Stimulation of Glucose Transport: Potential Role of Aktlffrotein Kinase Ba Elena Bogdanovic A thesis submitted in conformity with the requirements for the degree of Master of Science, Graduate Department of Physiology, University of Toronto O Copyright by Elena Bogdanovic (2000)
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Stimulation of Glucose Transport: Potential Role of Aktlffrotein Kinase Ba
Elena Bogdanovic
A thesis submitted in conformity with the requirements for the degree of Master of Science,
Graduate Department of Physiology, University of Toronto
O Copyright by Elena Bogdanovic (2000)
National Library 191 of Canada Bibliothbque nationale du Canada
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Stimulation of Glucose Transport: Potential Role of Aktl/Protein Kinase B a
A thesis by Elena Bogdanovic submitted in conformity with the requirements for the degree of Master of Science, Graduate Department of Physiology, University of
Toronto, January 2000.
ABSTRACT
Aktlffrotein Kinase Bu (AktUPKBa) has been shown to lie downstream of
phosphatidylinositol 3-kinase (PI3K) in the insulin action pathway and has been
implicated in the stimulation of glucose transport and Glut4 translocation by insulin. To
obtain further insight into this role of Akt l/PKBa, two models were used.
Vanadate and pewanadate (pV) are protein tyrosine phosphatase inhibitors that
can stimuiate glucose uptake and Glut4 translocation in L6 myotubes independently of
PI3K. The present study showed that while insutin and pV increase AktlPKBu activity
in a PI3K dependent manner, vanadate did not. These data suggest that Aktl/PKBu is
important for insulin but not for vanadate or pV stirnulated glucose uptake.
The second mode1 exarnined Akt l/PKBcr activation in insuIin resistant rat
adipocytes. Rat adipocytes rendered insulin resistant by chronic exposure to
hyperglycemia and hyperinsulinemia (High GA) showed decreased insulin-stimulated
glucose transport and Glut4 translocation and AktlPKBa activation. This was
associated with normal IRS- 1 tyrosine phosphorylation and IRS- 1 associated p85. Co-
incubation with NAC (N-acetyl-cysteine) prevented the insulin resistance in glucose
uptake, Glut4 translocation and Akt l/PKBa.
Taken together, these findings suggest a role for Akt l/PKBa to mediate insulin-
stimulated glucose transport. However, additional pathways must also exist that cm
promote glucose uptake and Glut4 translocation.
ACKNOWLEDGEMENTS
1 would like to thank my supervisor Dr. George Fantus for giving me the
opportunity to compIete a Master of Science degree in his laboratory under his
supervision, and for his support, both academic and non-academic.
I would like to thank Dr. Amira KJip and individuals in her laboratory, especially
Jean-Francois St. Denis, Toolsie Ramlal, and Romel Somwar for support and technical
assistance which were of immeasurable value.
In addition, I would also like to thank Dr- Jim Woodgett and his laboratory for
technical assistance.
Finally 1 would li ke to acknowledge the individuals in my own laboratory for
training and help.
III
TABLE OF CONTENTS
ABSTRACT ACKNO WLEDGEMENTS TABLE OF CONTENTS LIST OF FIGURES LIST OF ABBREVIATIONS
II III IV
VI1 vnr
............................................................................................................... CHAPTER ONE. 1 INSULIN STIMULATION OF GLUCOSE TRANSPORT....... ...............................W... L
7 1.1 Overview of Glucose Uptake Stimulation by Insulin ............................................. - ............................................................ 1.1.1 The lnsulin Receptor and IRS Proteins 4
INTRODUCTION TO CHAPTER 2 A N D CHAPTER 3 ............................................. 9
CHAPTER TWO ............................................................................................................. 12 ...... Raie of Aktl/PKBa in Vanadate and Pervanadate Stimulated Glucose Uptake 12
........................................................................................................ CHAPTER THREE 26 The Antioxidant N-Acetyl-Cysteine Prevents the Impairment in Glucose Transporter Translocation and AktlmKBa Activation in Hyperglycemia and ...................................................................... Glucosamine Induced Insulin Resistance 26
................................................................. 3.2.5 Preparation of Whole Cell Lysates 41 3.2.6 P rotein Determination .................................................................................... 41 3.27 Determination of Glut4 Translocation and Glut 1 Levels ............................... 42
............................................................................ 3.2.8 IRS- 1 Immunoprecipi tation 42 3.2.9 Determination of IRS-1 Tyrosine Phosphorylation and Association with p85 ..
3.3 Results ................................................................................................................... 46 3.3.1 Effect of NAC on Glut4 Translocation in Cells Treated with Hïgh GA and
.................................................................................................. Glucosamine -46 3.3.2 Effect of NAC on Glutl Expression in Cells Treated with High GA and
................................................................................................... Glucosami ne 49 3.3.3 Effect of High Gfl and NAC on Akt lPKB a Activation and Phosphorylation .
...................................................................................................................... -49 3.3.4 Effect of High GA and NAC on IRS-1 Tyrosine Phosphorylation and
Association with p85 in the LDM .................................................................. 59 3.3.5 Effect of High G/I and NAC on IRS-1 Tyrosine Phosphorylation in Total
FIGURE S. 1 Effect of Insulin, Vanadate, and pV on Akt l/PKBu Activity FIGURE 2.2 Serine 473 Phosphorylation of Akt 1/PKBa
CHAPTER THREE
FIGURE 3.1 The Hexosamine Biosynthesis Pathway FIGURE 3.2 Glut4 Translocation in Hïgh GA and NAC Treated Adipocytes FIGURE 3.3 Glut4 Translocation in Glc and NAC Treated Adipocytes F I G W 3.4 Effect of NAC Alone on Glut4 Translocation FIGURE 3.5 Effect of High G/I and NAC on Glutl Expression FIGURE 3.6 Effect of Glc and NAC on Glutl Expression FIGURE 3.7 Effect of High G/I and NAC on Aktl/PKBa Activity FIGURE 3.8 Effect of NAC on Aktl/PKBa Activity FIGURE 3.9 Effect of High G/I and NAC on Serine 473 Phosphorylation FIGURE 3.10 Effect of NAC on Serine 473 Phosphorylation of Akt l/PKBu FIGURE 3.11 Effect of High G/I and NAC on Total Levels of Akt l/PKBu FIGURE 3.12 Effect of High G/I and NAC on RS-1 Levels in the LDM FIGURE 3.13 Effect of High Gn and NAC on IRS-1 Tyrosine Phosphorylation
In the W M FIGURE 3.14 Effect of High GA and NAC on IRS-1 Assoçirited p85
In the LDM FIGURE 3.15 Effect of High GO and NAC on iRS-1 Tyrosine Phosphorylation
In Total Ce11 Lysates FIGURE 3.16 Effect of High G/I and NAC on iRS-1 Levels in Total Cell
Lysates
LIST OF ABBREVIATIONS
AMP ANOVA ATP BSA CAMP DAG DMEM DNA ECL EDTA EGTA FBS GFA Glc GLDH Glut GSH HBP HBS HDM HEPES High G/I IRS KRBH LDM MAPK MAPKAP cc-MEM MOPS NAC NAD NADH NIDDiM NIH NMR 8-OHdG PDGF PDK PH Pi PI3 K PKB
adenosine monophosphate analysis of variance adenosine triphosphate bovine serum albumin cyclic adenosi ne monophosphate diac ylgl ycerol Dulbecco's Modified Eagles Medium deoxyribonucleic acid enhanced cherniluminescence eth ylenediaminetetraacetic acid ethylenebis(oxyethylenenitrilo)tetraacetic acid fetal bovine semm Glutamine: fructose-6-phosphate amidotransferase gl ucosamine L-glutamate deh ydrogenase glucose transporter reduced glutathione hexosamine biosynthesis pathway HEPES buffered sali ne high-density microsome N-[2-Hydroxye<hyl]pipera~ine-N'-[2-ethanesulfonic acid] combined high glucose and high insulin concentrations insulin receptor substrate Kreb's Ringer Bicarbonate HEPES buffer low-densi ty microsome mitogen activated protein kinase mitogen activated protein kinase activated protein alpha Minimum Essential Medium (3-[N-Morpholino]propanesulfonic acid] N-acetyl-cysteine nicotinamide adenine dinucleotide reduced NAD non-insulin dependent diabetes me1 litus National Institute of HeaIth nuclear magnetic resonance 8-hydroxydeoxyguanosine platelet denved growth factor phosphatidylinositol dependent protein kinase plec kstrin homology inorganic phosphate phosphatidylinositol 3-kinase protein kinase B
protein kinase C plasma membrane phosphatidylinositol 3-phosphate phosphatidylinosi tol 3.4-diphosphate phosphatidylinositol3,4,5-triphosphate phosphatidylinositol3.5-diphosphate protein tyrosine phosphatase pervanadate nbonucleic acid sodium dodecyl sulphate-polyacrylamide gel electrophoresis standard error src hornotogy 2
CHAPTER ONE
INSULIN STIMULATION OF GLUCOSE TRANSPORT
CHAPTER ONE
INSULIN STIMULATION OF GLUCOSE TRANSPORT
Insulin, a peptide hormone, was first discovered by Frederick Banting and Charles
Best in 1921 (1). Insulin is an anabolic hormone and the principal regulator of energy
metabolism (2). One action of insulin is to promote glucose entry into muscle and
adipose tissue (2). Insulin increases glucose entry into these tissues by promoting the
translocation of glucose transporters from intracellufar low-density microsornes (LDM)
to the plasma membrane (PM) (3). Once recruited, the glucose transporters insert into the
PM and are likely activated (4) allowing the uptake of glucose into the cell. This action
of insulin is particularly important for glucose homeostasis in normal physiology. This is
evident in diseases such as Type 2 diabetes in which the decreased ribility of insulin to
promote glucose uptake in target tissues is a major defect and may contribute to the
development of the disease (2,4).
How the activation of the insulin receptor promotes glucose transporter
translocation and glucose uptake is not entirely known but several signahg proteins have
been i mpl icated.
1.1 Overview of Glucose Uptake Stimulation by Insulin
Full stimulation of glucose transport by insulin requires translocation of glucose
transporters (Gluts) from internai membranes to the PM (3), associated with an increase
in intnnsic activity of the transporters (4). Insulin stimulates both processes likely by
parallel pathways (4), although more is known about the pathway that leads to glucose
transporter translocation (Fig. 1.1).
IRS PROTEINS
INSULIN RECEPTOR
1 PIJ-KINASE 1
GLUT4
PKB/Akt ATYPICAL PKCs
GLUT4 TRANSLOCATION
FIG. 1.1 STIMULATION OF GLUT4 TRANSLOCATION BY INSULIN
3
1- 1- 1 The I~zsrtlNt Recepfor and IRS Proreins
The insulin receptor contains two a and two P subunits ( 5 ) . The u subunîts
are located entirely on the extracellular side of the plasma membrane and contain the
insulin binding sites (5). The B subunits are transmembrane proteins that contain tyrosine
kinase activity in the cytoplasmic portion (5). lnsulin binding to the cjr subunit leads to
the activation of the receptor tyrosine kinase. Once active. the insulin receptor kinase
phosphorylates numerous substrates on tyrosine residues which then function as docking
proteins (5). There are at least four insulin receptor substrates (IRSs) identified, IRS-1
(6), IRS-2 (7), IRS-3 (8), and IRS-4 (9). Once phosphorylated on tyrosine residues, the
IRS proteins recruit and activate Src homology 2 domain (SH2) containing proteins. For
the stimulation of glucose transport, the p85 subunit of Class IA phoshatidylinositol 3-
kinases appears to be the central protein recmited to the tyrosine phosphorylated IRS
proteins (l0,ll).
1.1.2 Plzospharidylit~ositol3- Kitzase
Phosphatidylinositol 3-kinases (PI3K) are a family of lipid kinases that catalyze
the phosphorylation of phosphoinositides on the 3-position of the inositol ring (12). Four
different lipid products of the P13K family have been identified; PtdIns-3-P, PtdIns-3,4-
PI, PtdIns-3,s-PI, and PtdIns-3,4,5-P; (12). The Class IA P13K have been demonstrated to
play a role in the stimulation of glucose uptake by insulin, most likely by regulating the
trafficking of glucose transporter containing vesicles (13). Class IA PI3K are composed
of two subunits: the p85 regulatory subunit, and the pl 10 catalytic subunit (12). The p85
subunit contains two SH2 domains which bind to specific tyrosine phosphorylated motifs
on the R S proteins (10) leading to the activation of the pl IO catalytic subunit (14). in
vitro, p l 10 can catalyze the formation of the above mentioned lipids but i)t vivo only
PtdIns-3.4-Pz and PtdIns-3,4,5-P3 levels are increased by insulin and other extracellular
signals (12). Ptdns-3-P is constitutively present inside the ce11 (12).
1.1.3 Downsrrearn Targets of Pl1osllaridylinosiral3-Kinase
There are 2 important serinehhreonine kinases that have been shown to act
downstream of PI3K and may play a rote in insulin-stimulated glucose transporter
translocation. The first kinase is AktPPKB (15). There are 3 isoforms discovered to date:
Akt 1PKBa (16- 18), Akt2/PKBB (19), and Akt3PKBy (20). AI 1 isoforms possess an N-
terminal pleckstrin homology (PH) domain, followed by a catalytic domain and a C-
terminal taii (21). Akt3/PKBy is truncated at the C terminal compared to the other 2
isoforms (20). Full activation of Akt/PKB requires phosphorylation on threonine
(Thr308 on Akt l/PKBcc, Thr309 on AktZPKBP, Thr305 on Akt3RKBy) and senne
residues (Ser473 on AktlPKBa, Ser474 on Akt2PKBQ) (22). Phosphorylation of
these residues has been shown to be dependent on PUK (21). The enzyme which
phosphorylates the threonine residue has been purified from rabbit skeletal muscle and
shown to be dependerit on PtdIns-3,4-Pz and PtdIns-3.4,5,-P3 for activation. It was
therefore termed phosphoinositide-dependent protein kinase 1 (PDK 1 ) (23). Li ke
Akt/PKB, PDKl also contains a PH domain (34). The kinase which phosphorylates the
senne residue, termed PDK2, has not yet been identified but it will likely be regulated in
the same manner as PDKl and also contain a PH domain (23). The mechanism of
Akt/PKB activation via PI3K is not entirely understood, but a mode1 has been proposed
for the activation of Akt/PKB irl vivo. In this mode!, Ptdns-3,4-Pz and PtdIns-3,4,5-P3
recruit Akt/PKB to the plasma membrane (24). This is accomplished by the presence of
the PH domain in Akt/PKB (24). Once at the PM, PtdIns-3,4-Pz and Ptdns-3,4,5-P3 may
promote conformational changes in Akt/PKB which would allow PDKl and PD= to
phosphoryiate and activate the kinase (12).
The second serinekhreonine kinase that has been implicated in the stimulation of
glucose transporter translocation by insulin is a group of enzymes known as the atypical
PKCs, PKCE and PKCh (25). Insulin activation of PKCC and PKCh has been shown to
be dependent on increased PtdIns-3,4,5-P3, and PDK- 1 dependent phosphorylation of
activation loop sites in PKCC and PKCA (26). Adipocytes transiently transfected with
kinase inactive mutants of PKCC and PKCA displayed a 40-60% inhibition of insulin-
dependent protein kinase inhibitor peptide (lOpM), and the substrate peptide
(RPRAATF) (lOOpM), which is a relatively specific substrate for Akt i/PKBa because it
is not phosphorylated to a significant extent by MAPKAP-Ki or p70 S6 kinase (60).
Following the 10 minute incubation, the samples were centrifuged and the supernatants
were spotted on P8 1 phosphocellulose paper. Radioactivity was determined by
scintillation counting.
2.2.5 Ir~znrrrrzoblor Analysis
Cells were serum starved for 3 hours prior to stimulation. Cell lysates (60pg) were
subjected to SDS-PAGE (10% polyacrylamide) followed by electro transfer of proteins to
nitrocellulose. Following the transfer, the membranes were incubated for 1 hour at 22°C
in Tris buffered saline (TBS) (25mM Tris, pH 7.5, 154m.M NaCl) containing O. L%
Tween 20 and 5% non-fat dry milk. The membrane was then incubated overnight with a
polyclonal phospho-specific anti4473 antibody to detect active Akt l/PKBa, or with an
antibody against the full length of the kinase to quantify total levels. Following the
overnight incubation the membrane was treated for 1 hour wi th an anti-rabbit secondary
antibody conjugated to horseradish peroxidase. Proteins were then visualized by
enhanced cherniluminescence and quantified by NIH Image.
3.26 Srarisrical Analysis
Results are expressed as MEANS k SE. The significance between groups was
determined using analysis of variance (ANOVA, ~ 4 . 0 5 ) .
2.3 Results
Stimulation with IOOnM insulin for 5 minutes resulted in a 2.30M.37 fold activation
of Akt l/PKBct above basal (Fig. 2.1). Treatment of cells with 5mM vanadate for 30
minutes resulted in a slight but insignificant activation of AktlPKBa, 1.15s. 17 above
control, while stimulation with 5mM vanadate for 60 minutes did not activate
AktUPKBa, 0.90d. 10. pV was a potent stimulator of Akt l/PKBu, 17e.11 fold
activation was observed after incubating the cells with 100pM pV for 15 minutes.
15 - O- wortmannin :
I + wortmannin
Con Ins Van30 Van60
FIG. 2.1 Effect of insulin, vanadate, and pV on Akt l/PKBa activity. L6 myotubes were inc ubated in the presence or absence of 1 OOnM wortmannin for 1 0 minutes. The cells were then stirnulated with either lOOaM insulin for 5 minutes, 5mM vanadate for 30 or 60 minutes, or 100pM pV for 15 minutes. Cells were lysed and Aktl/PKBa activity was determined using the irnrnunoprecipitation b a s e assay described in Section 2.2.4. Insulin and pV increased Akt l/PKBa activity which was inhibited by wortmannin pretreatment. Vanadate had no significant effect. Results are MEANkSE of three to five expenments (Op < 0.05 compared to untreated Control).
Pretreatment with lOOnM wortmannin for 10 minutes blocked Akt l /PKBa activation by
insulin (1 .O8M.X), vanadate (30 minutes, O.86-CI.l l), and pV (L.8Odl.57). Although
not statistically significant, pretreatment of control cells with wortmannin reduced
Akt l/PH3u activity in the basal state (O.88d. 19). Similar results were obtained by
immunoblotting with an anti phospho-Ser473 anti body which recognized the
phosphorylated form of Aktl/PKBa (Fig.2.2).
2.4 Discussion
Insulin stimulates Akt 1 P K B a activity in a time and dose dependent manner (6 1).
The stimulation protocol used in this study (100nM insulin for 5 minutes) would produce
maximal activation of the kinase by the hormone. Pretreatment of cells with wortmannin
inhibited subsequent activation of the kinase by insulin. This finding is well docurnented
and supports Aktl/PKBu as a downstream target of PI3K. Kohn er al. (38) have shown
that inhibition of insulin stimulated Aktl/PKBu activity by using several concentrations
of wortmannin closely paralied the inhibition of PI3K in anti-phosphotyrosine
immunoprecipitates, indicating that Akt l/PKBa activation by insulin is closel y
associated with the activation of P13K.
pV was a potent activator of Akt l/PKBa and induces phosphorylation of
Senne373. This observation is consistent with other reports (62,63). pV has been s h o w
to potently activate and phosphorylate Aktl/PKBa by a wortmannin sensitive mechanism
Wortmannin - O - - + + + + Treatment C 1 V p V C I v PV
FIG. 2.2 L6 myotubes were pretreated in the presence or absence of 100nM wortrnannin followed by the addition of either 1OOn.M insulin for 5 minutes, 5mM vanadate for 30 minutes, or 100pM pV for 15 minutes. Cells were then lysed and proteins (60pg) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with (A) an anti-Ser473 antibody to examine phosphorylated Akt l/PKBa, or (B) an anti-Akt 1 /PKBa antibody to determine total levels. Insulin and pV increased the Ser473 phosphoryiation of Akt l/PKBa, which was prevented by wortmannin. Vanadate treatment had a minimal eEect. Total levels of AktlPKBa were similar in al1 conditions. Relative intensities of stimulation corrected for total Aktl/PKBa detennined by densitometry in arbitrary units; insuiin, 0.64; vanadate, 0.065; pV, 1.32.
in both 3T3 fibroblasts (62) and isolated rat adipocytes (63). In isolated rat adipocytes
(63) pV stimulated Aktl/PKBa activity more potently than vanadate. Taken together, the
results of the present study and previous studies indicate that pV, and to a much lesser
extent vanadate, induce phoshorylation and activation of Akt l/PKBa by ri PI3K
mechanism.
The difference in activation of AktlffKBu by vanadate and pV may be due to the
different mechanism of PTP inhibition by each agent. pV is a much more potent PTP
inhibitor than vanadate (5 l,52). Vanadate, a completely reversi ble inhibitor, inhi bits
PTPs by competing with phosphate for binding at the active site (64). In contrast. pV
inhibits PTPs irreversibly (64) by oxidizing the cysteine residue Iocated at the catalytic
site (65). The irreversible inhibition by pV may account for the fact that pV is much
more potent in mimicking the actions of insulin than vanadate. Fantus et al. (52) have
shown that pV is 10'-l0' times more potent than vanadate in stimulating lipo, uenesis,
protein synthesis, and inhibiting epinephrine-stirnulated lipolysis in isolated rat
adipocytes. In isolated rat skeletal muscle, pV treatment stimulated glucose utilization
significantly more than vanadate alone (66). In addition, due to the different mechanisms
of inhi bition, vanadate and pV may have different FTP specificities. Fantus et al. (52)
have shown that vanadate, but not pV, inhibited tyrosine dephosphorylation of the insulin
receptor P-subunit by alkaline phosphatase. In the same experiment, pV powerful ly
inhibited tyrosine dephosphorylation of the insulin receptor by a crude preparation of
phosphotyrosine phosphatase, while vanadate had minimal effects. In the case of the
present study, pV may be inhibiting a key phosphatase(s) important in regulating the
activation of Akt l/PKBa.
Pretreatment of L6 myotubes with wortmannin inhibited insulin stimulated Glut
translocation and glucose uptake (43). Pretreatment with wortrnannin at the
concentration and duration used in this study effectively inhibited PI3K but did not block
vanadate and pV induced glucose transport or Glut4 translocation (43). suggesting that
Akt i/PKBa is also not essential for these agents to increase glucose transport.
Tt is interesting to note that other stimuli can also increase cellular glucose uptake
by a pathway(s) that does not involve P13K or Akt l/PKBa. Examples are muscle
contraction (67,68), hypoxia (67) and osmotic shock (69). Muscle contraction and
hypoxia have been shown to stimulate Glut4 translocation and glucose uptake as potently
as insulin (67). Lee et al. (67) have shown that wortmannin pretreatrnent blocked insulin
but not contraction or hypoxia stimulated glucose uptake in rat skeletal muscle.
Furthermore, Brozinick et al. (68) have shown that muscle contraction does not activate
Akt l/PKBa suggesti ng that contraction induced increases in glucose transport are not
mediated by Akt/PKB. Chen et al. (69) have shown that osrnotic shock increases glucose
transport and Glut translocation in 3T3-LL adipocytes by a wortmannin insensitive
tyrosine kinase pathway that did not involve Akt/PKB.
In summary, pV, but not vanadate, potently activates AktlPKBa in L6 myotubes
by a wortmannin sensitive mechanism, In contrast to insulin, both vanadate and pV
stimulate glucose transport and Glut translocation by a mechanism independent of PUK
and Aktl/PKBa in L6 myotubes. Since glucose transport is not further stimulated by
these agents above that achieved by maximum concentration of insulin, this mechanism
rippears to involve a separate signaling pathway that converges with the insulin action
pathway (53). This is supponed by the finding that cytochalasin D, an actin cytoskeleton
dismpting agent, inhibited glucose transport stimulation by al1 three agents (53).
CHAPTER THREE
The Antioxidant N-Acetyl-Cysteine Prevents the Impairment in Glucose Transporter Translocation and Aktl/PKBa Activation in Hyperglycemia and
Glucosamine Induced Insulin Resistance
ABSTRACT
To examine the potential role of oxidative stress in high glucose/insulin and
glucosamine induced insulin resistance, nt adipocytes were incubated for 18 h in media
containing 20mM glucose/lOOnM insulin (High Gn) or 5mM glucosamine (Glc). Each
incubation was done in the presence or absence of 5mM N-acetyl-cysteine (NAC). High
GfI and Glc impaired the translocation of Glutl from low-density microsornes (LDM) to
the plasma membrane (PM). which could be prevented by NAC. High GR. but not Glc
treatment, decreued Glut 1 expression in the PM, which was also prevented by NAC.
Funher examination of the insulin signaling cascade in High G/I treated cells revealed
that [RS-1 tyrosine phosphorylation and association with the p85 subunit of P13K in
response to insulin was not impaired, whether examined in the LDM or in total ce11
lysates. In contrast, Aktl/PKBu activation and Ser173 phosphorylation in response to
insulin were signi ficantl y impaired in resistant cells, which was prevented upon co-
incubation with NAC.
In conclusion, NAC. an antioxidant and glutathione (GSH) precursor was able to
prevent both h yperglycemia and glucosamine induced defects in insu1 in action. The
results from this study suggest a role for oxidative stress in the induction of insulin
resistance in this model. NAC may serve as a potential design for novel therapeutic
agents.
CHAPTER THREE
The Antioxidant N-Acetyl-Cysteine Prevents the Impairment in Glucose Transporter Translocation and Aktl/PKBct Activation in Hyperglycemia and
Glucosamine Induced Insulin Resistance
3.1 Introduction
Diabetes mellitus is a metabolic disease affecting 5% of the population
worldwide (2,701- Type 2 diabetes or non-insulin dependent diabetes mellitus (NIDDM)
accounts for over 90% of diabetic patients (2,70) and is charactenzed by (1) insulin
resistance in peripheral target tissues, namely, muscle and fat. (2) decreased pancreatic P
ce11 function, and (3) increased hepatic glucose production (7 1). Chronic complications
of diabetes include kidney failure, lower Iirnb amputations, blindness, and increased risk
of cardiovascular disease and stroke (2,70)-
Genetic and environmental factors contribute to the development of Type 2
diabetes (70). One of the earliest defects observed. and likely a key factor in the
progression of the disease, is insulin resistance (70). Insulin resistance c m be genetic or
acquired or both, and is chardcterized by impaired stimulation of glucose uptake by
insulin in target tissues (71). The exact cause or mechanism of insulin resistance is not
entirely known. Genetic analysis of insulin resistance examined the effects of mutations
in the insulin receptor gene and genes encoding the proteins in the insulin action pathway
(2). Mutations in the insulin receptor gene result in severe insulin resistance, but this
defect accounts for only ri very small percentage of Type II diabetic patients (2) . Type II
diabetes has not been shown to be associated with mutations in genes encoding proteins
in the insulin action pathway (2). At the present time, the genetic cause of insulin
resistance is not known and multiple inhented traits may be important (2). Numerous
factors have been shown to be able to induce insulin resistance in target tissues such as
hormones, cytokines, and metabolites (72). One of them is hyperglycemia (73). in Type
II diabetic patients, hyperglycemia is present, and may exacerbate the already present
insulin resistance (7 i).
3 1. i Pathogenesis of Hyperglycenlia- Induced Insulin Resisfurtce: The Hexosamine
Biosyn thesis Parlz ivay
In t h e 1 ate 1980s, Garvey et al . (73) showed using primary cultured rat adipocytes
that insulin and glucose CO-regulate the glucose transport system. In these experiments,
isolated adipocytes were incubated in media for 24 h in the absence or presence of
various glucose and insulin concentrations. At the end of the incubation, basal and
maximal insuli n stimulated gtucose transport was assayed. Glucose alone had no effect
on basal or maximal insulin stimulated glucose transport. Incubation of adipocytes with
increasing concentrations of insulin in the absence of glucose slightly decreased insulin
sti mulated glucose transport but on1 y at the highest insulin concentration tested, wi th no
decrease in basal uptake. In contrast, CO-incubation with glucose and insulin impaired
subsequent basal and insulin stimulated glucose uptake in a dose-dependent manner. At
the highest glucose and insulin concentration tested (20mM glucose, 50ng/ml insulin),
basal and insulin stimulated glucose transport were decreased by 89% and 63%
respectively. In a subsequent study by Traxinger et al. (74), it was discovered that insulin
resistance induced by glucose and insulin required the presence of the amino acii: L-
glutamine. Together these findings led to the hypothesis that the hexosamine
biosynthesis pathway (HBP) was imponant in the induction of insulin resistance by high
glucose (75). The HBP is thought to act as a 'glucose sensor' coupled to a negative
feedback system which would sense high glucose availability and subsequently prevent
excess glucose from entering the ce11 (75).
When adipocytes are exposed to high concentrations of glucose and insulin,
(insulin facilitates glucose entry into cells), the incoming glucose saturates the glucose
utilizing pathways such as glycogen s ynthesis and glycol ysis. The rernaining glucose
(usually 2-3% of total incoming glucose) fluxes through the HBP. Glucose enters the ce11
by the glucose transporters and is phosphorylated to glucose-6-phosphate which can enter
the glycogen synthesis pathway or be further processed to fructose-6-phosphate.
Fructose-6-phosphate then enters the glycolytic pathway or the HBP. The first step of the
HBP is the conversion of fructose-6-phosphate in the presence of L-glutamine to
glucosamine-6-phosphate by the rate-Iimiting enzyme Glutamine: fructose-6-phosphate
amidotransferase (GFA). Glucosamine-6-phosphate then undergoes a series of
biochemical steps to form UDP-N-acetyl-hexosamines as the end products. The main
product of this pathway, UDP-N-acetyl-glucosamine, is used in the synthesis of
gl ycoproteins, proteogl ycans, and gl ycolipids (Fig. 3.1). Several lines of evidence
support the role of the HBP in high glucose induced insulin resistance. First, GFA
inhi bi tors, azaserine and 6-diazo-5-oxonorleucine, which inhibit the conversion of
fructose-6-phosphate to glucosamine-6-phosphate, also prevented insulin resistance
induced by high glucose (75). This inhibition of GFA by azasenne closely paralleled the
prevention of glucose induced insulin resistance (75). Second, overexpression of GFA in
GLUCOSAMINE GLUCOSE
N-ACETYL-GLUCOSAMINE
GLYCOPROTEINS PEPTIDOGLYCANS GLYCOLLPKDS
I I
? ' + IMPAIRED GLUCOSE TRANSPORT
FIG. 3.1 THE HEXOSAMINE BIOSYNTHESIS PATHWAY
s keletal muscle and adipoc ytes in transgenic mice resulted in decreased insulin mediated
glucose disposa1 in vivo in the absence of hyperplycemia (76). Third, adipocytes treated
wi th glucosamine become insulin resistant. Glucosamine enters adipocytes via the
glucose transporters. Once inside the ceIl, glucosamine is phosphorylated to
glucosamine-6-phosphate and enters the HBP distal to GFA, thereby bypassing the
enzyme (75). Glucosamine is 40x more potent than glucose in inducing insulin resistance
(75). Lastly, hyperglycemia and glucosamine induced insulin resistance are non-additive
suggesting that both agents operate through the same pathway (77).
In addition to primary cultured rat adipocytes, chronic hyperglycemia and
glucosamine treatment have been shown to induce insulin resistance in 3T3-Ll
adipocytes (78) and in skeletal muscle preparations irt vitro (79,80). In vivo. studies
using the euglycemic hyperinsulinemic clamp technique have shown that prolongeci
h ypergl ycemia (8 1) or glucosamine infusions (8 1 $2) markedl y impair insulin mediated
olucose uptake at the whole body level and in skeletal muscle and adipose tissue (82). b
3.12. Poterztial Defects in the Insrilin Sigrzaiir~g P a r / w q
Insulin resistance induced by chronic hyperglycemia and glucosamine treatment
in isolated rat adipocytes is associated with impaired translocation of Glut4 to the plasma
membrane without a change in total transporter nurnber (73). Impaired Glut4
translocation has also been demonstrated in skeletal muscle (83), and in GFA
overexpressing transgenic mice (76). This type of defect is also seen in Type II diabetic
patients. Using NMR spectroscopy, C h e et al. (84) have shown that glucose transport
stimulation by insulin is defective in these patients, which was not due to decreased
levels of Glut4 (85) . These findings suggest that impaired translocation of Glut4 to the
plasma membrane is due to defective insulin signaling (83).
Studies examining the more proximal steps in the insulin signaling cascade in
hyperglycernia and glucosarnine induced resistance have led to controversial results.
Isolated rat adipocytes exposed to chronic hyperglycemia displayed decreased activation
of the receptor tyrosine kinase and IRS-1 tyrosine phosphorylation in response to insulin
in one study (86). Another study using the sarne cells and sirnilm incubation conditions
showed no defect in insulin receptor kinase activity or IRS-1 phosphorylation. Prolonged
exposure of rat skeletal muscle to 25mM glucose in vitro impaired Akt/PKB activation
and phosphorylation by insulin while PI3K activation was unaffected (79). Glucosamine
treatment of 3T3-L1 adipocytes led to impaired insulin receptor tyrosine kinase acti8/ity
and IRS- 1 tyrosine phosphorylation. impaired IRS- 1 associated PI3K activity and
decreased AktlPKB activation by insulin (87). Ir1 vivo, chronic glucosarnine infusion in
rats did not decrease insulin receptor tyrosine phosphorylation in skeletül muscle (88, 89).
IRS-1 tyrosine phosphorylation in response to insulin was reduced in one study (90) but
not in another (88). Glucosamine treatment has been shown to reduce IRS-1 associated
PI3K activity while having no effect on Akt/PKB activation or phosphorylation (88,89).
Similar findings were recently shown in skeletal muscle of obese Type II diabetic
patients (90). Obese Type II diabetic subjects displayed normal Aktl/PKBa and
Akt2/PKBP activation and phosphorylation in response to insulin while IRS-1 and IRS-3
associated PI3K activities were significantly reduced compared to lean controls and
obese non-diabetic subjects. The reason for the discrepancies among the various studies
and treatments is presently not clear but may be due to the length of the treatment times
or the presence or absence of insulin during the treatment.
Samples (lûûpI) of whole ceIl lysates, LDM and PM fractions were added to 900~1 of 5 ' -
ND reagent containing 3.2mM adenosine monophosphate (AMP), 0.2mM reduced NAD
(NADH). 3.7mM 2-Oxoglutarate, 1 1 000U/L L-glutamate deh ydrogenase (GLDH),
adenosine deaminase (ADA) 400 Un, fi-glycerophosphate, buffer, and stabilizers. Once
added, the reaction mixture was mixed by inversion and left at room temperature for 5
minutes. After 5 minutes the absorbance (A) was read using a spectrophotometer
(Beckman DU4O) at 340nm versus water as a reference (Initial A). The mixtures were
then placed in a 37°C incubator for 5 minutes after which the absorbance was read again
(Final A). 5'ND activity was calculated as follows:
*A per 5 min (W) = Initial A - Final A
5'-ND = *A per 5 min X 322 X 1.35
Where: Factor 322 = (1 X 1000)/(6.22 X O. 1 X 5)
1 = Total reaction volume (ml) 1000 = Conversion of activity per ml to activity per L 6.22 = Millimolar absorptivity of NADH at 340nm 0.1 = Volume of sample (ml) 5 = Conversion of *A per 5 min to *A per min 1.35 = Temperature correction factor
Enzyme activities were then corrected for total protein in the reaction mixtures and
expressed as U U m g protein. Results are: Whole cell lysate 10, LDiM 6, PM 8 1.
3.2.12 Statistical A~iulysis
Results represent the ME.4N-tSE. To analyze the data, a one-way analysis of variance
(ANOVA) was used. Post hoc comparisons of means were performed using Scheffe's
test and Tukey's test. Statistical signi ficance was defined as p < 0.05.
3.3.1 Ef ic t of NAC on Glirt3 Transfocarion in Cells Treared roith High GA? and Glitcosarnine
Glut4 levels in each fraction are expressed as a percentage of the total GIut4
found in the LDM and PM fractions combined. designated in each experiment as 100%.
In basal cells, almost al1 of the Glut4 was found in the LDM with very little found in the
PM. This was seen in al1 conditions (Fig. 3.2 and Fig. 3.3). In controf cells, insulin
treatment resuIted in the translocation of Gtut4 from the LDM to the PM, increasing the
plasma membrane concentration of Glut4 with a corresponding decrease in the LDM
content (Fig. 3.2 and Fig. 3.3). In cells treated with High GA, Glut4 translocation to the
PM was impaired (InsuIin stimulated PM; Control 74.16.3%, High GA 35.7k5.596,
p~O.05, n=3) with Glut4 being retained in the LDM (Insulin stimulated LDM; Control
35.9I6.3%, High GA 64.3f 5.5%. p<0.05). Co-incubation with NAC prevented the
impairment in Glut4 translocation induced by High GA (Insulin stimulated; PM
69.4_+2.1%, LDM 30.6I2.196). In cells treated with High G/I + NAC, the distribution of
Glut4 between the LDM and PM compartments in response to insulin was simihr to the
control (Fig. 3.2). Similar results were seen in cells treated with glucosamine ( F i g 3.3).
Glucosamine treatment impaired the translocation of Glut4 to the PM in response to
insulin (Insulin stimulated PM; Control 71.3&4.4%, Glc 46.8f6.6%, ~ ~ 0 . 0 5 , n=3) with
Glut4 remaining in the LDM (Insulin stimulated LDM: Control 28.7&4.4%, Glc
53.2I6.6%, p<0.05). Co-incubation with NAC prevented the impairment in Glut4
LDM 20.73+6.01%). Incubation with NAC alone had no effect on Glut4 translocation in
LDM PM LDM P M LDM P M LïBM PM LDM PM LDM PM
INSULIN - + + - - + + - + + Control High G/I High G/I +NAC
LDM P M LDM P M LDM P M LDM PM LDM P M LDM P M
INSULIN - + + - - + + - + + Control High GD High G/I + NAC
FIG. 3.2 Glut4 translocation in High GA and NAC treated rat adipocytes. Adipocytes were incubated for 18 h in media containing 20mM glucose/lOOnM insulin in the absence and presence of 5rnM NAC (High GA and High Gn + NAC respectively). Cells were washed, and incubated for 30 min in the presence or absence of 1 OOnM insulin. LDM and PM samples (30pg) were separated by SDS-PAGE, transferred to nitrocelluIose, and immunoblotted using an anti-Glut4 antibody. (A) Representative immunoblot. (B) Data are MEANSE of 3 independent experiments ( > < 0.05, High GA vs others).
LDM P M U>M PM LDM PM
INSULIN - O + Control
+ - - Glc
LDM LDM P M LDM P M
- - + + Glc + NAC
LDM PM LDM P M LDM PM LDM P M LDM PM LDM P M
INSULIN - - + + + + - - + + Control Glc Glc + NAC
HG. 3.3 Glut4 translocation in Glc and NAC treated rat adipocytes. Adipocytes were incubated for 18 h in media containing 5mM glucosamine/4.2nM insulin in the absence and presence of 5rnM NAC (Glc and Glc + NAC respectively). Cells were washed, and incubated for 30 min in the presence or absence of 100nM insulin. LDM and PM samples (30pg) were separated by SDS-PAGE, transferred to nitrocellulose, and inmunoblotted using an anti-GLut4 antibody. (A) Representative immunoblot. (B) Data are MEAN* SE of 3 independent experiments ( *p < 0.05, Glc vs others).
3.3.3 Effecr of NAC orr Glrttl Expressiort in CeIls Treared with Higli GA? and Gltr cosam irz e
Glutl was mainiy detected in the PM (Fig. 3.5 and Fig. 3.6), consistent with
previous reports (27). Since Glutl translocation to the PM in response to insulin was not
obsenled, on1 y basal cells were used in the calculations. Ali Glut 1 data is expressed as
fold of control basal which was designated a value of 1.0. In cells treated with High Gn,
Glut 1 levels were sigificantly reduced (Control 1 .O, High G/I 0.2kû. 1, p<O.OS, n=3)
(Fig. 3.5). This finding was consistent with the decrease in basal glucose uptake seen in
cells treated with High GA. Co-incubation with NAC prevented the downregulation in
Glut 1 induced by High GA (High G/T + NAC 1.2t0.1) (Fig. 3.5). In contrast to High G/I,
18 h glucosamine treatment did not significantly reduce Glutl levels and CO-incubation
with NAC had no further effect (Control 1 .O, Glc 1.1S.2, Gic + NAC 1.0k0.1, p = NS,
n=3) (Fig. 3.6). Glucosamine treatment did not significantly affect basal glucose uptake.
Treatment with NAC alone had no effect on Glutl levels (Control 1.0, NAC l.OS)(Fig.
3.4).
3.3.3 Effect of High G/I and NAC on Aktl/PKBaActivution and Phosplioryfation
Treatment of control cells with IOOnM insulin for 10 minutes resulted in a 1422.8
fold increase above basal in Akt lfPKBa activation (Fig. 3.7). In cells treated with High
Ga, Aktl/PKBa activation by insulin was reduced by 44% compared to the insulin
LDM PM LDM PM LDM P M LDM P M
INSULIN - - + + - - + + Control NAC
LDM PM LDM
INSULIN - - + Control
PM LDM PM LDM PM
+ - - + + NAC
FIG. 3.4 Effect of NAC alone on Glut4 translocation and Glutl expression. Adipocytes were incubated for 18 h in media containing SmM NAC. Cells were washed, and incubated for 30 minutes in the presence or absence of lOOnM insulin. LDM and PM samples (30pg) were separated by SDS-PAGE, transferred to nitrocellulose, and immuooblotted using (A) an anti-Glut4 antibody, or (B) an anti-Glut 1 antibody.
LDM PM LDM PM LDM P M LDM PM LDM PM LDM PM
INSULIN - - + + - - + + - - + + Control High G/I High G/I + NAC
Control High G/I High G/I + NAC
FIG. 3.5 Glut content of plasma membranes in High G/I and NAC treated rat adipocytes. Adipocytes were incibated for 18 h in media containhg 20mM glucose/lOOnM insulin in the absence and presence of 5mM NAC (High GA and High GA + NAC respectively). CelIs were washed, and incubated for 30 min in the presence or absence of lOOnM insulin. LDM and PM samples (30pg) were separated by SDS-PAGE, t rans ferred to r,itrocellulose, and immunoblotted using an anti-Glut 1 antibody . (A) Represeatative imrn~moblot. (B) Data are MEANISE of 3 independent experiments ( *p < 0.05, Hi& GA vs others). Results are expressed as the fiaction of total GIutI distributed in the PM in each condition, PM of Control was given the value 1 .O.
LDM P M LDM PM LDM P M LDM PM LDM PM LDM PM
INSULIN - - + + - - + + - t + Control Glc GIc + NAC
Control Glc Glc + NAC FIG. 3.6 Glut l content of plasma membranes in Glc and NAC treated rat adipocytes. Adipocytes were incubated for 18 h in media containing S r n M glucosamuie/4.2nM insulin in the absence and presence of 5mM NAC (Glc and Glc + NAC respectively). Cells were washed, and incubated for 30 min in the presence or absence of l OOnM insulin. LDM and PM samples (30pg) were separated by SDS-PAGE, transferred to nitroceilulose, and immunoblotted using an anti-Glut 1 antibody. (A) Representative irnmunoblor. (B) Data are MEAN* SE of 3 independent experirnents. Results are expressed as the fiaction of total Glut 1 distributed in the PM in each condition. Control PM was assigned the value 1 .O.
i
O L CI
r: 1 4 . U cLir
O 1 2 - = O z E ' O - .- > .- Y
Y 8 -
INSULIN - +
Control
- + High GD
- + Higb G/I + NAC
H G . 3.7 Effect o f High GA and NAC on Akt l/PKBa activity. Adipocytes were incubated for 18 h in media containing 20mM glucose/lOOnM insulin in the absence and presence of 5rnM NAC (High G/I and High G/I + NAC respectively). Following the incubation, cells were washed, and incubated for 10 min in the presence or absence of 1OOn.M insulin. Cells were then lysed, Akt lPKBa immunoprecipitated,and an N> vitro b a s e assay was performed using the substrate Crosstide. Results represent MEANSE of 4 independent experiments ( *p<0.05, High GA vs others).
stimulated control (High GA; 7.8W.95, pc0.05, n=4). Co-incubation with NAC
prevented the decrease in AktlPKBa activation induced by High G/I (High G/I + NAC:
14.7k3.1) (Fig. 3.7). Basal Akt l/PKBu activity was not different mong conditions.
Incubation with NAC alone reduced insulin stimulated AktlIPKBa activation by 34%,
while having no effect on basal AktliPKBa activity (Basal; Control0.09'10.03, NAC
0.05kO.01, p = NS, n 4 . Insulin stimulated; control 1 .O, NAC 0.66f0.05, p < 0.05, n d )
(Fig.3.8).
lmrnunoblot analysis of Ser473 phosphorylated AktlPKBu showed similar results
(Fig. 3.9). In basal cells, Ser473 phosphorylated AktUPKBa was not detected. but upon
stimulation with insulin a prominent band appeared on the irnmunoblot. Therefore, only
insulin treated conditions were used in the calculations. The insulin stimulated control
was designated a value of 1.0. In cells treated with High GA. AktllPKBu
phosphorylation on Ser473 was significantly decreased compared to the control (Control;
1 .O, High GA; 0.3 lkO.15, p<0.05, n=3) (Fig. 3.9). Co-incubation with NAC prevented
the impairment in AktlPKBa phosphorylation seen in cells treated with High G/I @gh
G/I + NAC 1.18M.07) (Fig. 3.9). Incubacion with NAC alone did not have any effect on
the insulin-induced phosphorylation of Ser473 (Control 1 .O, NAC 1.1410.02, p = NS,
n=3)(Fig. 3.10). Total levels of Akt l/PKBa were unchanged by the treatments (Basal;
Control 1.0. High GA 1-04 I 0.23, High G/I + NAC 1.0 r 0.08, p = NS. Insu!in
stimulated; Contro10.97 dl.08, High G/I 0.82 a .05 , High G/I +NAC 0.90 H.08, p =
NS, n=3) (Fig. 3.10 and Fig. 3.11).
INSULIN - + Control
- + NAC
FIG. 3.8 Effect of NAC on Aktl/PKBa activity. Adipocytes were incubated for 18 h in media containing 5mM NAC. Following the incubation, cells were treated with 1OOnM insulin for 10 minutes. Cells were subsequently lysed, Akt l/PKBa irnmunoprecipitated and in vitro kinase activity determined using the substrate Crosstide. Results represent MEANkSE of 4 independent experiments. The insulin treated control was assigned the value 1 .O (Basal; Control0.09f0.03, NAC O.O5+O.O 1, p = NS. Insulin; Control 1 .O, NAC 0.66I0.05, *@.05).
INSULIN - + Control
- + - + High GA High G/I + NAC
Control High G/I High G/I + NAC
FIG. 3.9 E ffect of High G/I and NAC on Ser473 phosphory lation of Akt 1 /PKBa. Adipocytes were incubated for 18 h in media containing 20mM glucose/lOOnM insulin in the absence and presence of SmM NAC (High G/I and High G/I + NAC, respectively). Following the incubation, the cells were washed, stimulated for 10 minutes with 1 OOnM insulin, and lysed. Samples ( 6 0 ~ g ) were separated on SDS-PAGE, transferred to nitrocellulose, and probed for (A) Ser473 phosphorylated Akt LPKBa. (B) Represents the MEANkSE of 3 independent experiments. The insulin treated control was designated the value 1 .O since Ser473 phospho- Akt l/PKBa was only detected with insulin treatment (Control 1 .O; High GA 0.3 1 M. 15; High G/I + NAC 1.17fl.07, *@.OS, Control vs others).
INSULIN - + Control
I + NAC
Control NAC
FIG. 3.10 Effect of NAC alone on Ser473 phosphorylation of Akt l/PKBa. Adipocytes were incubated for 18 h in media containhg 5mM NAC. Following the incubation, the cells were washed, stimulated for 10 minutes with lOOnM insulin, and lysed. Samples (60pg) were separated on SDS-PAGE, transferred to nitrocellulose, and probed for (A) total Akt l/PKBa, or (B) Ser473 phosphorylated AktlPKBa. (C) Represents the MEANHE of 3 independent experiments represented in (B), the insulin treated control was designated the value 1 .O since Ser473 phospho- Akt l/PKBa was only detected with insulin treatment (Control 1 .O; NAC 1.1+0.02, p = NS).
INSULIN - + - + - + Control High G/I High G/I +NAC
O --
INSULIN Control High G/I High G/I + NAC
FIG. 3.11 Effect of High G/I and NAC on total levels of Aktl/PKBa. Adipocytes were incubated for 18 h in media containing 20mM glucose/lOOnM insulin in the absence and presence of 5mM NAC (High GA and High G/I + NAC, respectively). Following the incubation, the cells were washed, stimulated for 10 minutes with 1 OOnM insulin, and lysed. Samples (60pg) were separated on SDS-PAGE, transferred to nitrocellulose, and probed for (A) total AktlPKBa. (B) Represents the MEANSE of 3 independent experiments (Basal; control 1 .O; High GA 1 .O4 + 0.23;High GA + NAC 1 .O + 0.08 p = NS. Insulin stimulated; control0.97 50.08; High GA 0.82 F0.05, Hi& GA +NAC 0.90 k0.08, p = NS).
3.3.4 Eflect of High G/I and NAC on IRS-I Tyrosine Phasphorylatiorr and Associariorz
irpith p85 in the LDM
Several studies have suggested that insulin resistance is associated with
increased serine/threonine phosphorylation of IRS- 1. which may prevent its interaction
with, or render it an inhibitor rather than a substrate of the insulin receptor kinase (94).
This would decrease insulin-stirnulated tyrosine phosphorylation (93, 109). To determine
whether the High GO treatment caused such a defect, iRS-1 was immunoprecipitated
from the LDM and tyrosine phosphorylation rneasured. In rat adipocytes, it has been
dernonstrated previously that the rnajority of IRS-1 is located in the LDM and this
localization was unchanged after insulin stimulation (36). This was confirmed in this
study. Total levels of IRS-1 in the LDM were unchanged by any of the treatments
(Basal; Control 1.0, High G/I 0.75kO.05, High GA + NAC 0.8 lH.07, p = NS. Insulin;
Control 0.97k0.11, High GA 0.78H.02, High GA + NAC 0.99%. 13, p = NS, n=3) (Fig.
3.13). Insulin treatment of control cells led to a 13-fold increase in tyrosine
phosphorylation compared to basal cells (ControI; Basal 0.06kO.03, Insulin treated
control was designated 1 .O) (Fig. 3.13). Treatment cf cells with High G/I for 18 h did not
have an effect on insulin-stimulated tyrosine phosphorylation of IRS-1 compared to the
control (Insulin; High G/I 1 .O3M.35, p = NS, n=3). There was a slight increase in brisa
tyrosine phosphorylation of iRS-1 compared to the basal controI but this was not
statistically significant (Basal; High GA 0.22M.07, p = NS). In cells CO-incubated with
NAC and Hiph GA, tyrosine phosphorylation of IRS-1 in basal and insulin treated cells
was similar to the control (High G/I + NAC; Basal 0.03M.01, Insulin 0.89H.32, p = NS)
(Fig. 3.13).
- + - + - + Control High G/I High GA + NAC
0 ---
INSULIN
Control High Gn High GD + NAC
FIG. 3.12 Effect of High GA and NAC on IRS-1 levels in the LDM. Adipocytes were incubated for 18 h in media containing 20mM glucose/100nM insulin in the absence and presence of 5mM NAC (High GA and High G/I + NAC, respectively). Cells were subsequently washed and stimulated with 1 OOnM insulin for 5 minutes. LDM samples (8Opg) were resolved by SDS-PAGE, transferred to nitrocellulose and blotted with an anti-IRS- 1 antibody. (A) Representative immunoblot. (B) Represents the MEANSE of 3 independent experiments. Basal control was assigned the value 1 .O (Basal; Control 1 .O, High GA 0.75k0.05, High GA + NAC 0.8 1k0.07, p = NS. Insulin; Control 0.97k0.11, Hi& G/X 0.78M.02, High GA + NAC 0.99k0.13, p = NS).
INSULIN 9 + - + 9 + Control Higb GA High G/I + NAC
INSULIN - + + + Control High G/I High GD + NAC
HG. 3.13 Effect of High GA and NAC on IRS-1 tyrosine phosphorylation in the LDM. Adipocytes were incubated for 18 h in media containing 20mM glucose/ 100nM insulin in the absence and presence of 5rnM NAC (High GA and High GA + NAC, respectively). Followùig the incubation, cells were washed and treated with lOOnM insulin for 5 minutes. IRS- 1 was imrnunoprecipitated fiom LDM samples (SOOpg), resolved by SDS-PAGE, transferred to nitrocellulose and probed with anti-phosphotyrosine antibodies (pY99). (A) Representative immunoblot. (B) Total IRS-1 imrnunoprecipitated in (A). (C) Results represent MEANkSE of 3 independent experiments representedin (A). The insulin treated control was assigned the value 1 .O (Basal; Control0.06+0.03, High GA 0.22k0.08, High Gd+ NAC 0.03M.01 p = NS. Insulin; Control 1 .O, High G/I 1.03+0.35, High GA + NAC 0.89-tO.32, p = NS).
61
The p85 subunit of PI3K binds to tyrosine phosphorylated IRS-1. Therefore, the amount
of p85 associated with RS-1 was determined. RS-1 was immunoprecipitated from the
LDM, resolved by SDS-PAGE and immunoblotted with p85 antibodies. In control cells,
insulin increased the amount of p85 bound to ES-1 approxirnately 5- fold above basal
(Control; Basal O. 17H.06, Insulin 1.0) (Fig. 3.14). Ceils treated with High G/I displayed
similar ievels of p85 associated with IRS-1 in both basai and insulin treated cells (High
GA: Basal O - Z k O . 19, Insulin i.26kû. 19, p = NS, n=3). In ce1 1s CO-incubated with NAC
and High GA, the amount of p85 associated with RS-1 in basal and insulin treated cells
was similar to the control and High GA treated cells (High G/i + NAC; Basal 0.30kû. 19.
Insulin 0.8 1 s . 16, p = NS) (Fig. 3.14).
3.3.5 Effect of High G/I and NAC on I R S - I Tvrosine Phosphory farion in Total Ce11
Lysares
IRS- 1 tyrosine phosphoryiation was measured in total ce11 lysates. since recent
studies have suggested that disruption of IRS-1 function may Vary among cellular
compartments ( 1 15). In control cells, insulin increased tyrosine phosphorylation of IRS-
1 (Control; Basal 0.04&0.02, Insulin 1 .O) (Fig. 3.15). Cells treated with High GA,
displayed higher basal leveis of tyrosine phosphorylation but lower levels after insulin
treatmenr compared to the control. These differences did not reach statistical significance
(High G/I; Basal 0.30N. 14, Insulin 0.65f0. Il, p = NS, n=3). Ceils CO-incubated with
High GA and NAC displayed a tyrosine phosphorylation pattern similar to the control, in
both basal and insulin treated cells (High G/i + NAC; Basal 0.15M.08; Insulin
1.07k0.43, p = NS). Due to variability, tyrosine phosphorylation of IRS-1 in response to
INSULIN - + + + Control High GA High GD + NAC
cf- INSULIN - + - + - +
Control High G/I High G/I + NAC
FIG. 3.14 Effect of High GA and NAC on IRS-1 associated p85 in the LDM. Adipocytes were incubated for 18 h in media containhg 20mM glucose/lOOnM insulin in the absence and presence of 5mM NAC (High GA and High G A + NAC, respectively). Following the incubation, cells were washed and treated with 100nM insulin for 5 minutes. IRS-1 was immunoprecipitated h m LDM samples (500pg), resolved by SDS-PAGE, transferred to nitrocellulose and probed with an anti-p85 antibody. (A) Representative imrnunoblot. (B) Results represent MEAMSE of 3 independent experiments. The insulin treated control was assigned a value of 1 .O (Basal; Control 0.17t0.06, High GA 0.25k0.19, High G/I + NAC 0.3020.19,p = NS. Insulin; Control 1.0, High GD 1.2620.19, f igh GII +NAC 0.81k0.16, p = NS).
63
Control High G/I High G/I + NAC B
INSULIN - + - + - + Control High G/I High GII + NAC
FIG. 3.15 Effect of High GA and NAC on iRS-1 tyrosine phosphorylation in total ce11 lysates. Adipocytes were incubated for 18 h in media containing 20m.M glucose/100nM insulin in the absence and presence of 5mM NAC (High GA and High G/I + NAC, respectively). Followïng the incubation, cells were washed and treated with l0On.M insulin for 5 minutes. XRS-1 was immuno- precipitated fkom ceIl lysates (lmg), resolved by SDS-PAGE, transferred to nitrocellulose and probed with anti-phosphotyrosine antibodies (pY99). (A) Representative immunoblot. (B) Results represent M E W S E for 3 independent experirnents. The insulin treated control was assigned the value 1 .O (Basal; Control O.O4f 0.02, High G/I 0.30kO. 14, High G/I + NAC O. 15f 0.08, p = NS. Insulin; Control 1 .O, High GA 0.65kO. 1 1, High GA + NAC 1.07H.43, p = NS).
insulin was not significantly different among conditions (Fig. 3.15). 18 h treatment with
NAC alone had no effect on insulin-stimulated IRS-1 tyrosine phosphorylation (Basal;
Controt 0.16, NAC 0.1 1 , insulin; Control 1 .O. NAC 0.88). Total levels of IRS-1 in ce11
l ysates were unchanged by the treatments (Control 1.0, High G/I 1 . O 8 S . Z ? High G/E +
NAC 1.01k0.15, p = NS. n = 3, NAC alone 0.78) (Fig. 3.16).
- Control High Gfl High G/l + NAC NAC
Control High Gfï High GA + NAC NAC
FIG. 3.16 Effect of High GA and NAC on IRS-1 levels in total ce11 lysates. Adipocytes were incubated for 18 h in media containing 20mM glucose/lOOnM insulin in the absence and presence of 5mM NAC (High GA and High GA + NAC, respectively), or with 5mM NAC alone. Ce11 lysates (80pg) were resolved by SDS-PAGE, transferred to nitrocellulose and blotted with an anti-IRS- 1 antibody. (A) Representative immunoblot. (B) Represents the MEANkSE of 3 independent experiments. The control was assigned the vaIue 1 .O (Control 1 .O, High G/I 1.0820.22, High GA + NAC 1 .Ol+O. 15, p = NS, NAC; 0.78).
3.4 Discussion
Previous work in our laboratory has shown that isolated rat adipocytes chronically
treated with high glucose/insulin, or glucosamine, dispiayed marked decreases in insulin-
stimulated glucose uptake which could be prevented by NAC. The present study has
showed that insuiin resistance induced by high plucose/insulin or glucosmine is
associated wi th impaired translocation of Glut4 to the plasma membrane, as reported
previously (73). Co-incubation with NAC prevented the impairment in GIut4
translocation, suggesting that NAC prevents insulin resistance. The fact that NAC
prevented both high glucose/insulin and glucosamine induced insulin resistance implies
that the site of action of NAC is at a common point in the two pathways. In addition, the
ability of NAC to prevent insulin resistance induced by high glucose/insulin or
glucosamine suggests a potential roIe of oxidative stress in the induction of insulin
resistance in rat adipocytes.
In contrast to Glut4 translocation, high gIucose/insulin and glucosamine had
different effects on Glutl expression. High glucose/insulin decreased Glutl levels in the
plasma membrane. High glucose and insulin have shown to have independent effects on
Glut 1 expression. In ceIl cultures prolonged treatment with high glucose decreases Glut 1
expression (27). El-Kebbi et al. (1 10) have shown that treatment of BC3H-1 cells, a
cultured skeletal muscle ceIl line, for 24 h in 25mM glucose resulted in an 80% decrease
in Glutl content. AIthough chronic insulin treatment has been shown to increase Glutl
expression in L6 muscle cells and 3T3-LI adipocytes (1 1 1,112). this effect was not seen
in isolated rat adipocytes (73). Therefore, it appears that high glucose has a dominant
effect on the levels of Glutl in rat adipocytes incubated with high glucose/insulin.
G m e y er al. (73) have shown that high glucose and insulin together decreased Glutl
levels, but chronic insulin treatment alone had no effect suggesting that increased
metabolism of glucose causes a reduction in Glutl protein levels. In contrast to high
gIucose/insulin, glucosamine treatment, either in the presence or absence of NAC, had no
effect on Glutl protein levels. The difference between the effects of high glucose/insulin
and glucosamine may be explained by the different point of entry into the hexosarnine
biosynthesis pathway by these agents. For high glucose to enter the hexosamine
pathway, it first has to saturate glycolysis and glycogen synthesis before being converted
to glucosamine-6-phosphate by GFA (75). Intermediates from any of these pathways
could potentially be involved in the regulation of Glut 1 levels (1 13). Glucosamine
bypasses these steps and enters the hexosarnine pathway distal to GFA (75). NAC was
able to prevent the decrease in Glut 1 induced by high glucose/insulin. How NAC
prevents the decrease in Glutl is not clear. NAC may be acting upstream of GFA thereby
blocking the generation of the glucose signal. In contrast to the present study, Rudich et
ul. (104) have shown that prolonged oxidative stress, induced by incubating cells with
micromolar concentrations of H2O2 increases Glutl levels in 3T3-L1 adipocytes.
IRS-1 tyrosine phosphorylation in response to insulin was not altered with high
glucose/insuiin treatment whether measured in the LDM or in total ce1 1 l ysates,
sugesting that, in this modei, there was no significant serinehhreonine phosphorylation
of IRS-I preventing tyrosine phosphorylation. In addition, the association of p85 with
RS-1 in response to insulin was not altered in the LDM fraction. Due to technical
difficulties, IRS-1 associated p85 in total cell lysates was not measured. Since almost al1
of the IRS-1 is located in the LDM in rat adipocytes (36), the results obtained from the
experiments examining RS- t in the LDM fraction should accurately reflect the status of
these proteins under these conditions. Lima et al. (1 L4), have s h o w that glucose/insulin
induced desensitization of glucose transport i n rat adipocytes occurred without effect on
IRS- 1 tyrosine phosphorylation. High glucose was also without effect on IRS-1 tyrosine
phosphorylation and PI3K activity in rat skeletal muscle (79)- In conuast, treatrnent of
3T3-L1 adipocytes with H202, inhibited insulin stirnulated IRS-1 tyrosine
phosphorylation and IRS-1 associated P13K activity in the LDM (1 15).
Although not statistically significant, basal levels of IRS-1 tyrosine
phosphorylation and association with p85 in high glucose/insuIin, and high
gIucose/insulin + NAC, treated cells tended to be higher compared to control cells. One
possibility for this effect coulci be that following the 18 h incubation there was some
residud effect of insulin remaining after the washing steps (1 14).
In the high glucose/insulin mode1 of insulin resistance, total levels of IRS-1 were
unchanged. Sorne insulin resistance States have shown to disptay decreased levels of
IRS-1 expression, for example, chronic insulin treatrnent of 3T3-Ll adipocytes ied to a
significant reduction in iRS-1 protein levels ( 1 17).
In addition to IRS-1, rat adipocytes also contain IRS-2, which is also located in
the LDM fraction (36). IRS-2 binds to p85 and can mediate the action of insulin to
stimulate Glut4 translocation (1 18). Therefore, it is possible that in rat adipocytes treated
with high glucose/insulin, IRS-2 tyrosine phosphorylation in response to insulin rnay be
impaired. Anai et al. (36) have shown that in rats rendered insulin resistant by high fat
feeding, expression of RS-2, and to a Iesser extent RS-1, was reduced in adipose cells.
. AktllPKBa activation by insulin was impaired in cells treated with high
giucose/insulin which was prevented by NAC. The decrease in kinase activity with high
glucose/insulin treatment was accompanied by decreased phosphorylation of Ser473
suggesting an upsueam signaling defect. PI3K activity and lipid products were not
measured in the present study, therefore the possibility rernains that there might be a
defect at this sep. There may also be a defect in the upstream kinase that phosphorylates
Ser473. Although not measured in this study, phosphorylation of Th308 may also be
reduced with high glucose/insulin treatment which wouid implicate defective PDK-1
activity. High glucose/insulin may activate protein phosphatase 2A (PPZA), a
serinehhreonine phosphatase which could dephosphorylate and inactivate AktlffKBu
(62). Co-incubation with NAC prevented the defect in Aktl/PKBcz activation and Ser473
phosphorylation suggesting that one point at which NAC is acting is upstream of
Akt l/PKBu and downstream of IRS- 1. A discordance between ES- 1fPI3K and
Akt l/PKBa activation has previously been shown in ceramide induced insulin resistance.
Summers et al. (1 19), have shown that exposing 3T3-LI adipocytes to the short-chain
translocation, and Akt l/PKBcr and ~kt2/PKBp activities. These defects occurred despite
normal insulin stimulated IRS- 1 tyrosine phosphorylation, association with p85, and
PI3K activity.
High glucose has previously k e n reported to impair Akt l/PKBa activation in
skelctal muscle (79). Hyperglycemia could impair Akt lPKBa by a PKC dependent
rnechanism (79). Bsuthel er al. (1 16) have shown that pnor activation of PKC by PMA
inhibited the subsequent ability of insutin to stimulate A k t l P K B a and Akt3PKBy
activities in 3T3-L1 fibroblasts and adipocytes. Hyperglycemia has been shown to
acti vate PKC (86). Interestingly, Hz02, has also been shown to activate PKC (120). If
oxidative stress is operative in the present high gIucose/insulin model, this rnechanism of
Akt l/PKBcx inactivation remains possible.
lmpaired AktIPKB activation has also been shown to occur in 3T3-L1 adipocytes
treated with HzOz Lipoic acid, an antioxidant like NAC, was able to prevent the
impairment in Aktl/PKBa activation induced by H301. These findings suggest that
Akt l/PKBa activation may be sensitive to the oxidation state of the cell.
Activation of A k t l P K B a has been associated with the translocation of Gfut4 to
the plasma membrane in response to insulin. Insulin resistant cells (high glucose/insulin
treated) displayed approximately 50% lower Akt 1 P K B u activation than the control o r
NAC treated cells. How much of a reduction in activity is necessary to impair Glut4
translocation is not yet known. Wang et al. (4 1) compared various dominant-negative
mutants of AktlPKBa on Glut4 translocation and showed that a 2- fold increase in
Akt l /PKBa activity was enough to fully translocate Glut4 to the plasma membrane. In
the present study, the resistant cells displayed a 7-foid increase in Akt l / P K B a activity
above basal. Whether the decrease in Akt l/PKBcc activity can account for the impairment
in Glut4 translocation is not certain.
In addition to Aktl/PKBa, rat adipocytes also express Akt2/PKw (121). Most
studies in the past have focused mainly on Aktl/PKBa. Recently, numerous studies have
implicated Akt2/PKBP in the stimulation of Glut4 translocation by insulin in rat
adipocytes (29,42). Preliminary data from our laboratory suggest that AktYPKBB
activation by insulin is not impriired with high glucose/insulin treatment.
Treatment with NAC alone led to a reduction in Aktl/PKBa activation by insulin
wi th no change in Ser473 phosphorylation. Why NAC reduced Akt l/PKBa activation is
presently not ciear. There are two possibilities that may explain the discordance between
Aktl/PKBu activation and Ser473 phosphorylation. First, full activation of AktlPKBa
requires phosphorylation on both Ser473 and Thr308 (21). Phosphorylation on either
residue alone results in only partial activation of the kinase (21). The possibility exists
that phosphorylation on Thr308 may be defective. Second, the kinase assay may be a
more sensitive measure that Ser473 imrnunoblotting.
There are several possibilities that may explain the different signaling defects
observed between the present study, from those observed in 3T3-L1 adipocytes exposed
to H?O1 (lO4,lO5,ll5). First, the cells used were different. Although both types represent
adipocytes, the two celi types may respond differently to various treatments. For
example, chronic incubation of 3T3-L1 adipocytes with insulin was able to markedly
decrease insulin-stimulated glucose uptake (78), white having a minimal effect in isolated
rat adipoc ytes (73). In addition, chronic incubation of 3T3-L 1 adipocytes wi th glucose
and insulin decreased Glut4 levels (78), something not seen in isolated adipocytes (73).
Second, the method of generating reactive oxygen species was different between the two
models of insulin resistance. In the HIOz model, reactive oxygen species were generated
in the media surrounding the cells, whereas the high glucose/insulin model suggests that
reactive oxygen species are generated inside the ce11 from metabolism. Reactive oxygen
species generated outside the ce11 may activate different stress pathways. It is possible
that cells may respond differently to various forrns of oxidative stress.
Overall, whife various modets of insulin resistance have a11 shown impaired
translocation of Glut4 to the PM in response to insulin, there have been no consistent
signahg defects observed. One possibility may be that different factors which induce
insulin resistance generate different signals resulting in differential defects in insulin
signaling. Another possibility may be the duration of the treatments. Some defects rnay
occur early on, while others take longer to manifest.
To ssummarize and conclude, NAC. an antioxidant and glutathione (GSH)
precunor, was able to prevent the impairment in Glut4 translocation induced by high
glucose/insuiin and glucosamine. NAC was also able to prevent the decrease in Glutl
expression and Akt l/PKBu activation and phosphorylation observed in ce1 1s treated wi th
high glucoselinsulin. These findings suggest a role for oxidative stress as a potential
mechanism in the induction of insulin resistance in this model. In addition, these findings
are relevant for Type II diabetic patients where hyperglycemia and hypennsulinemia are
IikeIy present and may worsen penpheral insulin resistance. Finally, NAC may provide
the potential design for novel therüpeutic agents.
GENERAL DISCUSSION
The studies described in this thesis (Chapters 2 and 3) were designed to
investigate the potential role of AktUPKBa in the stimulation of glucose uptake and
Glut4 translocation using 2 different models. In the first model (Chapter 2) using insulin,
vanadate, and pV to stimulate glucose uptake, the data support a role for AktlPKBa in
Glut3 translocation mediated by insulin. Thus wortmannin pretreatment of cultured rat
skeletal muscle cells (L6 cells) inhi bited P13K and subsequent activation of Akt l/PKBu
by insulin i n association with inhibition of insuiin-mediated Glut4 translocation and
glucose uptake (43). In the second model, further support for Akt l/PKBu in insulin
stimulated Glut4 translocation came from the study of Akt l/PKBa in rat adipoocytes
rendered insulin resistant by treatment with high glucose/insulin, In these cells insulin
stimulated Glut4 translocation and glucose uptake were impaired by 50% and 60%
respectively. In these same cells, Aktl/PKBa activation by insuIin was reduced by 44%.
Important1 y, CO-incubation with NAC, which prevented the impairment in Glut4
translocation and glucose uptake, also corrected the defect in Akt l/PKBa activation. The
close association between these parameters in both models strongly supports a role for
Akt l/PKBa in mediating insulin induced Glut4 translocation and subsequent glucose
uptake. Chronic treatment of rat adipocytes with glucosamine also resulted in impaired
Glut4 translocation and glucose uptalie stimulated by insulin. Co-incubation with NAC
prevented these defects. The activation of Akt l/PKBa in this third model has not yet
been studied but will be important to detennine. These data strongly suggest but do not
prove that the Akt lPKBa defect is responsible for the insulin resistance of glucose
transport. To funher examine this question, it would be worthwhile to activate
AktlPKBu in these insulin resistant cells. This could be accomplished by using okadaic
acid (56) or by transfecting a constitutively active forrn of the enzyme (38110). The
effects on GIut4 translocation could then be observed. To date, a specific inhibitor of
Akt l/PKBa has not been found (22). The majority of studies implicating AktlPKBu in
Glut4 translocation have used consti tutivel y active or dominant negati ve forms of the
enzyme. The development of a specific pharmacological inhibitor would also contribute
significantty to Our understanding of the role of this enzyme in the stimulation of Glut4
translocation by insulin.
In these studies the site of the glucose transport defect in the insulin resistant
(High GA treated) adipocytes appears to lie at least in part at the leveI of Aktl/PKBoc.
However, the cause of the defect in this enzyme is not yet clear. Thus, insulin-stimulated
tyrosine phosphorylation of IRS-1 and the association of IRS-1 with the p85 regulatory
subunit of PI3K was found to be normal. In general, this association implies an intact
ability of insulin to stimulate PUK and generate the lipid second messengers PtdIns-3,4-
PI and PtdIns-3,4,5-P3. The steps between PI3K and Akt l/PKBa include activation of
PDKl and PD= which are immediately downstrearn of PI3K and upstream of
Akt IPKBa. Future studies must determine whether these enzymes are normally
activated in response to insulin or are defective in the insuiin resistant adipocytes.
Another possibility to explain such a defect is increased activity of a lipid phosphatase
such as SHIP (SH2-domain containing inositol 3' phosphatase). This could potentially
result in a reduction in the active PI3K Iipid products in the face of normal PI3K activity.
Finally, the data presented here (Chapter 2) using vanadate and pV implicate that
at least one novel pathway independent o f PI3K and Aktl/PKBa exists by which glucose
uptake and Glut4 translocation may be stimulated.
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