fold (Figure 7), which was comparable to previous studies
(Beale, Andreone et al. 1984; Imai, Miner et al. 1993). Following treatment of insulin
after cAMP a 25% decrease was observed in the cAMP induced gene expression. In cells
treated with glucosamine, cAMP still caused an increase in the expression of PEPCK by
23 fold, results comparable to those observed without glucosamine. As expected, the
inhibition of cAMP induced PEPCK expression by insulin was not observed in the
glucosamine treated cells (Figure 7).
57
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Figure 7: Effect of glucosamine on PEPCK gene expression. Primary rat hepatocytes
were incubated in DMEM low glucose media with or without ImM
glucosamine for 18hrs in the presence or absence of 44nM insulin and
500uM cAMP for lhr. The cells were then processed and the PEPCK
mRNA was measured using qRT PCR. Data represent the mean +/- S.E.M
(n=8), "n" is the number of animals used. Statistical analysis was performed
by one-way analysis of variance (ANOVA) followed by Student -Newman
Keuls test (PO.05). * = Significant increase compared to NA. A =
Significant decrease compared to insulin, it it = no significance. NA- No
addition, Ins- Insulin, Gin- Glucosamine, Gln+lns- Glucosamine +Insulin,
cAMP- Cyclic adenosine monophosphate.
58
Effect of glucosamine on insulin regulation of Foxa2
The transcriptional regulation of the gene encoding PEPCK involves complex
interactions of a variety of transcription factors and other proteins. The PEPCK promoter
contains an insulin response sequence (IRS). This sequence has been functionally
characterized, and the transcription factors binding to this region have been studied. The
PEPCK promoter has hepatic nuclear factor-3 (HNF-3 also known as Foxa) binding sites,
and members of the Foxa family are involved in the glucocorticoid-stimulated expression
of PEPCK. Furthermore, in the PEPCK promoter, an HNF-3 binding site is located in
close proximity to the IRS with a partial overlap of both structures. Therefore, HNF-3 has
also been discussed as a transcription factor that may mediate insulin's effect on the
PEPCK promoter. So our next objective was to examine whether or not the insulin
mediated reduction in PEPCK expression could be attributed to reduced levels of Foxa2
(HNF-3P) and if so what effect it would have.
An approximately 40% decrease in Foxa2 expression was observed following
treatment of insulin after cAMP. Glucosamine did not affect Foxa2 expression or alter the
effect of cAMP (Figure 8). With glucosamine treatment, there was no significant
difference between cAMP induced Foxa2 gene expression with or without insulin. This
result shows that under glucosamine treatment insulin no longer was able to inhibit the
induction of cAMP induced gene expression of Foxa2.
59
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(S
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o
D)
§1.5
O
s
1
U
S0.5
^
^
/
&*
&
/
V
Figure 8: Effect of glucosamine on Foxa2 gene expression. Primary rat hepatocytes were
incubated in DMEM low glucose media with or without ImM glucosamine
for 18hrs along with or without 500uM for lhr followed by insulin at 44nM
at for lhr. The cells were then processed and the Foxa2 mRNA was
measured using qRT PCR. Data represent the mean +/- S.E.M (n=5), "n" is
the number of animals used. Statistical analysis was performed by one-way
analysis of variance (ANOVA) followed by Student -Newman Keuls test
(PO.05). it = Significant increase compared to NA. A = Significant
decrease compared to insulin. ^ ^ = no significance.
60
Effect of selenium on Akt phosphorylation
Selenium, an essential trace element, has been shown to mimic some of insulin's
actions both in vivo and in vitro. To determine whether selenium can act as an
antidiabetic agent in this model system, the effect of selenium on Akt phosphorylation
was first investigated. If selenium truly acts as a mimetic, then an increase in
phosphorylation of Akt with selenium should occur. Our results show that selenium
treatment increased the phosphorylation of Akt by 13 fold as compared to the basal level
in control (Figure 9). Selenium induced phosphorylation of Akt is unaffected in the
presence of glucosamine unlike what was observed with the quenching effect of
glucosamine on the insulin induced phosphorylation of Akt. This suggests that selenium
can act as a potent insulin - mimetic not only under normal/ control conditions but also
under insulin resistant condition as well.
NA
Se
Gin
61
Gln+Se
B
Figure 9. Effect of glucosamine on selenium induced Akt phosphorylation. A.
Representative western blot showing phospho-Akt (Ser-473). B.
Quantitation of phospho-Akt blots. Primary rat hepatocytes were incubated
in DMEM low glucose media with or without ImM glucosamine for 18hrs
followed by media change with low glucose media and then treated with
selenium at 500uM for 3 hr. The cells were lysed and processed and
immunoprecipitated using Phospho-AKT (Ser 473) antibodies. Western blot
was run with samples and blots were quantified. Data represent the mean +/S.E.M (n=4), "n" is the number of animals used.. Statistical analysis was
performed by one-way analysis of variance (ANOVA) followed by Student
-Newman Keuls test (PO.05). ic = Significant increase compared to NA.
itit= no significant difference between selenium and glucosamine + selenium.
62
The next objective of the study was to investigate whether or not similar selenium
effect could be found on the expression of key metabolic enzymes in glucosamine
induced insulin resistance. Our lab had shown that like insulin selenium can affect the
expression of two key lipogenic enzymes, G6PDH and FAS. So we chose these two
metabolic enzymes as our first targets for study.
Effect of selenium on G6PDH gene expression
Our results show that selenium induces the gene expression of G6PDH by
approximate 1.5 fold at 6hr respectively. Unlike our findings with insulin in the presence
of glucosamine, selenium induction of G6PDH was similar in the presence or absence of
glucosamine (Figure 10).
63
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1
NA
Se-1hr
Se-3hr
Se-6hr
Gin
1
Gln+Se1hr
Gln+Se3hr
Gin+Se6hr
Figure 10: Effect of selenium on G6PDH gene expression. Primary rat hepatocytes were
incubated in DMEM low glucose media with or without ImM glucosamine
for 18hrs along with or without Selenium at 500uM concentration at various
time period ( 1, 3, 6 hrs). The cells were then processed and the G6PDH
mRNA was measured using qRT PCR. Data represent the mean +/- S.E.M
(n=4), "n" is the number of animals used.. Statistical analysis was performed
by one-way analysis of variance (ANOVA) followed by Student -Newman
Keuls test (PO.05). All the data in the figures are presented as fold changes
compared to the control (NA). it = significant increase compared to NA.^Ht
= No significant induction of G6PDH in the presence or absence of
glucosamine. NA- No addition, Se-Selenium, Gln-Glucosamine, Gln+seGlucosamine +Se
Effect of selenium on FAS gene expression
The results show that selenium induces the gene expression of FAS by 1.32 fold
at 6hr (Figure 11). There was no significant difference between selenium induced FAS
gene expression under glucosamine treatment compared to the selenium induced FAS
gene expression under control. Similar to the findings with G6PDH, the expression of
FAS is still responsive to selenium even when glucosamine is present.
64
Figure 11: Effect of selenium on FAS gene expression. Primary rat hepatocytes were
incubated in DMEM low glucose media with or without ImM glucosamine
for 18hrs along with or without Selenium at 500uM concentration at various
time period ( 1, 3, 6 hrs). The cells were then processed and the G6PDH
mRNA was measured using qRT PCR. Data represent the mean +/- S.E.M
(n=4) "n" is the number of animals used. Statistical analysis was performed
by one-way analysis of variance (ANOVA) followed by Student -Newman
Keuls test (PO.05). All the data in the figures are presented as fold changes
compared to the control (NA). it = significant increase compared to NA.^H^r
= No significant difference in selenium induction of FAS in the presence or
absence of glucosamine. C-NA- No addition, C-Se- Control selenium, GlnNA -Glucosamine No addition, Gln-Se.
Effect of selenium on PEPCK gene expression
As mentioned before, gluconeogenesis plays a key role in diabetes as it is the
process by which the liver makes glucose. We therefore examined expression of PEPCK,
the key enzyme of this pathway. In these studies, cAMP was used to induce the gene
65
expression of PEPCK. It was found that addition of 500uM of cAMP for 1 hr in control
group caused an increase in PEPCK gene expression by 27 fold (Figure 12). Selenium
treatment decreased cAMP induced gene expression of PEPCK significantly and
expression was near basal level by 6hr. Again, like with FAS and G6PDH, selenium had
similar effects on the expression of PEPCK in the presence of glucosamine suggesting its
actions are not sensitive to glucosamine.
66
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Figure 12: Effect of selenium on PEPCK gene transcription. Primary rat hepatocytes
were incubated in DMEM low glucose media with or without ImM
glucosamine for 18hrs along with or without selenium for various time
period (1, 3, 6 hrs) at 500uM followed with 500uM cAMP for lhr. The cells
were then processed and the PEPCK mRNA was measured using qRT PCR.
Data represent the mean +/- S.E.M (n=5), "n" is the number of animals used.
Statistical analysis was performed by one-way analysis of variance
(ANOVA) followed by Student -Newman Keuls test (PO.05). * =
significant increase compared to NA. irk = No significant difference in
cAMP induced selenium induction of PEPCK in the presence or absence of
glucosamine. • = significant decrease compared to cAMP induced PEPCK
gene expression. NA- No addition, Ins- Insulin, Gin- Glucosamine, cAMPcyclic AMP.
67
Effect of selenium on Foxa2 gene expression
Our result shows that the addition of 500uM of cAMP for 1 hr in control group
caused an increase in Foxa2 gene expression by approximately 2.3 fold. Selenium
treatment decreased cAMP induced gene expression of Foxa2 significantly compared to
cAMP induced gene expression of Foxa2. While under glucosamine treatment, cAMP did
increase the expression of Foxa2 by 2.5 fold. Selenium treatment under insulin resistant
state could inhibit the induction of cAMP induced gene expression of Foxa2. Again, like
with FAS, G6PDH, PEPCK, selenium had similar effects on the expression of Foxa2 in
the presence of glucosamine suggesting its actions are not sensitive to glucosamine
(Figure: 13).
68
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Figure 13: Effect of selenium on Foxa2 gene transcription. Primary rat hepatocytes were
incubated in DMEM low glucose media with or without ImM glucosamine
for 18hrs along with or without selenium for various time period (1, 3, 6 hrs)
at 500uM followed with 500uM cAMP for lhr. The cells were then
processed and the Foxa2 mRNA was measured using qRT PCR. Data
represent the mean +/- S.E.M (n=5). Statistical analysis was performed by
one-way analysis of variance (ANOVA) followed by Student -Newman
Keuls test (PO.05). i* = significant increase compared to NA. irk= No
significant difference in cAMP induced selenium induction of PEPCK in the
presence or absence of glucosamine. 0 = significant decrease compared to
cAMP induced PEPCK gene expression. NA- No addition, Se-Selenium,
Gin- Glucosamine, cAMP- cyclic AMP.
69
Lactate dehydrogenase assay
Lactate dehydrogenase is a cytosolic enzyme present within all mammalian cells.
The normal plasma membrane is impermeable to LDH, but damage to the cell membrane
results in a change in the membrane permeability and subsequent leakage of LDH into
the extracellular fluid (Rae 1977). In vitro release of LDH from cells provides an accurate
measure of cell membrane integrity and cell viability. As a result, the release of lactate
dehydrogenase has proved to be a reliable test for cytotoxicity.
In order to assess whether or not there was any effect of glucosamine on cell
viability, primary rat hepatocytes were incubated in DMEM low glucose media with or
without ImM glucosamine for 18hrs. The release of LDH into the culture supernatant
correlates with the amount of cell death and membrane damage, providing an accurate
measure of the cellular toxicity induced. We used the direct spectrophotometric assay
(Wroblewski and Ladue 1955) which is based upon the ability of LDH to catalyze the
reaction:
Lactate + NAD > Pyruvate + NADH
Changes in optical absorbance, measured at 340nm, reflect changes in the
concentration of NADH and hence the level of LDH in the sample. The result shows that
there is no significant difference in glucosamine treated cells versus control which
suggests the cells are not compromised with glucosamine treatment (Figure 14).
70
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Glucosamine
Figure 14 Effect of glucosamine on lactate dehydrogenase release into the media
Results are expressed as percent of LDH activity in the media from control
Data represent the mean ±SEM (n=3), Statistical significance was tested at
pO05
Effect of glucosamine on O-GlcNAc modification
Protein modification through post-translational events like phosphorylation,
acylation or glycosylation can dramatically alter a proteins activity Since glucosamine
increases the flux through the HBP and O-GlcNAc is a product, a possible consequence
is increased O-GlcNAc modified proteins We first examined O-GlcNAc modified
71
proteins were increased using western blot. For this, primary rat hepatocytes were
incubated in DMEM low glucose media with or without imM glucosamine for
i8hrs in the presence or absence of 44nM insulin for lhr. After treatment, cells were
lysed and lysate was collected for each sample. The sample was then immunoprecipitated
using O-GlcNAc specific monoclonal antibody 110.6 and subjected to electrophoresis.
Proteins were transferred onto the membrane and the membrane was exposed to the film.
The results indicate that the glucosamine and glucosamine plus insulin treatments showed
an increase in proteins that are O-GlcNAcylated compared to control (Figure 15). Since
more proteins are modified, it is attractive to speculate that this modification of key
proteins is linked to the induction of insulin resistance.
72
NA
Ins
Gin
Gln+lns
200
140
100
W
60
SO
40
30
20
ure 15: Effect of glucosamine on O-GlcNAc modification. Primary rat hepatocytes
were incubated in DMEM low glucose media with or without ImM
glucosamine for 18hrs followed by media change with low glucose media
and then treated with 44nM insulin for 1 hr. The cells were lysed and
processed and immunoprecipitated using O-GlcNAc specific monoclonal
antibody 110.6 and subjected to electrophoresis. Proteins were transferred
onto the membrane and the membrane was exposed to the film.
73
Identification of O-GlcNAc modified proteins by LC-MS
We observed O-GlcNAc modified proteins were increased in the presence of
glucosamine using western blot analysis. The next goal was to identify the OGlcNAcylate- modified proteins. For this study primary rat hepatocytes were incubated in
DMEM low glucose media with or without ImM glucosamine for 18hrs in the presence
or absence of 44nM insulin for lhr or 500uM selenium for 3hr. O-GlcNAcylated proteins
were isolated and purified using Wheat Germ Agglutinin (McGarry, Kuwajima et al.)
column chromatography. Purified proteins were then subjected to SDS PAGE and
proteins were visualized by silver staining (Figure 16).
74
^fggg
Figure 16 Representative SDS-P AGE of O-GlcNAcylated proteins Primary rat
hepatocytes were incubated in DMEM low glucose media with or without
ImM glucosamine for 18hrs in the presence or absence of 44nM insulin for
lhr or 500uM selenium for 3hr After treatments, cells were lysed and lysate
was collected for each sample O-GlcNAcylated proteins were isolated and
purified proteins were then subjected to SDS PAGE and proteins were
visualized by silver staining Lane 1 -Marker, Lane 3- C-NA (control-No
addition), Lane 4- C-Ins (control msulm), Lane 5- Gln-NA (GlucosammeNo addition), Lane 6- Gin-Ins (Glucosamine- Insulin), Lane 7 - C-Se
(Control- selenium), Lane 8- Gln-Se (Glucosamine -selenium)
After purification of O-GlcNAcylated proteins, samples were submitted to the
Mass Spectrometry and Proteomics Lab at the Van Andel Research Institute for LC-MS
75
analysis for label free quantitative protein profiling. This system combines a quadrupole
time-of-flight Premier mass spectrometer (Qtof-P) with a NanoAcquity ultra-high
pressure liquid chromatography (UPLC) system for nanoscale separations with on-line
electrospray ionization. The system represents a paradigm shift in proteomics because it
employs a novel acquisition method termed LC-MS. Complex protein samples are
digested into peptides using trypsin (or other enzymes) and without chemical or stable
isotope labeling (label-free) peptides are analyzed to obtain both quantitative and
qualitative data in a single LC-MS run. Throughout an LC-MS analysis, the Qtof-P mass
spectrometer alternates the collision energy every 1 second in a high-low manner such
that peptide mass, retention time, and intensity are recorded during low collision energy
acquisition and peptide fragmentation is generated with high collision energy. Peptide
mass and retention time are used to compare across samples while peptide intensity
recorded by the mass spectrometer is used for both relative and absolute quantitation as
described by Silva (Silva, Denny et al. 2005; Silva, Gorenstein et al. 2006). High
collision energy acquisition generates peptide fragmentation data used to identify the
protein from which the peptide originated. However, unlike all other mass spectrometers
that perform conventional MS/MS data acquisition for protein identification and
quantification one peptide at a time, the Qtof-P does not filter peptide ions at a given m/z
value prior to fragmentation. Instead, all ions transmitting the Qtof-P, including coeluting peptides, are fragmented simultaneously, hence the term LC-MS. Using
innovative software called Identity, peptide fragments within a mixture of fragment ions
from many different peptides can be assigned to the parent peptide. This approach relies
on the simple premise that peptide fragments generated from a given peptide in the high76
energy mode will have the same shape and retention time as its parent peptide recorded in
the low energy mode. Once fragments are associated with a given peptide, the data is
used to identify the protein from which the peptide originated.
While the methodology described above should yield both qualitative and
quantitative data in one run, attempts to receive quantitative analysis between control and
experimental group did not succeed. For unexplained reasons, before trypsin digestion
the amount of protein quantified was sufficient to carry out the experiments but after
trypsin digestion the protein amount was too low. The digestion procedure was
attempted on several sample sets and similar results were obtained. While sufficient
protein was not available for quantitative analysis, a qualitative identification of protein
O-GlcNAc modified was made. Since our attempt to perform quantitative analysis did
not succeed, the data that was generated were analyzed comparatively. This comparative
analysis led us to identify variations in glycosylation based on treatment. The following
table lists the glycosylated proteins that were uniquely found in the glucosamine treated
cells compared to the control, untreated cells (Table 4).
77
Table 4: O-GlcNAc modified proteins found uniquely in the glucosamine treatment as
compared to control (no addition).
1 Functional
Subgroup
1 Cytoskeleton
1 Actin based
Intermediate
filaments
Microtubule
based
Chaperones
Oxido reductase
Process
Accession
Protein
IPI00373136
IPI00365944
IPI00207014
Ankyrin repeat domain 5
Myosin light polypeptide 6
Keratin type I cytoskeletal 19
IPI00370073
IIPI00370090
IIPI00371003
IPI00421791
IIPI00421793
IIPI00421795
IIPI00421855
IIPI00454479
IPI00551558
IPI00565487
IPI00882425
IPIOO188666
IPI00471523
Keratin type I cytoskeletal 17
Keratin type I cytoskeletal 15
Keratin type I cytoskeletal 25
Keratin type I cytoskeletal 42
Type I keratin KA16
Keratin type I cytoskeletal 13
Keratin type I cytoskeletal 14
Type I keratin KA11
Keratin type II cytoskeletal 2 epidermal
Keratin type I cytoskeletal 12
Keratin K6 Fragment
Keratin type I cytoskeletal 27
Tubulin alpha 3 chain
IPIOO189795
IPI00339167
IPI00364046
IPIOO199636
IPI00211100
Tubulin alpha 1A chain
Tubulin alpha IB chain
Tubulin alpha 1C chain
Calnexin
3 alpha hydroxysteroid dehydrogenase
IPIOO197770
IPI00205018
Aldehyde dehydrogenase mitochondrial
Methylmalonate semialdehyde dehydrogenase acylating
mitochondrial
MHC class I RT 1 Ac heavy chain
MHC class I RT1 Au heavy chain
MHC class IRT1 Au heavy chain Fragment
Mature alpha chain of major histocompatibility complex class I
antigen Fragment
Annexin A5
Basic transcription factor 3
Myoglobin
Eukaryotic translation elongation factor 1 delta
Immune response
IPI00551631
IPI00855239
IPI00551567
IPI00896766
Apoptosis
IPI00471889
IPI00372004
IPI00214517
IPI00471525
Heme binding
Elongation factor
78
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
|
Table 4: Continued
1 Growth factor
activity
Metal binding
Cell diviion
Protease
Other
IPI00204130
Hepatoma derived growth factor related protein 3
IPIOO195036
IPI00231107
IPI00210009
IPIOO191748
IPI00763910
IPI00765011
IPI00566672
IPI00210090
IPI111 111 13
IPI00365582
Metallothionein 2
Parathymosin
Nuclear migration protein nudC
Proteasome subunit alpha type 1
Similar to 60 kDa heat shock protein mitochondrial precursor
Similar to Actin cytoplasmic 2
Similar to heat shock protein 8
SP120
Serum albumin precursor Allergen Bos d 6
1
Carbamoyl phosphate synthetase 2 aspartate transcarbamylase and 1
dihydroorotase
Isoform 2 of Basigin
1
Isoform 2 of Haptoglobin
1
IPIOO193425
IPI00382202
Surprisingly, the analysis of the proteins that were glycosylated under the
glucosamine conditions did not reveal any modification to signaling proteins or metabolic
enzymes but interestingly literature search on all of the above proteins shows that none of
the above mentioned proteins have so far been identified to be glycosylated. So, to the
best of my knowledge all the above mentioned proteins have been identified as
glycosylated proteins for the first time in this study. Eventhough, the identified proteins
did not reveal any modification to signaling proteins or metabolic enzymes, some
functions were of interest. For example, 3 alpha hydroxysteroid dehydrogenase is an
enzyme that catalyzes the following chemical reaction
Androsterone + NAD (P) + ^5alpha-androstane-3, 17-dione + NAD (P) H + H+
79
It belongs to the family of oxidoreductases, and participates in 3 metabolic pathways: bile
acid biosynthesis, c21-steroid hormone metabolism and androgen and estrogen
metabolism. The literature suggests that 3-a-hydroxysteroid dehydrogenase is involved in
regeneration of L-Ascorbic acid (AA) in diabetic animals. L-Ascorbic acid is an
antioxidant that is responsible for the scavenging of toxic free radicals in both plasma and
tissues. AA is oxidized to monodehydroascorbic acid and dehydroascorbic acid (DHA). It
has been reported that AA levels in plasma and tissues are lower than normal in diabetic
animals (Will and Byers 1996). AA also increases the stability of blood vessels in
patients with diabetes and a decrease in AA might contribute to the diabetic
complications found at the late stages of the disease. The levels of AA are maintained in
plasma and tissues by the intracellular reduction of an oxidized form of AA. This
reductive regeneration is catalyzed by NADPH-dependant regenerating enzymes such as
3-a-hydroxysteroid dehydrogenase (AKR) and thioredoxin reductase (TR). The study
carried out by Kashiba revealed that low AA levels in plasma and tissue resulting from a
decrease in AA regeneration may be caused by a decrease in 3-a-hydroxysteroid
dehydrogenase and this might increase oxidative stress in Goto - Kakizaki (GK) diabetic
rats (Kashiba, Oka et al. 2000). Another protein modified is aldehyde dehydrogenase
mitochondria (ALDH2) and is responsible for acetaldehyde oxidation in ethanol
metabolism. It is involved in the oxidation and detoxification of aromatic and aliphatic
aldehydes such as 4-hydroxy-2-nonenal (Bosron and Li 1986; Ohsawa, Nishimaki et al.
2003; Vasiliou and Nebert 2005). Association of aldehyde dehydrogenase mitochondrial
with inheritance of non-insulin dependent diabetes mellitus (NIDDM) was shown by
Matsuoka. In that study, they investigated the influence of the ALDH2 genotype on
80
diabetes. The preliminary study on Japanese patients with NIDDM suggests a
relationship between alcohol intolerance and inheritance of diabetes (Suzuki, Muramatsu
et al. 1996). Recent work carried out by Wang has shown that hyperglycemia-induced
oxidative stress could reduce the activity and expression of ALDH2 in streptozotocininduced diabetic rat heart, and antioxidant could ameliorate these effects. They suggested
that ALDH2 inhibition aggravates mitochondrial impairment in response to
hyperglycemia, which could be the mechanism underlying left ventricle contractile
dysfunction in diabetic rats.
Further we compared the glucosamine plus insulin treatment to insulin only control. The
following table lists the proteins that are uniquely glycosylated in the glucosamine plus
insulin treatment compared to insulin only control (Table 5).
81
Table 5: O-GlcNAc modified proteins found uniquely in glucosamine plus insulin
treatment compared to insulin
1 Functional Subgroup
Accession
Protein
IPI00197888
Isoform 1 of Tropomyosin alpha 1 chain
IPIOO187731
Isoform 2 of Tropomyosin beta chain
IPI00207964
Isoform 4 of Tropomyosin alpha 1 chain
IPI00365944
Myosin light polypeptide 6
IPI00370090
Keratin type I cytoskeletal 15
IPI00480679
Keratin type I cytoskeletal 18
IPI00207014
Keratin type I cytoskeletal 19
1
IPI00421802
Keratin type I cytoskeletal 40
1
IPI00421791
Keratin type I cytoskeletal 42
1
IPI00393340
Keratin type II cytoskeletal 6A
1
Microtubule based
IPI00213299
Chaperones
IPI00215463
Microtubule associated protein RP EB 1
family member 1
Calmodulin like protein 3
1
IPIOO196751
Heat shock 70 kDa protein 1A IB
1
IPI00870885
Cdk5 and Abl enzyme substrate 1
1
Elongation factor
IPI00471525
Metal binding
IPI00231107
Eukaryotic translation elongation factor 1
delta
Parathymosin
1
IPI00230788.6
Carbonic anhydrase 3
1
Protease
IPI00191748
Proteasome subunit alpha type 1
1
Other
IPI00371266
Nascent polypeptide associated complex
alpha polypeptide Predicted isoform CRA b |
1 Cyto skeleton
1 Actin based
1 Intermediate filaments
1
82
In order to assess whether or not selenium could potentially reverse or prevent the
glycosylation of proteins that might be responsible for the glucosamine induced insulin
resistance, the glycosylation of proteins with selenium treatment was evaluated. The
following table lists the proteins that are found uniquely in glucosamine plus selenium
treatment compared to selenium (Table 6).
Table 6: O-GlcNAc modified proteins found uniquely in glucosamine plus selenium
treatment compared to selenium
Functional Subgroup
Accession
Protein
Cyto skeleton
Actin based
IPI00360356
Actin beta like 2
IPI00454431
Brain specific alpha actinin 1 isoform
IPI00231563
Isoform 3 of Tropomyosin alpha 1 chain
IPI00421782
Keratin 75
IPI00421802
Keratin type I cytoskeletal 40
IPI00421857
Keratin type II cytoskeletal 1
IPI00421788
Keratin type II cytoskeletal 7
IPI00421784.1
Type II keratin Kb24
IPI00558057
Similar to keratin Kb40
Ligase
IPIOO188989
Long chain fatty acid CoA ligase 1
Chaperones
IPI00206660
DnaJ homolog subfamily C member 3
IPI00207355
Heat shock related 70 kDa protein 2
IPI00566672
Similar to heat shock protein 8
IPIOO197770
Aldehyde dehydrogenase mitochondrial
IPI00369579
Isoform 1 of ERO1 like protein alpha
Intermediate filaments
Oxid reductase Process
83
Table 6: Continued
1 Immune response
IPI00551567
MHC class I RT1 Au heavy chain Fragment
1 Initiation factor
IPI00206152
Eukaryotic translation initiation factor 1A
I Protease
IPI00758468
Plasma glutamate carboxypeptidase
IPIOO191748
Proteasome subunit alpha type 1
IPI00369093
Cytochrome b cl complex subunit 6 mitochondrial
IPI00421517
Desmin
IPI00231827
Nucleolin
IPI00361827
Pgm2 protein
IPIOO192140
Protein Niban
IPI00363569
RAN binding protein 1
IPI00734740
Similar to 60S acidic ribosomal protein P2
1
IPI00559713
Similar to Ferritin light chain 2
1
IPIOO189682
Similar to Glycine cleavage system H protein 1
mitochondrial precursor
IPI00362649
Similar to paladin
IPI00368613
Similar to ring finger and KH domain containing 3
IPI00476033
Ubc protein
IPI00207657
Ubiquitin specific peptidase 5
Other
1
1
After comparing the list of O-GlcNAc modified proteins uniquely present in
the glucosamine plus insulin treatment with those uniquely found with glucosamine
84
treatment, we found the following proteins are present in both glucosamine and
glucosamine plus insulin treatment (Table 7).
Table 7: O-GlcNAc modified proteins found in both glucosamine and glucosamine
plus insulin treatment
Functional Subgroup
Accession
Protein
Elongation factor
IPI00471525
Eukaryotic translation elongation factor 1 delta
Metal binding
IPI00231107
Parathymosin
Protease
IPIOO191748
Proteasome subunit alpha type 1
Among the O-GlcNAcylated proteins under glucosamine selenium treatment,
the following two proteins that are present in glucosamine plus insulin treatment are
not present in glucosamine plus selenium treatment (Table 8).
Table 8: O-GlcNAc modified proteins found uniquely in glucosamine plus insulin
treatment but not present in glucosamine plus selenium treatment
Functional Subgroup
Accession
Protein
Elongation factor
IPI00471525
Eukaryotic translation elongation factor 1 delta
Metal binding
IPI00231107
Parathymosin
It is attractive to speculate that this glycosylation reversal facilitates in some
way the regulation of key metabolic enzymes even in an insulin resistant state.
85
However, further investigation is required to show that selenium can decrease or
reverse glucosamine induced O-GlcNAcylation.
Functions of the identified O-GlcNAc modified proteins
Eukaryotic translation elongation factor delta 1 encodes a subunit of the
elongation factor-1 complex, which is responsible for the enzymatic delivery of
aminoacyl tRNA to the ribose. This subunit functions as a guanine nucleotide
exchange factor (Sanders, Brandsma et al. 1996). The next protein parathymosin is
also called 11.5KDa zn 2+ binding protein (ZnBP) or macromolecular translocation
inhibitor II (MTI-II). This protein is almost ubiquitous. It has the highest
concentration in liver, brain, adrenal gland and smooth muscle and the lowest level in
skeletal muscle. It inhibits the glycolytic enzyme phosphofructokinase-lin vitro. This
inhibition is reversible and results from a dissociation of the tetrameric enzyme into
its inactive protomers and is dependent on the presence of zinc (Brand, Heinickel et
al. 1988). Four clusters of acidic amino acid residues responsible for zinc binding and
inactivation of phosphofructokinase (PFK) were identified between positions 35 and
78 of parathymosin. By further proteolytic cleavage the two specific zinc-binding
sites were located to amino acid residues between positions 51 and 72, whereas the
region between positions 35 and 50 is necessary for binding and inactivation of
phosphofructokinase (Brand, Heinickel et al. 1988). Phosphofructokinase is a key
regulator of glycolysis. It is a kinase that phosphorylates fructose-6-phosphate (F6P)
in glycolysis. As mentioned before, one of the sites by which glucose enters into
86
hexosamine pathway is by conversion of F6P to glucosamine-6-phosphate. So,
inhibition of PFK may shift fructose-6-phosphate from the glycolytic pathway to
HBP. So, if parathymosin inhibits PFK it can increase the carbon flux into the HBP.
It has also been shown by parathymosin-Sephrose affinity chromatography
that the interaction of parathymosin with many enzymes of carbohydrate metabolism
is zinc specific. From liver cytosol the following enzymes were retained:
hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, phosphofructokinase-1,
aldolase, glycerol-3-phosphate dehydrogenase, glyceral-3-phosphate dehydrogenase,
fructose-1, 6-bisphosphatase, and the phosphorylated form of pyruvate kinase L
(Brand, Heinickel et al. 1991). So it is possible that parathymosin, due to interaction
with enzymes at the branching points of carbohydrate metabolism, could shift the
glycolytic flux in the directions to other metabolic pathways.
However, more experiments are required to support this idea. These include
demonstration of parathymosin containing complexes in intact cells, study of kinetic
effects of complex formation on defined metabolic fluxes.
No reports exist on potential phosphorylation or any other posttranslational
modifications of parathymosin except the acetylation of its N terminus (Brand,
Heinickel et al. 1991). So we have shown for the first time the glycosylation of
parathymosin. So our result shows that parathymosin is glycosylated under
glucosamine treatment as well as glucosamine plus insulin treatment but not in
control. But glycosylated parathymosin is not present in glucosamine plus selenium
87
treatment which again attracts us to speculate that this glycosylation reversal
facilitates in some way the regulation of PFK.
It is also shown to enhance glucocorticoid dependent transcription via a direct
interaction with the glucocorticoid receptor in vivo. But so far there is no literature
report on influence of parathymosin on glucocorticoid regulation of metabolic genes.
Without glucocorticoid hormone, the glucocorticoid receptor (GR), a member of the
nuclear receptor family, is localized in the cytosol through its association with a
variety of heat shock/chaperone proteins like heat shock protein 90 (hsp90), heat
shock protein 60 and protein FKBP52. These chaperone proteins were not identified
in our study. Upon hormone binding, GR dissociates from the chaperone proteins and
translocates from the cytosol to the nucleus. In the nucleus, the ligand-bound GR
binds to specific DNA sequences termed glucocorticoid response elements (Elchebly,
Payette et al. 2000), where it recruits a coactivator complex containing the pi60
steroid receptor coactivator (SRC), acetyltransferases (CBP, p300, and p/CAF), and
methyltransferases (CARM1 and PRMT1). In association with this coactivator
complex, GR directly activates gene transcription (Okamoto and Isohashi 2000). The
next protein is proteasome subunit alpha type 1.There is not much information on the
function of proteasome subunit alpha type 1. No reports exist on potential
glycosylation modification of these proteins. So, we have shown here for the first
time the glycosylation of the proteasome subunit alpha type 1
88
CHAPTER 4
DISCUSSION
The discovery of insulin by Banting and colleagues in the early 1920s, stands
as one of the greatest scientific achievements of the 20th century. Few scientific
endeavors have had such scientific impact. Insulin was first isolated in 1921, by
Banting, Best, Collip and Macleod following the observations of Von Mering and
Minkowski that removal of the pancreas made dogs severely diabetic (Banting, Best
et al. 1991). Banting and Best made a pancreatic extract that reduced glucose levels
when injected into these diabetic dogs. Since then, insulin administration has become
a common treatment for both Type 1 and Type 2 diabetes. Prior to this discovery the
prospects for patients afflicted with diabetes were grim. It is one of the most
decorated molecules in biology and the work on insulin (directly or indirectly) has
yielded six Nobel prizes. Even though 90 years have passed since the discovery of
insulin, the molecular actions of insulin have only begun to be understood over the
past 25 years with the cloning of the insulin receptor, the start of transgenic
technology and the development of phosphotyrosine-specific antibodies.
89
The primary metabolic action of insulin is to facilitate the postprandial
disposition of glucose via its actions on three key target tissues, suppression of
glucose output from the liver and stimulation of glucose uptake and metabolism in
skeletal muscle and adipose tissue. Defects in insulin secretion and insulin action on
its target tissues manifest clinically as diabetes mellitus. Diabetic Mellitus is
classified primarily as either Type 1 or Type 2. Type 1 diabetes, comprises only 10%
of those affected by the disease and results from autoimmune destruction of the Pcells of the pancreas. The remaining 90% of patients, classified as type 2, are affected
by a complex disorder characterized by insulin resistance.
Insulin resistance is a condition in which normal insulin secretion from the
pancreas is insufficient to induce a biological response in the peripheral tissues (liver,
muscle, adipose). In the early stage of the disease progression, the pancreas enhances
insulin secretion to maintain normal blood glucose levels; however at later stages
pancreatic function becomes impaired, which leads to hyperglycemia. Sustained
hyperglycemia has been shown to induce insulin resistance and this has been
suggested to be an adaptive mechanism in protecting cells from the oxidative stress
that results from excess nutrients (Buse 2006). How hyperglycemia mediates insulin
resistance at the cellular level remained unanswered for many years. One of the
mechanisms that appear to be involved in the adverse effects of hyperglycemia is the
O-GlcNAc modification of intracellular proteins on serine and threonine residues. OGlcNAc modification of proteins are dependent on the concentration of UDP-GlcNAc
90
produced by the HBP, which itself depends on the concentration of glucose in the
cell. An increase in glucose uptake is thought to lead to increased flux through the
HBP, resulting in increased UDP-GlcNAc levels which directly cause an increase in
protein O-GlcNAcylation.
O-GlcNAc was discovered in 1983, when purified bovine milk
galactosyltransferase and its radiolabeled donor substrate (UDP-[3H] galactose) were
used to probe for GlcNAc-terminating glycoconjugates in living murine thymocytes,
splenic B- and T-lymphocytes, and macrophages (Torres and Hart 1984). As
mentioned before, enzymes catalyzing O-GlcNAc addition are referred to as OGlcNAc transferases (OGT) and enzymes that remove O-GlcNAc are referred to as
O-GlcNAcases. Both are found in the cytosol and nucleus.
O-GlcNAcylated proteins have been identified from all cellular compartments
representing nearly all functional class of proteins. O-GlcNAc modified proteins are
found in the nuclear pore complex (Holt and Hart 1986) and on many cytoplasmic,
mitochondrial and membrane associated proteins (Love, Kochan et al. 2003). These
proteins belong to functional groups like transcription factors (c-myc) (Chou, Hart et
al. 1995), polymerases (RNA Pol II) (Kelly, Dahmus et al. 1993), chaperones (Heat
shock protein 70 (HSP70) (Walgren, Vincent et al. 2003), Heat shock protein 27
(HSP 27) (Roquemore, Dell et al. 1992), cytoskeletal proteins (Synapsin) (Cole and
Hart 1999), phosphatases (Nuclear tyrosine phosphatases p65) (Meikrantz, Smith et
91
al. 1991) and kinases and adapter proteins (casein kinase II) (Lubas and Hanover
2000); (Insulin receptor substrate 1 and 2) (Vosseller, Wells et al. 2002).
This dynamic addition of O-GlcNAc to proteins has been involved in
modulating protein behavior via different mechanisms. For example, in vitro studies
on RNA polymerase II (RNA pol II) showed that reciprocal glycosylation and
phosphorylation of RNA Pol II may regulate transcription to prevent premature
transcriptional initiation (Comer and Hart 2001). Similarly, the reciprocal
modification by phosphorylation or O-GlcNAcylation at Ser 16 on the estrogen
receptor beta modifies the receptor's behavior by altering the rate of degradation.
When the estrogen receptor is phosphorylated, the protein is rapidly targeted for
degradation, while the O-GlcNAcylated form is degraded much more slowly (Cheng
and Hart 2001). Modification of the proteasome in vitro by O-GlcNAc reduces
degradation by inhibition of the ATPase activity (Zhang, Su et al. 2003). O-GlcNAc
can also modulate protein-protein interaction, for example, O-GlcNAc modification
of a zinc finger DNA-binding transcription factor, YY1 causes its dissociation from
retinoblastoma protein (pRb) allowing it to bind DNA(Hiromura, Choi et al. 2003).
The major work so far to test the hypothesis, that increased flux through the
hexosamine biosynthetic pathway can induce insulin resistance has been carried out
in adipocytes, muscles and pancreatic P cells and less attention has been focused on
liver. As mentioned in the introduction, the liver plays an important role in
coordinating the whole body metabolism, including carbohydrate, lipid and protein
92
metabolism and detoxification. The various functions of the liver are carried out by
the liver cells or hepatocytes. Some of the major metabolic functions of the liver
include gluconeogenesis, glycogenolysis, glycogenesis, lipogenesis (Leclercq, Da
Silva Morais et al. 2007). Hepatic insulin resistance is due to impaired suppression of
hepatic glucose production, which accounts for hyperglycemia and glucose
intolerance (Reaven 1995). Failure of insulin to inhibit hepatic gluconeogenesis and
glycogenolysis, is responsible for the development of fasting hyperglycemia and
persistent stimulation of insulin production by pancreatic P-cells. For example, mice
lacking the insulin receptor (IR) in hepatocytes exhibit insulin resistance, severe
glucose intolerance and failure of insulin to regulate hepatic gene expression and to
suppress hepatic glucose output (Michael, Kulkarni et al. 2000). In contrast, normal
glucose and insulin levels are found in mice with a deletion of IR in skeletal muscle
(Bruning, Michael et al. 1998). Deletion of the IR in the adipose tissue was shown to
be associated with low insulin levels suggesting improved insulin sensitivity (Kim,
Michael et al. 2000; Bluher, Michael et al. 2002). When the IR is simultaneously
knocked down in fat and muscle, there is no change in insulin or glucose levels. Thus,
hepatic insulin resistance, but not peripheral insulin resistance, is necessary to
develop hyperglycemia and glucose intolerance.
McClain was the first to examine the effect of increased hexosamine flux in
liver. They overexpressed GFAT in transgenic mice using the PEPCK promoter.
93
They observed that older transgenic mice gained more weight compared to the control
mice, and also developed glucose intolerance with a significant decrease in glucose
disposal rates (Veerababu, Tang et al. 2000).
In our study, we have used glucosamine to induce insulin resistance in
primary rat hepatocytes. Glucosamine enters the hexosamine biosynthetic pathway
downstream of GFAT, bypassing the rate limiting step. It has been used to evaluate
the role of hexosamine flux in the development of insulin resistance. For example, in
vitro studies carried out in rat skeletal muscle indicates that glucosamine induces
insulin resistance and may modulate insulin and glucose effects on pyruvate kinase
and glycogen synthase (Traxinger and Marshall 1992; Robinson, Sens et al. 1993).
Similarly, infusion of glucosamine in Wistar rats induces insulin resistance of skeletal
muscle through decreased phosphorylation of insulin receptor substrate-1 (IRS-1) and
its association with PI3-Kinase and inturn results in impaired translocation of GLUT4
to the plasma membrane (Baron, Zhu et al. 1995). In a similar experiment in rat
adipocytes incubated in 5mM glucosamine for 4h the activity of GLUT4 is decreased
and longer incubation (16h) leads to a decrease in the amount of GLUT4 in the
plasma membrane (Chen, Ing et al. 1997). In a recent study, incubation of HEK293
cells with 25mM glucose for 24hr or 5mM glucosamine for 2hr resulted in increase in
FoxOl O-GlcNAc modification which in turn increased the expression of G6pase
gene (Kuo, Zilberfarb et al. 2008).
94
The major pitfall to earlier experiments using glucosamine was the use of very
high concentrations (5mM-50mM) of glucosamine that could obscure its main action.
For example studies carried out by Mueckler et al concluded that glucosamineinduced insulin resistance in 3T3-L1 cells is caused by depletion of intracellular ATP
(Hresko, Heimberg et al. 1998). Subsequent investigators such as those described by
Gulve et al have shown that this conclusion was derived from a toxic adverse effect
of glucosamine that could be minimized by the judicious use of glucosamine (Ross,
Chen et al. 2000). In our study, we utilized ImM glucosamine and evaluated whether
or not there was any effect of glucosamine on cell viability or cytotoxicity using the
LDH assay. Our results show that there is no significant difference in the level of
LDH in glucosamine treated cells versus control, which suggests the cells are not
compromised by glucosamine treatment.
One of the molecular mechanisms responsible for the insulin resistant
condition in type 2 diabetes is alteration at one or more levels of the insulin-signaling
cascade in the insulin sensitive tissues: skeletal muscles, adipocytes, and liver. The
protein kinase Akt, downstream to PI3-kinase, is an important mediator of insulin
signaling in the regulation of cell survival and metabolism (Chang, Chiang et al.
2004; Zdychova and Komers 2005). Several studies have demonstrated linkage
between the HBP, the development of insulin resistance, and altered Akt signaling.
For example, treatment of 3T3-L1 adipocytes with PUGNAc (an inhibitor of OGlcNAcase) reduces Akt phosphorylation, resulting in reduction of insulin stimulated
95
2-deoxyglucose uptake (Vosseller, Wells et al. 2002; Park, Ryu et al. 2005).
However, the involvement of Akt in insulin resistance from O-GlcNAc modification
is controversial. In a study carried out by Patti et al, it was observed that glucosamine
treatment in rat skeletal muscle caused reduced tyrosine phosphorylation of IRS-1
and 2 but attenuation of insulin stimulated phosphorylation of Akt was not observed
(Patti, Virkamaki et al. 1999). Similarly, the elevated O-GlcNAc modification by
PUGNAc induced insulin resistance in skeletal muscle was found to be independent
of attenuated phosphorylation of Akt and GSK3a/p (Kim, Zhu et al. 1999; Arias, Kim
et al. 2004). These contradictory results among insulin sensitive tissues led us to
further investigate the effect of glucosamine on the insulin induced phosphorylation
of Akt in primary rat hepatocytes. Hence, this was the first target for the
investigation.
In our study we found that the insulin stimulated phosphorylation of Akt (Ser
473) was drastically decreased by 4 fold in the presence of glucosamine when
compared to the phosphorylation of Akt with insulin which is in agreement with the
study carried out by Vosseller et al, which reported that PUGNAc decreased insulin
stimulated activation of Akt in 3T3-L1 adipocytes. In our study the treatment of
glucosamine caused drastic reductions in insulin stimulated phosphorylation of Akt,
clearly suggesting that one of the mechanisms through which glucosamine interferes
with insulin signaling is through inhibiting insulin stimulated Akt phosphorylation at
96
Ser-307 in primary rat hepatocytes. It is possible that different cells or tissues respond
differently under various experimental conditions, explaining the controversial
results. Difference in HBP induced changes in Akt phosphorylation or activity could
be attributed to changes in the insulin signaling upstream of Akt. For example, a
study in rat insulinoma P-cells has shown that HBP flux can impair insulin-induced
tyrosine phosphorylation of IRS-1, PI 3-kinase activity, and subsequent Akt activation
demonstrating that impaired upstream signaling may lead to impaired Akt activation
(Andreozzi, DAlessandris et al. 2004). In contrast to the above study, McClain
showed an increase in Akt signaling resulting from overexpression of O-GlcNAcase;
however they did not observe an increase in PI 3-kinase activity (Soesanto, Luo et al.
2008).
Selenium, an essential trace element, has been shown to mimic some of
insulin's actions both in vivo and in vitro. For example, incubation of rat adipocytes
with selenium stimulated glucose transport activity, due to the translocation of two
types of glucose transporters (GLUT1 and GLUT2) to the membrane surface (Ezaki
1990). In addition to glucose uptake, selenium has been shown to regulate the activity
and expression of various enzymes involved in the processes of glycolysis,
gluconeogenesis, fatty acid synthesis and the pentose phosphate pathway. Oral
administration of selenate to diabetic animals partly reversed abnormal liver
expression of both glycogenic and gluconeogenic enzymes (Becker, Reul et al. 1996).
Regulation by insulin-mimetics of the expression of lipogenic enzymes was also
97
found to be similar to insulin. Treatment of the diabetic animals or rat hepatocytes in
culture with selenate restored the expression of both FAS and G6PDH, demonstrating
that selenate was capable of stimulating lipogenesis in the liver (Berg 1995; Ghosh
1994). The literature indicates that like insulin, selenium treatment also triggers a
variety of phosphoproteins of insulin signaling pathway. For example, treatment of
NIH3T3 HIR 3.5 cells with selenate was found to stimulate phosphorylation of the
insulin receptor (Pillay and Makgoba 1992). In a later study, incubation of either
primary rat hepatocytes or 3T3-LI adipocytes in culture with selenate caused not only
a concentration and time dependent increase in phosphorylation of the P subunit of
the insulin receptor but also IRS-1 as determined through immunoprecipitation
studies (Stapleton, Garlock et al. 1997).
To determine whether selenium can act as an antidiabetic agent in this model
system, we first investigated the effect of selenium on Akt phosphorylation.
Previously we had shown selenium caused increases in phosphorylation of signal
proteins early in the PI3 kinase cascade (Stapleton, Garlock et al. 1997). If selenium
truly acts as a mimetic, then an increase in phosphorylation of proteins downstream of
PI3 kinase such as Akt with selenium should also occur. Our results show that
selenium treatment increased the phosphorylation of Akt by 13 fold as compared to
the basal level in control. Unlike what we observed with the quenching effect of
glucosamine on the insulin induced phosphorylation of Akt, selenium induced
phosphorylation of Akt is unaffected in the presence of glucosamine. This suggests
98
that selenium can act as a potent insulin - mimetic not only under normal/ control
conditions but also under insulin resistant condition as well. Selenium was first
shown to stimulate PI3 kinase activity in 3T3-L1 adipocytes by Heart and Sung. They
reported that selenium stimulates Akt phosphorylation and this selenium action is
downstream of PI3 kinase as in insulin signaling pathways. They showed this by
using the PI3 kinase inhibitor Wortmanin which completely abolished Akt
phosphorylation by selenium (Heart and Sung 2003). Further studies carried out in
non-obese diabetic mice (NOD), showed that expression level of Akt and phosphoAkt significantly increased in selenium treated diabetic NOD mice (Hwang, Seo et al.
2007).
Increase in the flux through HBP has been shown to cause an increase in OGlcNAc modified proteins (Vosseller, Wells et al. 2002; DAlessandris, Andreozzi et
al. 2004; Park, Ryu et al. 2005; Akimoto, Hart et al. 2007). Studies from various labs
suggest that numerous components of the insulin signaling pathway that are involved
in glucose metabolism are O-GlcNAcylated. For example, studies carried out by
Gandy et al showed that Aktl was modified by O-GlcNAc. In this study it was
observed that treatment of neuroblastoma cells with 0-(2-acetamido-2-deoxy-dglucopyranosylidene) amino-iV-phenylcarbamate (PUGNAc), which inhibits the
enzymatic removal of O-GlcNAc from proteins, increased cytosolic O-GlcNAc Aktl
levels (Gandy, Rountree et al. 2006). A similar study carried out in rat primary
99
adipocytes showed that treatment with PUGNAc increased O-GlcNAc modification
of IRS-1 and Akt2, accompanied by a partial reduction of insulin stimulated
phosphorylation of IRSl and Akt2. PUGNAc also decreased insulin stimulated 2deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes (Park, Ryu et al.
2005). Other signaling proteins that have been shown to be O-GlcNAcylated are:
Glutl(Buse, Robinson et al. 2002), casein-kinase II, glycogen synthase kinase 3
(Lubas and Hanover 2000) and IRSl/2(Patti, Virkamaki et al. 1999). We found that
glucosamine and glucosamine plus insulin treatment showed an increase in proteins
that are O-GlcNAcylated compared to control by immunoblotting with anti OGlcNAc antibody. In a recent report, Yang et al. demonstrates that upon insulin
stimulation OGT can be translocated to the plasma membrane through interaction
with PI(3,4,5)P3, demonstrating a potential mechanism by which Akt can be OGlcNAcylated (Yang 2008). We were unable however to demonstrate anti O-GlcNAc
antibody cross reactivity with immunoprecipitated Akt (data not shown). However,
this does not exclude the possibility that it can be modified by O-GlcNAc. The
stochiometry of the modification might have been below the limit of detection by the
specific antibody chosen for identifying O-GlcNAcylation, 110.6. Since we found an
increase in proteins that are O-GlcNAcylated under glucosamine and glucosamine
plus insulin treatment, we speculated that this modification of key proteins might be
linked to the induction of insulin resistance. In order to verify that hypothesis, we
attempted to identify the O-GlcNAc modified proteins. For this, O-GlcNAcylated
100
proteins were isolated and purified using wheat germ agglutinin column. Purified
proteins were verified using SDS PAGE and samples were submitted to the Mass
Spectrometry and Proteomics Lab at Van Andel research institute, Grand Rapids for
LC-MS analysis. Our attempt to receive quantitative analysis between control and
experimental group was unsuccessful. However, this experiment generated a list of
O-GlcNAc modified proteins. We generated a list of proteins that are uniquely found
in the glucosamine treated cells compared to control, untreated cells. Surprisingly,
analysis of the proteins that were glycosylated under the glucosamine conditions did
not reveal any modification to signaling proteins or metabolic enzymes. Most of the
proteins that were identified are chaperones and cytoskeletal proteins. Selenium as
well as insulin alone also causes unique glycosylation events. It was attractive to
speculate that proteins glycosylated under insulin plus glucosamine are reversed with
selenium treatment. We observed in the glucosamine plus insulin treatment the
following uniquely identified proteins, eukaryotic translation elongation factor 1
delta, parathymosin and proteasome subunit alpha type 1. Among these three OGlcNAcylated proteins, eukaryotic translation elongation factor 1 delta and
parathymosin were the two proteins that are not glycosylated under glucosamine
selenium treatment. Given observation it is attractive to speculate that selenium might
be reversing the glycosylation event allowing regulation of metabolism to occur.
However, further quantitative studies are required to validate our observation.
101
Insulin, for example regulates many cellular processes and the expression of
several genes through the activation of various signaling proteins and pathways. If the
activation of the signal proteins is affected, then the effect on gene expression should
also be altered. So, we examined the effect of glucosamine on the insulin regulated
expression of key metabolic enzymes of pentose phosphate pathway, fatty acid
biosynthesis and gluconeogenesis.
G6PDH is a lipogenic enzyme that catalyses the first reaction in the pentose
phosphate pathway. The pentose phosphate pathway is an anabolic pathway that
utilizes excess glucose present in the liver. The primary function of this pathway is to
generate reducing equivalents in the form of NADPH, which is necessary for
biosynthetic reactions including fatty acid metabolism, and to produce ribose-5phosphate (R5P), which is necessary for the synthesis of nucleotides, the building
blocks of nucleic acids. Thus G6PDH is important in both carbohydrate and fatty acid
metabolism. The G6PDH gene has been considered to be a "housekeeping" gene, but
several studies have shown its expression to be highly regulated. The expression of
G6PDH has been shown to be increased by insulin in primary rat hepatocytes (Kurtz
and Wells 1981; Stapleton, Stevens et al. 1993) and by oxidative stress in cells in
culture (Ursini, Parrella et al. 1997; Xu, Maki et al. 2003).
Our result shows that under glucosamine treatment, insulin stimulated gene
expression of G6PDH was decreased significantly compared to insulin stimulated
102
G6PDH gene expression in control, suggesting that insulin is no longer able to fully
regulate G6PDH expression.
Since the pentose phosphate pathway is responsible for most of the NADPH
production, and NADPH is needed by fatty acid biosynthesis (Mourrieras, Foufelle et
al. 1997), it stands to reason that the decrease in the insulin induced gene expression
of G6PDH under glucosamine treatment might lead to a decrease in fatty acid
synthesis. If insulin sensitivity is truly impacted then insulin induced expression of
FAS should be altered. Our results show that insulin significantly increased the gene
expression of FAS by 1.6 fold when compared to control, which is comparable to
published reports (Paulauskis and Sul 1988; Katsurada, Iritani et al. 1990) On the
other hand, insulin stimulated expression of FAS under glucosamine treatment
decreased significantly compared to insulin stimulated FAS gene expression in
control. This indicates that glucosamine inhibits insulin stimulated FAS gene
expression. In contrast to our studies, Marshall studies in primary rat adipocytes
reported that in insulin treated primary rat adipocytes, glucosamine unregulated FAS
mRNA levels (Rumberger, Wu et al. 2003).
As mentioned before, like insulin selenium can affect the gene expression of
lipogenic enzymes, G6PDH and FAS in streptozotocin induced type 1 diabetic model
(Berg, Wu et al. 1995). In the presence of glucosamine we investigated the effect of
selenium on the gene expression of G6PDH and FAS. Our results show that selenium
induces the gene expression of G6PDH by approximate 1.5 fold and FAS by 1.3 fold
103
at 6hr respectively. Unlike our findings with insulin in the presence of glucosamine,
selenium induction of G6PDH and FAS was similar in the presence or absence of
glucosamine. This result is comparable to our previous study in which it was found
that administration of selenate or vanadate to streptozotocin- induced diabetic rats
restored the expression of FAS and G6PDH (Berg, Wu et al. 1995).
Hepatic glucose output is mainly from gluconeogenesis (Nordlie, Foster et al.
1999) and plays an important role in diabetes. As mentioned before, hepatic insulin
resistance is due to impaired suppression of hepatic glucose production (Reaven
1995). One of the key rate limiting enzymes of gluconeogenesis is PEPCK. The
control of hepatic gluconeogenesis is regulated, in large part, by altering the rate of
transcription of the PEPCK gene (Pilkis and Gramier 1992). Glucagon and
glucocorticoids induce transcription of the PEPCK gene and insulin inhibits its
induction (Sutherland, O'Brien et al. 1996). To address the importance of insulin
signaling in the control of gluconeogenesis in vivo, liver specific disruption of the
insulin receptor was performed in mice (LIRKO mice). Disruption of insulin action in
the liver of LIRKO mice lead to severe glucose intolerance, resistance to the blood
glucose lowering effect of insulin, and non inhibited hepatic glucose production due
to elevated liver G6Pase and PEPCK expression(Michael, Kulkarni et al. 2000). To
study the role of the expression of PEPCK gene in the development of type 2 diabetes
many transgenic mice expressing a PEPCK gene have been generated and studied.
For example, studies carried out by Valera et al, showed that overexpression of the
104
PEPCK gene leads to alterations in liver glycogen content and muscle glucose
transporter GLUT-4 gene expression and increased glucose production in hepatocytes
in primary culture. When intraperitoneal glucose tolerance tests were performed,
blood glucose levels were higher than those detected in normal mice. This study
clearly showed that primary alterations in the rate of liver glucose production may
induce insulin resistance and type 2 diabetes (Valera, Pujol et al. 1994).
We investigated the effect of glucosamine on the insulin regulation of this key
enzyme of gluconeogenesis. We used cAMP to induce the gene expression of
PEPCK. Glucagon increases PEPCK gene expression through the second messenger
cAMP (Lamers, Hanson et al. 1982). Upon glucagon binding to the glucagon
receptor, cAMP stimulates a series of reactions that leads to mobilization of glucose
from the liver: overall decreases in glycolysis and glycogen synthesis, and a increases
in glycogenolysis and gluconeogenesis. Our results show that the addition of cAMP
for 1 hr in the control group caused an increase in PEPCK gene expression by 27
fold, which was comparable to previous studies (Beale, Andreone et al. 1984; Imai,
Miner et al. 1993). Following treatment of insulin after cAMP a 25% decrease was
observed in the cAMP induced gene expression. In the glucosamine treated cells, the
inhibition of cAMP induced PEPCK expression by insulin was not observed, which
suggests that expression of PEPCK is resistant to down regulation by insulin.
It was recently demonstrated that adenoviral-mediated overexpression of OGT
in mouse liver attenuated the ability of insulin to suppress hepatic glucose production.
105
This study demonstrated a mechanism by which upon hormone stimulation OGT is
recruited to the plasma membrane in the vicinity of the proteins involved in early
steps of insulin signaling. In the basal state, OGT is localized to the nucleus and
cytoplasm (Kreppel, Blomberg et al. 1997). Upon insulin stimulation phosphatidylinositol 3-phosphate (PIP3) production by PI-3 kinase induces the recruitment of
OGT to the plasma membrane, causing GlcNAcylation of the insulin receptor and
insulin receptor substrate. It was observed that Akt phosphorylation and Akt activity
was decreased resulting in upregulation of the expression of gluconeogenic enzymes
PEPCK and glucose -6-phosphatase (G6pase). In the same paper it was reported that
the exposure of 3T3-L1 adipocytes to high glucose concentration or PUGNAc
decreases the phosphorylation of Akt at threonine 308. In contrast, the
phosphorylation level of the MAPK pathway, particularly the Erkl/2 kinases, is
unaffected. The phosphorylation of IRSl is also modified under these conditions,
since the treatment of adipocytes with PUGNAc or the adenovirus mediated
transfection of adipocytes and Fao hepatoma cells with Ad-OGT increases the
phosphorylation of IRSl at Ser307 and Ser632/635, three sites known to downregulate the insulin signaling pathway. Taken as a whole, this paper demonstrates that
OGT participates in the termination of the insulin signaling pathway by interacting
with PIP3 at the plasma membrane. OGT then O-GlcNAcylates numerous
components of the insulin signaling pathway, lowering the response to insulin; this
106
supports the role of OGT in the physiopathology of insulin resistance (Whelan, Lane
et al. 2008; Yang, Ongusaha et al. 2008).
Further validating the importance of O-GlcNAc on hepatic insulin signaling,
recent studies focusing on the transcription factors FoxOl and CRTC2/TORC2 have
shown that O-GlcNAcylation also leads to insulin resistance and glucose toxicity
through the control of gluconeogenesis (Dentin, Hedrick et al. 2008; Housley,
Rodgers et al. 2008; Kuo, Zilberfarb et al. 2008). FoxOl is responsible for the
expression of two enzymes of gluconeogenesis, G6pase and PEPCK. Recent data
demonstrated that FoxOl is O-GlcNAcylated in liver cells, resulting in an increase in
its transcriptional activity and in turn increasing the transcriptional activity of target
genes including PEPCK and G6pase.
In yet another report, it has been shown that the transcriptional co-activator
PGC-la, which is itself O-GlcNAcylated, regulates FoxOl O-GlcNAcylation, and
consequently its transcriptional activity, by recruiting and targeting OGT to FoxOl
(Housley, Udeshi et al. 2009). Interestingly, both glucose-6-phosphatase and PEPCK
are transcriptionally regulated by CRTC2, which is also controlled by
OGlcNAcylation unlike FoxOl, regulation occur through nuclear re-location since
the two major OGlcNAcylated sites that have been mapped sequester CRTC2 in the
cytoplasm by a phosphorylation-dependent mechanism. Under normal conditions,
CRTC2 is phosphorylated at Ser 70 and Ser 171 in the cytoplasm by 14-3-3- protein.
Upon high glucose treatment, CRTC2 becomes GlcNAcylated at Ser 70 and Ser 171,
107
allowing nuclear translocation and transcription of gluconeogenic genes in
hepatocytes (Dentin, Hedrick et al. 2008).
In the last several decades, most studies regarding the mechanism of hormone
action on PEPCK gene expression have focused on mapping out homione response
units (HRU) on this gene promoter. Studies in this field began to focus on the
identification of coregulators of the hormone-activated PEPCK gene promoter.
Afterward, the functional importance of certain transcription factors and coregulators
on mediating PEPCK gene expression were studied through the expression of
dominant negative forms of these proteins (Wang, Stafford et al. 2000).
In the early 1990s, studies from the Granner lab reported that insulin represses
glucocorticoid and cAMP-induced PEPCK gene transcription through a DNA
element located between -416bp and -402bp of this gene promoter, called the insulin
response sequence (IRS)(0'Brien, Lucas et al. 1990). Their group further
demonstrated that transcription factor Forkhead box a2 (Foxa2), also called hepatic
nuclear factor 1 beta (HNF3P) and CCAAT/enhancer-binding protein p (C/EBPP),
can associate with the IRS of the PEPCK gene promoter in vitro. However, only
Foxa2 is functionally required for glucocorticoid induced PEPCK gene expression
(O'Brien, Noisin et al. 1995).
O'Brien et al found that the insulin-like growth factor-binding protein
l(IGFBP-l) gene can be stimulated by glucocorticoids and inhibited by insulin.
108
Analyses of this promoter by transfection of IGFBP-1 chloramphenicol
acetyltransferase fusion genes into rat hepatoma cells has led to the identification of
insulin response sequences (IRSs) in this gene (O'Brien, Noisin et al. 1995). By
comparison, the IRS, T (G/A) TTTTG, is the same both in PEPCK and IGFBP-1
genes. The IGFBP-1 IRS and PEPCK IRS both bind the alpha and beta forms of
HNF-3p also called Foxal and Foxa2. Mutational analyses of the PEPCK and
IGFBP-1 IRSs revealed mutations that do not affect the binding of HNF3 proteins to
these elements but do eliminate the ability of the IRSs to mediate an insulin response.
These data led O'Brien et al to postulate that insulin mediates its negative effect on
glucocorticoid-induced PEPCK and IGFBP-1 gene transcription indirectly by
inhibiting HNF-3 binding (Foxa2) (O'Brien, Noisin et al. 1995). Later Wang et al
found that expression of a dominant negative Foxa2, in which the transactivation
domain is deleted, reduced glucocorticoid-mediated PEPCK gene expression and
hepatic gluconeogenesis (Wang, Stafford et al. 2000). Studies from the Stoffel group
showed that Foxa2 (HNF3P) physically interacts with Akt. Foxa2 is phosphorylated
at a single conserved site (T156). The insulin induced phosphorylation of Foxa2
through Akt mediates the translocation of this protein from nucleus to cytosol
(Wolfrum, Besser et al. 2003).
Our next objective was to examine whether or not insulin mediated reduced
expression of PEPCK could attribute to reduced expression level of Foxa2 and if so
what effect it would have. Our results show that the addition of 500uM of cAMP for
109
lhr in the control group caused a 2.3 fold increase in Foxa2 gene expression which is
in accord with studies carried out by Imae et al where they analyzed the effect of
insulin, dexamethasone and protein malnutrition on the hepatic mRNA levels of
HNF3a, HNF3P and HNF3y. They found that daily administration of dexamethasone
to Wistar rats caused the mRNA levels of HNF3a and HNF3p to increase (Imae,
Inoue et al. 2000). Following treatment of insulin after cAMP an approximately 40%
decrease in Foxa2 expression was observed. Glucosamine had no effect on Foxa2
expression or alter the effect of cAMP. With glucosamine treatment, there was no
significant difference between cAMP induced Foxa2 gene expression with or without
insulin. This result shows that under glucosamine treatment insulin no longer is able
to inhibit the induction of cAMP induced gene expression of Foxa2. Our results so far
indicate that under glucosamine treatment, all the key enzymes studied are
unresponsive to insulin.
The first animal study to investigate the antidiabetic mechanisms of selenate
was shown in type 2 diabetic db/db mice. They reported that selenate administration
to the db/db mice led to a marked downregulation of the gluconeogenic enzyme
PEPCK in comparison to selenium deficient mice (Mueller and Pallauf 2006). We
investigated the effect of selenium on the expression of PEPCK in our model system.
It was found that addition of cAMP for 1 hr in control group caused an increase in
PEPCK gene expression by 27 fold. Selenium treatment decreased cAMP induced
gene expression of PEPCK significantly and expression was near basal level by 6hr.
110
Again, like with FAS and G6PDH, selenium had similar effects on the expression of
PEPCK in the presence of glucosamine suggesting its actions are not sensitive to
glucosamine. We further found that selenium treatment under the insulin resistant
state could inhibit the induction of cAMP induced gene expression of Foxa2. Again,
as with FAS, G6PDH, PEPCK, selenium had similar effects on the expression of
Foxa2 in the presence of glucosamine suggesting its actions are not sensitive to
glucosamine.
In summary, we have established an insulin resistance model in primary rat
hepatocytes by using glucosamine, a precursor of HBP. We speculate this could be
due to increased O-GlcNAc modified proteins that were observed under glucosamine
compared to control however, the proteins we observed to be O-GlcNAcylated were
not as predicted. Another interesting observation of this study is that selenium is able
to act as an insulin-mimetic not only under the control conditions but also under
insulin resistant condition as well. We also observed glycosylation of proteins under
selenium treatment. However, further studies are necessary to determine if selenium's
actions are indeed mediated by altering the glycosylation state of the protein. Our
work is only the beginning of an intricate and interesting story. Further studies are
crucial in order decipher what other players are involved in glucosamine induced
insulin resistance.
Ill
APPENDIX
IACUC approval form for animal studies
WESTERN MICHIGAN UNIVERSITY
hstiiutiaiidl Animal Care antJ Use ComnuUoB
Dale
March 30, 2010
To
Susan Stapleton, Principal Investi/a/or/,
From Robert Evcrsole, ('hair
Re
IACUC Protocol No 10-02-01
Your piolocol tilled "Regulation of Gene Expression in Hepatoc) les"' ha.s received
npprovnl irora the Institutional Ajnmal Tare and Vw Committee. The conditions and
duration of this approved aie specified in the Policies of Western Michigan Umvcrail)
You may now begin to implement the research as described in the application
The Board wishes you success in the pursuit of youi research goals
Appi ovi«l Termination
Match 30,2011
Wa «od Hall Mim««» W mj% 54W
112
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