Rosiglitazone, an agonist of peroxisome-proliferator-activated receptor gamma (PPARgamma), decreases inhibitory serine phosphorylation of IRS1 in vitro and in vivo.

Abstract

Peroxisome-proliferator-activated receptor gamma agonists such as rosiglitazone, a thiazolidinedione, improve insulin sensitivity in vivo, but the underlying mechanism(s) remains unclear. Phosphorylation of IRS1 (insulin receptor substrate protein 1) on certain serine residues, including S307 and S612 in rodent IRS1 (equivalent to S312 and S616 in human IRS1), has been shown to play a negative role in insulin signalling. In the present study, we investigated whether rosiglitazone improves insulin sensitivity by decreasing IRS1 inhibitory serine phosphorylation. In HEK-293 (human embryonic kidney 293) cells stably expressing recombinant IRS1 and in 3T3L1 adipocytes, rosiglitazone attenuated PMA-induced IRS1 S307/S612 phosphorylation and decreased insulin-stimulated Akt phosphorylation. We observed increased IRS1 S307 phosphorylation and concomitant decrease in insulin signalling as measured by insulin-stimulated IRS1 tyrosine phosphorylation, and Akt threonine phosphorylation in adipose tissues of Zucker obese rats compared with lean control rats. Treatment with rosiglitazone at 30 mg/kg body weight for 24 and 48 h increased insulin signalling and decreased IRS1 S307 phosphorylation concomitantly. Whereas the 48 h treatment reversed hyper-phosphorylation (and activation) of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, the 24 h treatments only decreased hyper-phosphorylation of p38 mitogen-activated protein kinase. The treatment of the Zucker obese rats with rosiglitazone also reversed the high circulating levels of non-esterified fatty acids, which have been shown to be correlated with increased IRS1 serine phosphorylation in other animal models. Taken together, these results suggest that IRS1 inhibitory serine phosphorylation is a key component of insulin resistance and its reversal contributes to the insulin sensitizing effects by rosiglitazone.

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Biochem. J. (2004) 377, 339–346 (Printed in Great Britain)
339
Rosiglitazone, an agonist of peroxisome-proliferator-activated receptor γ
(PPARγ), decreases inhibitory serine phosphorylation of IRS1 in vitro
and in vivo
Guoqiang JIANG1, Qing DALLAS-YANG, Subarna BISWAS, Zhihua LI and Bei B. ZHANG1
Molecular Endocrinology – Diabetes, Merck Research Laboratories, RY80N-C31, P.O. Box 2000, Rahway, NJ 07065, U.S.A.
Peroxisome-proliferator-activated receptor γ agonists such as
rosiglitazone, a thiazolidinedione, improve insulin sensitivity
in vivo, but the underlying mechanism(s) remains unclear. Phos-
phorylation of IRS1 (insulin receptor substrate protein 1) on
certain serine residues, including S307 and S612 in rodent IRS1
(equivalent to S312 and S616 in human IRS1), has been shown to
play a negative role in insulin signalling. In the present study, we
investigated whether rosiglitazone improves insulin sensitivity by
decreasing IRS1 inhibitory serine phosphorylation. In HEK-293
(human embryonic kidney 293) cells stably expressing recom-
binant IRS1 and in 3T3L1 adipocytes, rosiglitazone attenuated
PMA-induced IRS1 S307/S612 phosphorylation and decreased
insulin-stimulated Akt phosphorylation. We observed increased
IRS1 S307 phosphorylation and concomitant decrease in insulin
signalling as measured by insulin-stimulated IRS1 tyrosine phos-
phorylation, and Akt threonine phosphorylation in adipose
tissues of Zucker obese rats compared with lean control rats.
Treatment with rosiglitazone at 30 mg/kg body weight for 24 and
48 h increased insulin signalling and decreased IRS1 S307 phos-
phorylation concomitantly. Whereas the 48 h treatment reversed
hyper-phosphorylation (and activation) of both c-Jun N-terminal
kinase and p38 mitogen-activated protein kinase, the 24 h treat-
ments only decreased hyper-phosphorylation of p38 mitogen-
activated protein kinase. The treatment of the Zucker obese rats
withrosiglitazonealsoreversedthehighcirculatinglevelsofnon-
esterified fatty acids, which have been shown to be correlated
with increased IRS1 serine phosphorylation in other animal
models. Taken together, these results suggest that IRS1 inhibitory
serine phosphorylation is a key component of insulin resistance
and its reversal contributes to the insulin sensitizing effects by
rosiglitazone.
Key words: insulin receptor substrate 1, insulin sensitization,
peroxisome-proliferator-activated receptor, rosiglitazone, serine
phosphorylation.
INTRODUCTION
TZDs (thiazolidinediones, e.g. rosiglitazone, troglitazone and
pioglitazone)areaclassofantidiabeticdrugsthatimproveinsulin
sensitivity in vivo [1,2]. The physiological effects of TZDs are
well documented. For instance, they decrease the levels of circu-
lating insulin, non-esterified fatty acids and triacylglycerols,
and increase insulin-stimulated glucose uptake and utilization
in both animal models and human subjects [1,2]. TZDs are
ligands of PPARγ (peroxisome-proliferator-activated receptor
γ), a member of a larger family of the ligand-activated nuclear
receptor transcription factors. Whereas it is generally thoughtthat
binding and activation of PPARγ is the predominant mechanism
by which TZDs mediate their antidiabetic efficacy, the precise
moleculareventsdownstreamofPPARγ activationthatarecritical
for TZDs-mediated insulin sensitization in vivo are less clear [3].
The action of insulin is mediated via the IR (insulin receptor).
IR is a heterotetramer consisting of two extracellular α-subunits
(IRα) and two transmembrane β-subunits (IRβ) with intrinsic
protein tyrosine kinase activity. The binding of the insulin
to IRα subunit leads to autophosphorylation and activation of the
IRβ subunit.Cytoplasmicsignalling moleculesincludingIRS1–4
(IR substrate proteins 1–4) bind to the phosphotyrosine residues
in the activated IRβ subunits and subsequently become tyrosine
phosphorylated by IRβ subunits. The phosphotyrosine residues
Abbreviations used: ECF, enhanced chemifluorescence; ECL®, enhanced chemiluminescence; IR, insulin receptor; IRS1–4, IR substrate proteins 1–4;
JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; mpk, mg/kg body weight; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C;
PPARγ, peroxisome-proliferator-activated receptor γ; TZD, thiazolidinedione; upk, units/kg body weight; for brevity the one-letter system for amino acids
has been used; S307, e.g., means Ser307.
1Correspondence may be addressed to either author (e-mail guoqiang jiang@merck.com or bei-zhang@merck.com).
in the IRS proteins provide docking sites for further downstream
signallingmoleculesincludingthep85subunitofthePI3K(phos-
phoinositide3-kinase).Thisbindingleadstoactivationofthep110
catalytic subunit of the PI3K, a key mediator required for insulin-
induced physiological effects. Activated PI3K catalyses the pro-
duction of phosphatidylinositide 3,4-biphosphate and phosphat-
idylinositide3,4,5-triphosphate,whichinturnbindtoandactivate
further downstream kinases including the phosphoinositide-
dependent serine/threonine kinase 1, Akt and PKCλ/ζ (protein
kinase C), all of which are known to play key roles in insulin-
induced metabolic effects [4,5].
There is emerging evidence that TZDs exert their insulin-
sensitizing effects at least partially by potentiating insulin signal-
ling. Insulin-stimulated PI3K and Akt activation is blunted in
diabetic and insulin-resistant states in animals and human beings
[6–10].TreatmentswithTZDspotentiateinsulin-stimulatedPI3K
and Akt activation in cultured cells in vitro [10–13]. Long-
termtreatmentwithrosiglitazoneincreasesinsulin-stimulatedAkt
phosphorylationintheskeletalmuscleofnormoglycaemichuman
beings at risk for Type II diabetes [14]. Long-term treatment
with rosiglitazone, particularly in combination with exercise, also
increased insulin-stimulated IRβ subunit tyrosine phosphoryl-
ation and Akt serine phosphorylation in Zucker obese rats [8]. We
reported previously that treatments with PPARγ agonists includ-
ing rosiglitazone acutely normalize impaired insulin signalling
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G. Jiang and others
intheZuckerobeseratsbyincreasingtheinsulin-stimulatedphos-
phorylation of IRβ subunits, IRS1, Akt and glycogen synthase
kinase 3 in both adipose and muscle tissues [10].
Thestudiesdiscussedaboveindicatethatpotentiationofinsulin
signalling is probably a part of the mechanisms underlying
PPARγ agonist-mediated insulin sensitization in vivo. The de-
tailedmechanismsatthesignallingmoleculelevelwarrantfurther
study. In addition to tyrosine phosphorylation, the IR and IRS
proteins undergo serine phosphorylation that attenuates insulin
signalling [15–19]. The inhibitory phosphorylation of IRS1 is
known to occur on multiple serine residues, including S307 and
S612 of rodent IRS1 (equivalent to S312 and S616 of human
IRS1; for convenience, S307 and S612 are used throughout the
paper for both human and rodent IRS1) [16,17,20–23]. To date,
several serine/threonine kinases have been implicated in this
process; these include PKC isoforms (i.e. PKCθ) [24,25], the
inhibitor of nuclear factor-κB kinase β [26–29] and JNK (c-Jun
N-terminal kinase) [17,30,31]. These inhibitory serine phos-
phorylation events function as negative feedback loops for insulin
signal transduction and provide a basis for cross-talk with other
pathways through which insulin resistance may be mediated [32].
Indeed, increase in IRS1 serine phosphorylation and/or activation
of serine kinases have been observed in tissues of obese animals
[31], animals infused with non-esterified fatty acids [25], and in
primary adipocytes of Type II diabetic human subjects [33].
To further our understanding of the mechanism underlying
PPARγ agonists-mediated insulin sensitization, the present study
was initiated to investigate whether increased IRS1 inhibitory
serine phosphorylation occurs under insulin-resistant states and,
if so, whether such inhibitory phosphorylation can be reversed by
treatments with rosiglitazone in vitro as well as in vivo.
MATERIALS AND METHODS
Materials
Insulin and PMA were purchased from Sigma. Rabbit polyclonal
antibodies against total IRS1 (IRS1-total) and IRS1-pS307
(IRS1 phosphorylated at S307) and monoclonal antibody against
tyrosine-phosphorylated proteins (clone 4G10) were purchased
from Upstate Biotechnology (Lake Placid, NY, U.S.A.). Rabbit
polyclonalantibodiesagainstIRS1,phosphorylatedatS612,were
purchasedfromBioSourceInternational(Camarillo,CA,U.S.A.).
Rabbit polyclonal antibodies against total Akt (Akt-total), and
Akt-pT308 or Akt-pS473 (Akt phosphorylated at T308 or S473),
total p38 (p38-total) MAPK (mitogen-activated protein kinase),
p38-pT180/Y182(p38MAPKphosphorylatedatT180andT182),
total p42/44 MAPK (p42/44-total), p42/44-pT202/Y204 (p42/44
MAPK phosphorylated at T202 and T204), total JNK (JNK-total)
and JNK-pT183/Y185 (JNK phosphorylated at T183 and T185)
were purchased from Cell Signaling (Beverly, MA, U.S.A.).
Cell culture, rosiglitazone treatment and protein extraction
HEK-293 cellsstably
IRS1 (HEK-293.IRS1 cells) were a gift from Dr R. Roth
(Stanford University, CA, U.S.A.). The cells were maintained
in Dulbecco’s modified Eagle’s medium with 10% (v/v) fetal
bovine serum, 100 units/ml penicillin and 100 µg/ml strepto-
mycin (Life Technologies, Grand Island, NY, U.S.A.) at 37◦C
in 10% CO2. 3T3L1 cells were maintained and differentiated
into adipocytes in an 8-day programme as described previously
[34]. For experiments, cells were first incubated with vehicle
expressingrecombinanthuman
(DMSO) or rosiglitazone in growth medium for 8 h and then
with vehicle or rosiglitazone in serum-free Dulbecco’s modified
Eagle’s medium overnight. DMSO or PMA (1 µM final con-
centration) was then added to the cells for 30 min. The cells were
exposed further to 100 nM insulin or vehicle for 15 min, washed
with ice-cold PBS and then subjected to lysis.
In vivo experiments with genetically obese Zucker (fa/fa) rats
Lean and obese Zucker female rats were purchased from
CharlesRiverLaboratories(Wilmington,MA,U.S.A.).Ratswere
kept on a 12 h light/dark cycle at constant room temperature.
Conventional laboratory diet and tap water were provided ad lib.
until 4 h before the animals were killed, when food was with-
drawn. At the time of experiment, the rats were approx. 11–
12 weeks old. There were eight rats per treatment group. The rats
were given a dose of 30 mpk (mg/kg body weight) rosiglitazone
orvehicle(0.5%methylcellulose)viaoralgavageinthemorning.
Twenty-four hour after the first dose (24 h treatment) or after the
seconddose(48 htreatment)undertheadlib.fedcondition,blood
samples were collected from the portal vein for determination
of the concentrations of non-esterified fatty acids in circulation
as described previously [35]. The animals were then subjected
to anaesthesia (intraperitoneal injection of nemtubal sodium
solution at the dose of 2.5 mg/animal) and approx. 200–300 mg
of abdominal fat was removed from each rat. Insulin was then
infused via portal veins at the dose of 0.5 upk (unit/kg body
weight). Similar amount of abdominal fat was removed again
from each of the rats 60 s after insulin infusion. Tissue samples
wereimmediatelyfrozeninliquidnitrogenandstoredat −80◦C.
The method for removing the tissue samples from intact animals
before and after insulin treatment has been used in earlier papers
[10,36]. The protocol for the animal studies was approved by
IACUC (Institutional Animal Care and Use Committee).
Immunoprecipitation and Western-blot analysis of cell
and tissue lysates
Proteins were extracted from HEK-293 cells and 3T3L1 adipo-
cytes in ice-cold lysis buffer [20 mM Hepes, pH 7.4/1% Triton
X-100/20 mM β-glycerophosphate/150 mM sodium chloride/
1 mMsodiumorthovanadate/10 mMsodiumfluoride/1×concen-
tration of a protease inhibitor cocktail (Roche Diagnostics,
Indianapolis,IN,U.S.A.)].Proteinsweresimilarlyextractedfrom
tissue samples of Zucker lean and obese rats but with the aid of a
Polytron homogenizer (Fisher Scientific, Pittsburgh, PA, U.S.A.).
Cell or tissue lysates were cleared by centrifugation. Protein
concentrationsweredeterminedusingBradfordreagent(Bio-Rad
Laboratories, Hercules, CA, U.S.A.). For immunoprecipitation,
lysates from adipose tissues were mixed with antiserum against
total IRS1 for 2–4 h and then mixed with Protein A/G–agarose
beads for another 1–4 h at 4◦C with gentle shaking. The beads
werewashedthreetimeswithlysisbuffers.Celllysatesorimmun-
oprecipitateswereresuspendedinSDSloadingbuffer(Invitrogen,
Carlsbad, CA, U.S.A.) and separated in precast 4–12% gradient
NuPAGE SDS/PAGE gels (Invitrogen). The proteins were then
transferred to PVDF membrane and probed with primary anti-
bodies. Detection was carried out using ECF (enhanced chemi-
fluorescence) or ECL®(enhanced chemiluminescence) Western
Blotting kit (Amersham Biosciences, Piscataway, NJ, U.S.A.).
WesternblottingwiththeECFmethodwasperformedbyscanning
withaStorm®gelandblotimagingsystem(MolecularDynamics,
Piscataway, NJ, U.S.A.) according to the manufacturer’s
recommendation. Western blotting using the ECL®method was
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Rosiglitazone decreases insulin receptor substrate 1 serine phosphorylation
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Figure 1
phosphorylation by rosiglitazone in HEK-293.IRS1 cells
Reversal of PMA-induced inhibition of insulin-stimulated Akt
Cells were treated with vehicle DMSO or 10 µM rosiglitazone (Rosi) and serum-starved
overnight. The cells were then treated with vehicle DMSO or 1 µM PMA for 30 min, and
thenexposedtovehicleaceticacidor100 nMinsulinasdescribedintheMaterialsandmethods
section.Equivalentamountsofcrudeproteinswereloadedontoeachofthelanes.Western-blot
analysis was performed using antibody against Akt-total or Akt-pS473. Similar abbreviations
are used in all the Figures. Similar results were obtained in more than three independent
experiments.
performed with a film according to the manufacturer’s recom-
mendation.
RESULTS
Rosiglitazone reverses PMA-induced inhibition of
insulin-stimulated Akt phosphorylation in HEK-293.IRS1 cells
PMAhasbeenshowntoinhibitinsulinsignallinginculturedcells
[15,37,38]. Akt phosphorylation (and activation) is a key event in
insulin signalling and has been commonly used as a surrogate
marker for assessing the levels of the activation of insulin-
signalling pathway [39]. Therefore we determined whether
PMA inhibits insulin-stimulated Akt phosphorylation, and if so,
whether such inhibition could be reversed by rosiglitazone. As
shown in Figure 1, stimulation with 100 nM insulin for 15 min
increased Akt-pS473 in HEK-293 cells stably expressing recom-
binant human IRS1 (HEK-293.IRS1 cells) (lane 5 versus lane 1).
Pretreatment with 1 µM PMA for 30 min decreased insulin-
stimulated Akt phosphorylation (lane 7 versus lane 5). Pretreat-
ment with 10 µM rosiglitazone overnight reversed the negative
effects by PMA (lane 8 versus lane 7). Taken together, these
results suggest that PMA inhibits insulin signalling in HEK-
293.IRS1 cells and that such inhibition of PMA can be reversed
by pretreatment with rosiglitazone.
Rosiglitazone reverses PMA-induced IRS1 S307 and S612
phosphorylation in HEK-293.IRS1 cells
PMA has been shown to inhibit insulin signalling by promoting
inhibitoryphosphorylationofIRS1onmultipleresiduesincluding
S307 and S612 [15,18,40,41]. Accordingly, we determined
whether the inhibitory phosphorylation of IRS1 on these sites
could be reversed by rosiglitazone in the HEK-293.IRS1 cells. As
shown in Figure 2, treatment with 1 µM PMA for 30 min resulted
in an approx. 3-fold increase in IRS1 S307 and S612 phos-
phorylation. Furthermore, such phosphorylation was largely re-
versed by overnight pretreatment with 10 µM rosiglitazone to a
levelindistinguishablefromthatinthevehicle-treatedcells.Taken
together, these results showed that PMA induces IRS1 serine
phosphorylation and that such inhibitory phosphorylation can
Figure 2
by rosiglitazone in HEK-293.IRS1 cells
Reversal of PMA-induced IRS1 phosphorylation at S307 and S612
Cells were maintained and treated with vehicle DMSO (Veh) or Rosi as described in Figure 1.
Western-blot analysis was performed using antibody against total IRS1 (IRS1-total) or IRS1-
pS307 or S612 (IRS1-pS612) (A). Quantification (B, C) was done using PhosphorImaging. In
the bar charts, bars labelled with different letters are statistically significantly different in t test
(P <0.05). Similar labelling was used for all the Figures. Similar results were obtained in two
independent experiments.
be effectively blocked by rosiglitazone treatment in the HEK-
293.IRS1 cells.
Rosiglitazone reverses the effect of PMA on Akt and IRS1
phosphorylation in differentiated 3T3L1 adipocytes
HEK-293.IRS1 cells were chosen for the above study because
these cells overexpress recombinant IRS1, facilitating the detec-
tion of IRS1 serine phosphorylation by Western-blot analysis. To
test our hypothesis in a more physiologically relevant setting,
we subsequently determined the effect of PMA and rosiglitazone
on Akt and IRS1 phosphorylation in differentiated 3T3L1 adipo-
cytes. As shown in Figure 3, stimulation with 100 nM insulin for
15 min increased Akt-pS473 in the adipocytes (lane 5 versus lane
1). Pretreatment with 1 µM PMA for 30 min decreased insulin-
stimulated Akt phosphorylation (lane 7 versus lane 5). Overnight
pretreatment with 10 µM rosiglitazone attenuated the negative
effects of PMA on Akt phosphorylation (lane 8 versus lane 7).
Taken together, these results suggest that, as in HEK-
293.IRS1 cells, PMA negatively affects insulin signalling in
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Figure 3
phosphorylation by rosiglitazone in 3T3L1 adipocytes
Reversal of PMA-induced inhibition of insulin-stimulated Akt
Cells were maintained and differentiated into adipocytes by an 8-day programme as described
in the Materials and methods section. As in Figures 1 and 2, the adipocytes were treated with
vehicle DMSO or 10 µM Rosi and serum-starved overnight. The cells were then treated further
withvehicleDMSOor1 µMPMAfor30 minandthenexposedtovehicleaceticacidor100 nM
insulin. Western-blot analysis was performed using antibody against Akt-total or Akt-pS473.
Similar results were obtained in three independent experiments.
Figure 4
rosiglitazone in differentiated 3T3L1 adipocytes
Reversal of PMA-induced IRS1 phosphorylation at S307 by
Cells were maintained and treated with vehicle (Veh), Rosi and PMA as described in Figure 3.
Western-blot analysis was performed using antibody against IRS1-total or IRS1-pS307 (A).
Quantification for the levels of IRS1-pS307 was done using PhosphorImaging (B). Similar
results were obtained in three independent experiments.
3T3L1 adipocytes and such inhibition can be reversed by
rosiglitazone.
As shown in Figures 4(A) and 4(B), PMA treatment resulted in
approx. 2-fold increase in IRS1 S307 phosphorylation in 3T3L1
adipocytes. Furthermore, overnight pretreatment with rosiglita-
zone largely blocked PMA-induced IRS1 S307 phosphorylation.
Taken together, these results suggest that PMA induces IRS1
S307 phosphorylation and that such inhibitory phosphorylation
can be effectively blocked by rosiglitazone treatment in 3T3L1
adipocytes.
Rosiglitazone acutely potentiates insulin signalling and decreases
IRS1 S307 phosphorylation in the tissues in Zucker obese rats
in vivo
We next determined whether insulin signalling (i.e. insulin-
stimulated Akt phosphorylation) is impaired and IRS1 serine
Figure 5
the adipose tissues of Zucker obese rats by rosiglitazone
Acute potentiation of insulin-stimulated Akt phosphorylation in
AsdescribedintheMaterialsandmethodssection,Zuckerobeseratsweregivenonesingleoral
dose of rosiglitazone at 30 mpk. As control, Zucker obese rats as well as lean littermates were
given a dose of vehicle (Veh). Twenty-four hours post dosing and after anaesthetization, tissues
fromtheanimalsweresampledimmediatelybeforeandafterinsulin(0.5 upk)infusion.Adipose
tissueswerecollectedfromtheanimalsimmediatelybeforeandafter insulininfusion.Eightrats
wereusedpertreatment.Fortheimageshown,crudelysatesfromtheeightindividualratsfroma
giventreatmentweremixedandthenloadedontoasinglelaneintheSDS/PAGEgel.Equivalent
amountsofproteinsfromeachofthetreatmentswereloadedontoeachlane.Resultsareshown
forWestern-blotanalysisonadiposetissuesusingantibodyagainstAkt-totalorAkt-pT308.The
t test was based on another analysis using eight animals per treatment. Similar results were
obtained in two independent experiments.
phosphorylation is increased in the adipose tissues of Zucker
obese rats relative to their lean littermates. We investigated
further whether rosiglitazone can improve insulin signalling and
decrease IRS1 serine phosphorylation in adipose tissues in a con-
certedfashion.AsdescribedintheMaterialsandmethodssection,
Zucker obese rats were given vehicle or rosiglitazone at 30 mpk
for24or48 h.Afterthetreatmentandanaesthesia,adiposetissues
from the animals were sampled immediately before and after
insulin infusion. As shown in Figure 5, infusion of insulin at
0.5 upk via portal vein increased Akt phosphorylation at T308
in the adipose tissue of the lean animals (lane 2 versus lane 1). In
contrast, no insulin-stimulated Akt T308 phosphorylation was
observed in the adipose tissues of the obese animals treated with
vehicle (lane 4 versus lane 3) and this defect was corrected in
the adipose tissue of the obese animals treated with rosiglitazone
(lane 6 versus lane 5). The extent of increase in Akt T308 phos-
phorylation by insulin in the obese animals treated with rosig-
litazone is similar to that in the lean animals (lanes 5 and 6
versus lanes 1 and 2). Taken together, these results suggest that
insulinsignallingisbluntedintheadiposetissuesofZuckerobese
rats and can be acutely normalized by treatment with rosiglit-
azone.
As shown in Figure 6, Western-blot analysis showed that, on
insulin stimulation, there was increased IRS1 S307 phosphoryl-
ation and decreased IRS1 tyrosine phosphorylation (lane 2 versus
lane1)intheadiposetissueoftheZuckerobeserats.Furthermore,
IRS1 S307 and tyrosine phosphorylation was normalized by the
24 h treatment with rosiglitazone (lane 3 versus lane 2). Taken to-
gether, these results indicate that, in adipose tissues, increased
IRS1 S307 phosphorylation is correlated with insulin resistance
and that the reversal of IRS1 S307 phosphorylation is correlated
with insulin sensitization by rosiglitazone in Zucker obese rats
in vivo.
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Rosiglitazone decreases insulin receptor substrate 1 serine phosphorylation
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Figure 6
IRS1 tyrosine phosphorylation in the adipose tissues of Zucker obese rats by
rosiglitazone
Reduction of IRS1 serine phosphorylation and potentiation of
For the image shown, crude adipose lysates from eight individual rats from a given treatment
after insulin infusion were mixed (1 mg of total crude proteins). The mixtures were subjected to
immunoprecipitation using antisera against IRS1-total. After SDS/PAGE, Western-blot analysis
was performed using antisera against IRS1-total, IRS1-pS307 or tyrosine-phosphorylated
proteins. Results of the Western-blot analysis are shown. Levels of IRS1-pS307 and IRS1-
pTyr (IRS1 phosphorylated tyrosine) were quantified using PhosphorImaging. The t test was
based on another analysis using eight animals per treatment. Owing to the large number of
samples (eight for each group, 24 in total), multiple gels were used for the quantitative analysis
and therefore the images are not shown. Similar results were obtained in two independent
experiments.
Rosiglitazone differentially inhibits p38 and JNK activation in the
adipose tissues of the Zucker obese rats in vivo
Tounderstandhowrosiglitazonetreatment decreasesserinephos-
phorylationofIRSproteins,wedeterminedwhetherrosiglitazone
treatments altered the activity of potentially relevant serine/
threoninekinasesintheadiposetissues.AsshowninFigures7(A)
and 7(B), MAPK p38 was hyper-phosphorylated (and therefore
hyper-activated) in the adipose tissues of Zucker obese rats
when compared with that in the lean control rats. Rosiglitazone
treatment for 24 h decreased p38 phosphorylation in the adipose
tissues of the obese rats to a level that was lower than that obser-
vedintheadiposetissuesoftheleanrats.AsshowninFigures7(A)
and 7(C), JNK was also hyper-phosphorylated (and therefore
hyper-activated) in the adipose tissues of Zucker obese rats when
compared with that in the lean control rats. Whereas 48 h rosigl-
itazone treatment decreased JNK phosphorylation in the adipose
tissues of the obese rats, 24 h rosiglitazone treatment did not. As
shown in Figure 7(A), in contrast with p38 and JNK, MAPK
p42/44 was not found to be hyper-phosphorylated in the adipose
tissues of the obese rats. Furthermore, the results showed that
rosiglitazone treatments did not affect p42/44 phosphorylation in
the adipose tissues of obese rats.
Figure 7
adipose tissue of Zucker obese rats by rosiglitazone
Inhibition of p38 and JNK phosphorylation (and activation) in the
Results of Western-blot analysis using adipose tissues from animals before insulin infusion
and antiserum against total p38 (p38-total), p38-pT180/Y182, p42/44-pT202/Y204, total JNK
(JNK-total) and pJNK-pT183/Y185 are shown. The image includes two representative animals
from each treatment. Quantification was done using PhosphorImaging. Similar results were
obtained in two independent experiments.
Rosiglitazone acutely decreases circulating non-esterified fatty
acid levels in the Zucker obese rats in vivo
It was reported that increased plasma non-esterified fatty acids
led to increased intracellular levels of certain fatty acyl-CoAs
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Figure 8
Zucker obese rats by rosiglitazone
Acute reduction of circulating non-esterified fatty acid levels in
Non-esterified fatty acid levels of the plasma in Zucker obese rats treated with vehicle for 24 h
(Veh), obese rats treated with rosiglitazone at 30 mpk for 24 h (Rosi) and control lean rats are
shown. The method used for measuring non-esterified fatty acids is mentioned in the Materials
and methods section. Similar results were obtained in two independent experiments.
and diacylglycerols, activation of serine/threonine kinases, in-
creased IRS1 serine phosphorylation, decreased IRS1 tyrosine
phosphorylation and insulin resistance [25]. To understand the
mechanism by which rosiglitazone potentiates insulin signalling
and decreases IRS S307 phosphorylation in vivo, we determined
the effect of rosiglitazone treatment on circulating non-esterified
fatty acid levels in Zucker obese rats. As shown in Figure 8,
Zucker obese rats had much higher levels of circulating non-
esterified fatty acids when compared with the lean control rats.
Furthermore, treatment with rosiglitazone at 30 mpk for 24 h
significantly decreased the level of non-esterified fatty acids in
the Zucker obese rats to the level seen in the lean control rats.
DISCUSSION
It has been shown previously that PPARγ agonists, including
rosiglitazone, exert their insulin-sensitizing effects at least par-
tially by improving insulin signalling in vitro and in vivo. For
instance, they promote insulin-stimulated tyrosine phosphory-
lation of IR and IRS1, PI3K activity associated with IRS proteins,
activating phosphorylation of Akt/protein kinase B and inhibitory
phosphorylation of glycogen synthase kinase 3 [10–13,42].
However, the mechanisms by which PPARγ agonists potentiate
insulin signalling remain unclear. One of the possibilities is that
treatment with PPARγ agonists regulates the expression of the
components of the insulin-signalling cascade, i.e. IRS1 [12],
the p85 subunit of the PI3K kinase [43] and c-Cbl-associated
protein [44]. However, different studies examining the expression
of these proteins by PPARγ agonists have yielded rather different
results. Serine phosphorylation of IRS proteins is known to in-
hibit insulin signalling and may represent a common molecular
mechanism for insulin resistance [32,45]. Accordingly, the
present study examined the possibility of whether the PPARγ
agonist rosiglitazone potentiates insulin signalling by reversing
inhibitory serine phosphorylation of IRS proteins such as IRS1.
WedemonstratedthatthePPARγ agonistrosiglitazonereverses
PMA-induced insulin resistance (Figure 1) and concomitantly
reverses PMA-induced S307 and S612 phosphorylation of re-
combinant human IRS1 in HEK-293.IRS1 cells (Figure 2). These
cells were chosen in the present study since the recombinant
human IRS1 protein was expressed at relatively high levels,
making it possible to detect readily IRS1 serine phosphorylation
by Western-blot analysis. We subsequently performed similar ex-
perimentsondifferentiated3T3L1adipocytes.Theresultsshowed
that, in both HEK-293.IRS1 cells and differentiated 3T3L1 adi-
pocytes, PMA inhibits insulin-stimulated Akt phosphorylation
and promotes IRS1 S307 and S612 phosphorylation and that such
effects of PMA can be reversed by rosiglitazone treatment (Fig-
ures3and4).Together,theseresultsprovidethefirstevidencethat
the ability of the PPARγ agonist to potentiate insulin signalling
is correlated with their ability to reverse IRS S307 and S612
phosphorylation in cultured cells.
Subsequent Western-blot analyses in the present study also
showed that, in adipose tissues, the levels of IRS S307 phos-
phorylation are significantly increased in the Zucker obese rats
comparedwiththeleancontrolrats(Figure6).Similarresultshave
been reported recently [31]. Taken together, these results strongly
suggest that increased inhibitory serine phosphorylation of the
IRSproteinsisassociatedwithandislikelytoplayaroleininsulin
resistance in multiple insulin responsive tissues in the disease
state. Most interestingly, Western-blot analysis showed that, in
adipose tissues, 24 and 48 h treatment with rosiglitazone acutely
reversed IRS1 S307 hyper-phosphorylation in Zucker obese rats
to the levels seen in the lean control rats (Figure 6). These results
provide important evidence that a PPARγ agonist decreases IRS1
inhibitory serine phosphorylation in one of the key insulin res-
ponsive tissues in vivo.
It is known that IRS isoforms (IRS1–4) are expressed differ-
entially and appear to play different roles in various insulin res-
ponsive tissues [32]. Owing to the lack of antibodies specific for
serine-phosphorylated proteins of other IRS isoforms (particu-
larlyIRS2),wehavenotinvestigatedwhetherincreasedinhibitory
serinephosphorylationoftheotherIRSproteinsalsooccursunder
insulin-resistant states in vivo or the effect of rosiglitazone on
such inhibitory phosphorylation. It will be of interest to study
the regulation of IRS2 serine/threonine phosphorylation in liver
since IRS1 serine phosphorylation was not detected in the present
study.
There is evidence suggesting that inhibition of IRS1 serine
hyper-phosphorylation may be physiologically relevant to
PPARγ-agonist-mediated insulin sensitization in vivo. We repor-
ted previously that, in Zucker obese rats, treatment with PPARγ
agonists promotes insulin-stimulated IRS1 tyrosine phosphory-
lation in vivo [10]. Serine phosphorylation is known to inhibit
IRS tyrosine phosphorylation [17,20–23,30]. Accordingly, a
decrease in IRS1 serine phosphorylation could directly contribute
to increased insulin signalling (i.e. increased IRS1 tyrosine
phosphorylation and Akt serine/threonine phosphorylation) by
rosiglitazone in the Zucker obese rats as we observed. Such a
proposal is supported further by the observation that the effect
on IRS1 serine phosphorylation and insulin signalling by
rosiglitazone in vivo occurred in a concomitant fashion, i.e. all
within 24 h after the initiation of the treatment.
The detailed underlying mechanism by which PPARγ agonists
reverse IRS1 serine phosphorylation remains unclear. It was
reported previously that increased plasma non-esterified fatty
acids lead to increased intracellular levels of certain fatty acyl-
CoAs and diacylglycerols, activation of PKCθ, increased IRS1
S307 phosphorylation, and decreased IRS1 tyrosine phosphory-
lation, PI3K activation and Akt activation [24,25,46]. Since 24 h
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Rosiglitazone decreases insulin receptor substrate 1 serine phosphorylation
345
treatment with rosiglitazone decreased the levels of circulating
non-esterifiedfattyacidsintheZuckerobeseratstothelevelsseen
intheleancontrolrats(Figure8),itisplausiblethatdecreasedIRS
S307 phosphorylation in the same time frame could result from
decreased non-esterified fatty acids and subsequent deactivation
of certain serine/threonine kinases that are responsible for phos-
phorylating IRS1 in the insulin-resistant state.
In the present study, we have demonstrated that rosiglitazone
treatment for 48 h also decreased JNK hyper-phosphorylation in
the adipose tissues of the obese rats (Figure 7). It was reported
that JNK is hyper-activated in obese animals [31] and that
rosiglitazonetreatmentdecreasesthelevelsofthephosphorylated
and activated JNK1 in the heart of diabetic rats [47]. JNK1 has
been shown to bind to IRS1 and also to phosphorylate IRS1 at
S307 directly in vitro [17,30]. JNK1 deficiency improves insulin
sensitivity in vivo [31]. However, since the decrease in JNK
hyper-phosphorylation in the adipose tissues was not observed
after 24 h rosiglitazone treatment (Figure 7) when potentiation of
insulin signalling was observed (Figure 6), these results suggest
that the insulin-sensitizing effect of rosiglitazone was not primar-
ily mediated by inhibition of JNK activity. On the other hand,
rosiglitazone treatment for 24 h decreased hyper-phosphorylation
of p38 MAPK in the adipose tissues of the obese rats (Figure 7).
Since the decrease in p38 phosphorylation and the potentiation of
insulin signalling in the adipose tissues occurred concomitantly
within 24 h after the initiation of rosiglitazone treatment, the
inhibition of p38 activation may play a role in decreasing IRS1
serine phosphorylation by rosiglitazone in the adipose tissue.
Since p38 has not been shown to phosphorylate directly IRS pro-
teins, further investigation is needed to understand the potential
role of p38 inhibition in rosiglitazone-mediated insulin sensitiz-
ation. It was reported that the mammalian target of rapamycin/
p70S6kinaseplaysapotentiallyimportantnegativeroleininsulin
signalling by affecting IRS1 [48,49]. It was also reported that p70
S6 kinase was activated in amino acids but not lipid-induced
insulin resistance in perfused rat hearts [50]. Whether p70 S6
kinase was hyper-activated in the tissues of Zucker obese rats and
inhibited by rosiglitazone treatments was not investigated in the
present study but warrants further investigation.
In summary, results from the present study suggest that hyper-
phosphorylation of IRS1 on the serine residues occurs in adipose
tissue in obese animals, and that treatment with rosiglitazone
decreases IRS1 serine phosphorylation in cultured cells and in
animal models, potentiates insulin signalling in vitro and in adi-
pose tissues in vivo, decreases circulating non-esterified fatty
acids in vivo and inhibits MAPK p38 activation in adipose tissues
in vivo.Takentogether,weproposethatthereverseofIRS1serine
phosphorylation is probably physiologically relevant to rosiglit-
azone-mediated insulin sensitization in vivo. It remains to be
seen whether similar effects can be observed with other PPARγ
agonists, particularly those that are structurally distinct from
rosiglitazone. Finally, the detailed mechanism(s) underlying the
ability of rosiglitazone to decrease IRS1 serine phosphorylation
also requires further investigation.
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Received 8 August 2003/24 September 2003; accepted 13 October 2003
Published as BJ Immediate Publication 13 October 2003, DOI 10.1042/BJ20031207
c ?2004 Biochemical Society