Am J Physiol Endocrinol Metab. Aug 2008; 295(2): E393–E400.
Published online Jun 10, 2008. doi: 10.1152/ajpendo.90355.2008
Effect of pioglitazone treatment on endoplasmic reticulum stress response in
human adipose and in palmitate-induced stress in human liver and adipose cell
Swapan K. Das,
Steven C. Elbein
Winston S. Chu, Ashis K. Mondal,Neeraj K. Sharma, Philip A. Kern, Neda Rasouli, and
Endocrinology Section, Medicine and Research Services, Central Arkansas Veterans Healthcare System, Little Rock; and Division of
Endocrinology and Metabolism, Department of Medicine, College of Medicine, University of Arkansas for Medical Sciences, Little Rock,
Address for reprint requests and other correspondence: S. C. Elbein or Swapan K. Das, Univ. of Arkansas for Medical Sciences,
Endocrinology 111J-1/LR, John L. McClellan Memorial Veterans Hospital, 4300 W. 7th St., Little Rock, AR 72205 (e-mail:
ud e . smau@cnev e t sn i eb l e or ud e . smau@sadks)
Received April 10, 2008; Accepted June 6, 2008.
Copyright © 2008, American Physiological Society
Obesity and elevated cytokine secretion result in a chronic inflammatory state and may cause the insulin
resistance observed in type 2 diabetes. Recent studies suggest a key role for endoplasmic reticulum stress
in hepatocytes and adipocytes from obese mice, resulting in reduced insulin sensitivity. To address the
hypothesis that thiazolidinediones, which improve peripheral insulin sensitivity, act in part by reducing the
endoplasmic reticulum stress response, we tested subcutaneous adipose tissue from 20 obese volunteers
treated with pioglitazone for 10 wk. We also experimentally induced endoplasmic reticulum stress using
palmitate, tunicamycin, and thapsigargin in the human HepG2 liver cell line with or without pioglitazone
pretreatment. We quantified endoplasmic reticulum stress response by measuring both gene expression and
phosphorylation. Pioglitazone significantly improved insulin sensitivity in human volunteers (P = 0.002)
but did not alter markers of endoplasmic reticulum stress. Differences in pre- and posttreatment
endoplasmic reticulum stress levels were not correlated with changes in insulin sensitivity or body mass
index. In vitro, palmitate, thapsigargin, and tunicamycin but not oleate induced endoplasmic reticulum
stress in HepG2 cells, including increased transcripts CHOP, ERN1, GADD34, and PERK, and increased
XBP1 splicing along with phosphorylation of eukaryotic initiation factor eIF2α, JNK1, and c-jun. Although
patterns of endoplasmic reticulum stress response differed among palmitate, tunicamycin, and thapsigargin,
pioglitazone pretreatment had no significant effect on any measure of endoplasmic reticulum stress,
regardless of the inducer. Together, our data suggest that improved insulin sensitivity with pioglitazone is
not mediated by a reduction in endoplasmic reticulum stress.
Keywords: insulin sensitivity, fatty acid, thiazolidinediones, type 2 diabetes, obesity
THE EARLY PATHOPHYSIOLOGY of type 2 diabetes remains uncertain. Three key defects mark the onset of
hyperglycemia in type 2 diabetes mellitus: increased hepatic glucose production, diminished insulin
secretion, and impaired insulin action (7). Obesity generally accompanies type 2 diabetes and is the key
modifiable risk factor. Recent data suggest that obesity leads to a state of chronic inflammation marked by
1,2 1,21,21,2 1,2 1,2
Page 1 of 18 Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipo...
presence of ER stress in obese humans, we examined subcutaneous adipose tissue from 86 no diabetic
individuals (body mass index 19–40 kg/m ) who participated in several previous studies. The sample
included 70 women and 16 men, aged 21–66 yr, of whom 32 individuals had impaired glucose tolerance.
Pioglitazone effects on ER stress were tested in 20 individuals with impaired glucose tolerance (IGT) who
were randomized to receive pioglitazone at 30 mg/day for 2 wk, then 45 mg/day for 8 wk (30). We report
only the pioglitazone arm of the study, which randomized individuals to pioglitazone or metformin (36).
Adipose and muscle biopsies and frequently sampled intravenous glucose tolerance tests were performed
before and after treatment. All subjects provided written informed consent under protocols approved by
University of Arkansas for Medical Sciences Institutional Review Board.
dimethyl sulfoxide (Sigma, St. Louis, MO). ER stress was induced as described below by tunicamycin
(Sigma), thapsigargin (Sigma), oleic acid (MP Biomedical, Solon, OH), or palmitic acid (Calbiochem, La
Jolla, CA). Tunicamycin was dissolved in DMSO, and thapsigargin was dissolved in ethanol. Palmitic and
elevated cytokine levels, which in turn may reduce insulin action. However, the proximal cause and
molecular mechanisms of this inflammatory response are uncertain.
Recently, Ozcan et al. (25) and others (15, 22, 24) proposed endoplasmic reticulum (ER) stress as the
proximal cause of chronic inflammation and reduced insulin action in adipocytes and hepatocytes. In mice,
both high fat diet and genetic obesity (ob/ob mice) activated proximal sensors of ER stress, including
increased transcription of GRP78 (also known as BiP, encoded by human gene HSPA5) and increased
phosphorylation of proximal ER stress proteins PERK and IRE1 (encoded by human gene ERN1) and
downstream factors including eukaryotic initiation factor eIF2α, JNK1, c-jun, and ultimately IRS1 (25).
Manipulation of chaperones further downstream such as ORP150 (encoded by human gene HYOU1) also
altered insulin sensitivity in mice (22, 24). Finally, this same mechanism may account for altered insulin
secretion (14, 33), thus linking the three key defects in the pathogenesis of type 2 diabetes.
Thiazolidinediones (TZDs), which include the presently available drugs rosiglitazone and pioglitazone, are
well established insulin sensitizing agents that act, at least in part, as agonists of the peroxisome
proliferator-activated receptor (PPAR)γ. These drugs appear to act as primarily peripheral insulin
sensitizers, but hepatic effects have been suggested also (1). Although the mechanism of action remains
controversial, expansion of adipocyte mass may reduce ectopic fat and hence improve hepatic insulin
sensitivity and β-cell function (37). TZDs reduce inflammatory cytokines and increase adiponectin (30,
31). Hence, TZDs affect the same pathways and genes that characterize diabetes pathogenesis and might be
expected to act on the proximal cause, such as ER stress. Indeed, Loffler et al. (20) found significant
downregulation of ER stress genes in livers from diabetic db/db mice treated with rosiglitazone, including
HSPA5, DNAJC3, and X-box protein-1 (XBP1). Similarly, Han et al. (12) showed reduced liver and white
adipose ER stress in diabetic db/db mice treated with the dual PPARα/γ agonist macelignan. Macelignan
also suppressed thapsigargin-induced ER stress in mouse hepatocytes and adipocyte cell lines (12). These
studies suggest that the beneficial effects of TZDs on insulin sensitivity may result in part from a reduction
of ER stress, but no human data are available to address this hypothesis.
We tested the hypothesis that pioglitazone acts in part by reducing ER stress in two systems. First, we
tested whether markers of adipocyte ER stress were reduced in human subjects with impaired glucose
tolerance treated with pioglitazone for 10 wk. To extend these human studies, we next tested whether
pioglitazone was able to protect the human hepatocyte HepG2 cell line or the human adipocyte Simpson-
Golabi-Behmel syndrome (SGBS) cell line from experimentally induced acute ER stress. In both studies,
we quantified the key indicators of the ER stress response.
MATERIALS AND METHODS
The human studies have been described in detail previously (29, 38). To establish the
Pioglitazone (a gift from Takada Pharmaceutical, Deerfield, IL) was dissolved in
Page 2 of 18 Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipo...
cultured in minimum essential medium (MEM) with Earle's balanced salt solution supplemented with 2
mM L-glutamine, penicillin, streptomycin, and 10% fetal bovine serum (all from Omega Scientific,
Tarzana, CA). At 80–90% confluence, cells were transferred to medium containing 2% fetal bovine serum
(FBS). Cells were preincubated for 16 h in pioglitazone (10 μM) or control (DMSO), after which
tunicamycin (1 μg/ml), thapsigargin (25 nM), or vehicle (DMSO or ethanol) was added to the culture plates
in the presence or absence of pioglitazone (10 μM). Palmitic acid and oleic acid experiments were
conducted similarly, except that after the preincubation period, cells were washed twice in serum-free
MEM and transferred to serum-free medium with or without pioglitazone (10 μM) and palmitic acid-BSA
conjugate (1 mM), oleic acid-BSA conjugate (1 mM), or BSA. Cells were harvested at 6 and 12 h
posttreatment for RNA or protein extraction.
RNA isolation and gene expression.
an RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA). Total RNA from HepG2 cells was isolated using
the RNeasy Mini Kit (Qiagen) and from SGBS cells by using the RNAqueous kit (Ambion, Austin, TX)
following the manufacturer's instruction. The quantity of isolated RNA was determined
spectrophotometrically and the quality by electrophoresis using an Agilent 2100 Bioanalyzer (Agilent
Technologies, Santa Clara, CA). Total RNA (0.4–1 μg) was reverse transcribed using random hexamer
primers with TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). Reverse-
transcribed RNA (cDNA) was amplified with 1× Sybr Green PCR Master Mix (Applied Biosystems) and
0.3 μmol/l gene-specific forward and reverse primers on an ABI 7500 Fast RT-PCR system (Applied
Biosystems) or a Rotorgene 2000 Real-Time PCR system (Corbett Life Science, Sydney, Australia).
Samples were normalized to 18S ribosomal RNA. Standard curves were generated using pooled cDNA
from the samples assayed. Primer sequences (Supplemental Table S1; supplemental data are available at
the online version of this article) were designed to capture all major known splice forms, to span an intron,
and to give a single band of the expected size. Experiments in HepG2 cells were conducted with three
technical replicates for each of two biological replicates, yielding six readings for each experiment. XBP1
splicing was examined by gel electrophoresis and quantified by densitometry for cell culture experiments.
Additionally, spliced XBP1 was specifically assayed from human adipose tissue cDNA using real-time
Cruz Biotechnology; 1× TBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium
azide) supplemented with Protease Inhibitor Cocktail (10 μl/ml; Santa Cruz Biotechnology),
oleic acids were conjugated to fatty acid-free bovine serum albumin (BSA, MP Biomedical) at a 2.5:1
molar ratio by dissolving them in ethanol and mixing with an aqueous BSA solution (BSA in MEM culture
medium) at 45°C until homogeneous, after which they were passed through a 0.2 μM filter (18). All
references to palmitic and oleic acids refer to the BSA conjugate. Antibodies for Western blot experiments
included phospho-eIF2α (BioSource Division of Invitrogen, Carlsbad, CA), total eIF2α (Santa Cruz
Biotechnology, Santa Cruz, CA), PERK (Santa Cruz Biotechnology), phospho-PERK (Santa Cruz
Biotechnology), phospo-JNK1 (Santa Cruz Biotechnology), total JNK1 (Santa Cruz Biotechnology),
phospho-c-jun (Cell Signaling, Danvers, MA), and β-actin (Chemicon Division of Millipore, Billerica,
MA). Mouse and rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from
Amersham Biosciences (Pittsburgh, PA).
Human hepatocyte HepG2 (American Type Culture Collection, Manassas, VA) cells were
Human SGBS preadipocytes were cultured as described previously (3). Briefly, SGBS cells were
morphologically differentiated (70%) into mature adipocytes and conditioned in serum-free basal medium
(DMEM-F12) for 24 h, followed by 16 h of pretreatment with pioglitazone. ER stress was induced by
treatment with palmitic acid (0.5 mM) for 6–12 h in serum-free basal medium, after which cells were
harvested for RNA extraction.
Total RNA from human subcutaneous adipose tissue was isolated using
HepG2 Cells were washed with cold PBS and harvested using 1× RIPA buffer (Santa
Page 3 of 18 Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipo...
Data analysis and statistics.
nonparametric Mann-Whitney U-test for unpaired samples or the Kruskal-Wallis test for three or more.
Human adipose samples obtained before and after pioglitazone were compared using the Wilcoxon signed
rank test. Correlations between the change in insulin sensitivity (ΔS ), change in body mass index (ΔBMI),
and change in gene expression were calculated using the Spearman correlation coefficient or partial
regression controlling for either ΔBMI or ΔS . All analyses were performed in SPSS for Windows v12.0
(SPSS, Chicago, IL).
ER stress is increased with obesity in human subcutaneous adipose tissue.
tissue from obese mice, but no data are available in humans. To address the role of ER stress in humans,
we examined subcutaneous adipose tissue from 86 individuals over a range of BMI (19–40 kg/m ) for the
ER stress marker HSPA5, which is generally viewed as the best single marker of ER stress (25). HSPA5
transcript levels rose from lean to overweight and obese individuals (mean and SD: 0.73 ± 0.17 for lean;
0.89 ± 0.22 overweight; 1.15 ± 0.31 obese; Fig. 1), with a 57% increase in obese when compared with lean
individuals (P = 0.00006). Additionally, in 83 subjects for whom data were available, HSPA5 levels
correlated significantly with insulin sensitivity (S ; r = −0.43, P = 0.00006). Age did not differ significantly
among groups and did not contribute to ER stress. Thus, as in mice, ER stress is activated in human
subcutaneous adipose tissue with obesity.
Pioglitazone treatment does not reduce ER stress in human adipose tissue.
reduced ER stress response in liver and white adipose in diabetic db/db mice (12, 20). To determine
whether TZDs reduce ER stress in humans with IGT, we examined subcutaneous adipose from 20 obese
individuals with IGT (30) before and after 10 wk of pioglitazone. We measured six key transcriptional
markers of ER stress: HSPA5, CHOP, ATF6, ERN1, PERK, and XBP1. Despite a significant improvement
in S (Table 1; P = 0.002), levels of ER stress gene transcripts were unchanged (P > 0.12; Fig. 2;
Supplemental Fig. S1A). Splicing of XBP1 is a marker of acute ER stress. We did not observe spliced
XBP1 before or after pioglitazone therapy using a gel-based assay (Supplemental Fig. S1B), and real-time
quantification of spliced XBP1 transcript was not changed with pioglitazone therapy (Fig. 2). Because
pioglitazone increased BMI, and BMI is closely associated with ER stress markers in mice and humans
(see data above), we examined the correlation of increase in BMI with the change in gene expression. No
significant correlation was noted (P > 0.1); similarly, we found no significant correlation between the
change in S and the change in gene expression before and after pioglitazone, even when controlling for the
change in BMI (P > 0.3).
Pioglitazone does not protect HepG2 cells from tunicamycin- and thapsigargin-induced ER stress.
may be a more important source of ER stress than subcutaneous fat (39), thus explaining the lack of
reduction in adipocyte ER stress with pioglitazone in humans. Because we were unable to evaluate
phenylmethylsulfonyl fluoride (10 μl/ml), and Phosphatase Inhibitor Cocktail 1 (10 μl/ml, Sigma) and
Cocktail 2 (10 μl/ml, Sigma). Cell lysates were homogenized, and protein content of clear lysates was
estimated by the Bradford method (6). Equal amounts (20–30 μg) of total protein were dissolved in
Lammeli buffer (50 mM Tris, pH 6.8, 100 mM β-mercaptoethanol, 2% SDS, 10% glycerol), separated on
8% SDS-polyacrylamide gel, and transferred to Trans-Blot Nitrocellulose membrane (Bio-Rad
Laboratories, Hercules, CA). Proteins were detected by immunoblot according to the manufacturer's
protocols, developed with SuperSignal Electro-Chemiluminescent reagent (Pierce Biotechnology Division
of Thermo-Fisher Scientific, Waltham, MA) and scanned in ChemiDox Image Analyzer (Bio-Rad).
Western blots were quantified using Quantity One Image Analysis software (v4.6.3; Bio-Rad). All total
proteins were normalized to β-actin, and phosphoproteins were normalized to total protein and/or to
β-actin. Data represent samples from three independent experiments with technical replicates.
Experimental conditions in in vitro experiments were compared using the
ER stress is activated in adipose
PPARγ agonists significantly
Page 4 of 18 Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipo...
Palmitate but not oleate induces ER stress in HepG2 cells.
circulating free fatty acids (16). Hence, we asked whether a more physiological stress might be ameliorated
by pioglitazone in HepG2 cells. Palmitic (C16:0) acid (1 mM) induced ER stress with 12 h of incubation,
marked by significant (P = 0.04–0.002) elevation of CHOP, ERN1, GADD34, and ATF4 and increased
XBP1 splicing. In contrast, equimolar oleic acid was identical to control (Fig. 4). Notably, palmitate
induction of ER stress differed from that observed with tunicamycin or thapsigargin, both of which
strongly upregulated HSPA5, EDEM1, and ATF6 (Fig. 3 compared with Fig. 4 and Supplemental Table
Pioglitazone does not reduce palmitate-induced ER stress.
10 μM) and the presence of pioglitazone during palmitate treatment failed to reduce ER stress markers
induced by 6 or 12 h of 1 mM palmitate treatment, measured by transcript levels (Fig. 5A), XBP1 splicing (
Fig. 5B), phosphorylation of eukaryotic initiation factor eIF2α (see Fig. 6, B and C), or phosphorylation of
factors downstream of ERN1, including PERK, JNK1, and c-jun (Fig. 6, A, C, and E). Previous studies
showed induction of ER stress by a 30–50 μM concentration of TZD. In our study, 10 μM pioglitazone
showed no induction of ER stress transcripts even after 28 h (16-h pretreatment followed by a 12-h
treatment period; Supplemental Fig. S2). However, we observed a significant increase (P = 0.014) in
eukaryotic initiation factor eIF2α phosphorylation (Fig. 6C). To examine another model of human adipose,
we repeated this experiment in the human adipocyte SGBS cell line. Palmitate (0.5 mM) modestly
increased apoptotic markers CHOP, GADD34, and XBP1 splicing by 6 h; pioglitazone pretreatment for 16
h did not reduce markers of ER stress (Supplemental Fig. S3).
hepatocytes from pioglitazone-treated humans, we tested whether pioglitazone could protect against
induced ER stress in HepG2 cells. We initially induced acute ER stress using either tunicamycin (1 μg/ml)
or thapsigargin (25 nM). Both classic inducers of ER stress increased HSPA5 transcript levels and XBP1
splicing in a time-dependent fashion, and preincubation with pioglitazone failed to protect against markers
of ER stress with either inducer (Fig. 3). These data thus support the results in human adipose tissue.
Pioglitazone did have a pharmacological effect in HepG2 cells, as demonstrated by upregulation of known
PPARγ target ApoA2 by 47% (1.08 ± 0.11 control, 1.59 ± 0.08 pioglitazone), as reported by others (28).
Obese individuals have elevated levels of
Pioglitazone pretreatment of HepG2 cells (16 h,
TZDs are among the most effective drugs in humans at improving glucose homeostasis (32). Although
TZDs appear to act primarily to improve peripheral S , the precise mechanism by which these PPARγ
agonists act is unknown. Many pathways have been proposed from stimulation of adipogenesis to
activation of AMP kinase or increased adiponectin secretion (5). Animal studies have suggested that ER
stress in adipose and liver is a key source of insulin resistance and impaired glucose homeostasis (25), and
indeed reduction of ER stress using small molecule chaperones markedly improved glucose homeostasis in
vivo in whole animals (26). If ER stress is central to the impaired insulin action and disordered glucose
homeostasis of type 2 diabetes, drugs that effectively improve S could be expected to reduce ER stress.
Indeed, recent studies provide some support for that hypothesis in db/db mice (12, 20), whereas opposing
data in cell lines suggest that at least some TZDs may induce ER stress by non-PPARγ-mediated
mechanisms involving ER calcium depletion (9). Hence we sought evidence that the TZD pioglitazone acts
in part to relieve adipocyte or hepatic ER stress in humans. The present study is to our knowledge the only
one to examine human tissues and human-derived cell lines with a TZD in active clinical use.
In the present study, we examined each of the major branches of the ER unfolded protein response pathway
downstream of the proximal sensors PERK, ATF6, and ERN1 in subcutaneous adipose tissue from humans
treated with pioglitazone. The key findings of our study are that pioglitazone neither reduced nor increased
adipocyte markers of ER stress in individuals treated for 10 wk with pioglitazone, although these
individuals experienced a marked improvement in S . The improved S with pioglitazone in a similar
Page 5 of 18Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipo...
population has been reported by our laboratory previously (30). Thus the mechanism of improved S does
not appear to be amelioration of ER stress in adipocytes. Furthermore, pioglitazone suppressed
inflammatory adipokines and increased adiponectin levels (31). Although ER stress and inflammatory
pathways are tightly connected (13), the failure of pioglitazone to reduce ER stress markers while
improving S and reducing inflammatory cytokines suggests that these pathways can be dissociated, and
that a reduction of ER stress is not essential to improve S and glucose homeostasis.
We recognized that induction of ER stress in hepatocytes might be of additional or larger physiological
significance. Unfortunately liver biopsies pre- and post-pioglitazone treatment cannot be justified in
humans. Hence we also sought to develop a model of ER stress using the human-derived HepG2
hepatocyte cell line to test whether pioglitazone might protect from ER stress in that system. To examine
the role in hepatocytes, we tested the ability of pioglitazone to protect cells from ER stress induced by
several mechanisms, including the classic stressors thapsigargin and tunicamycin. As in adipocytes,
pioglitazone failed to protect HepG2 cells from ER stress induced by blocking of glycosylation
(tunicamycin) or altering of ER calcium levels (thapsigargin). Because these chemical stressors might be
considered nonphysiological, we searched for inducers of ER stress in hepatocytes that were closer to the
human adipocyte model. Recent reports suggested that levels of circulating free fatty acids in humans are
in the range of 0.7–0.8 mM in obese humans and 0.3–0.4 mM in lean individuals (16), thus suggesting that
the 1 mM concentrations of palmitate and oleate are near physiological. These studies resulted in a second
key finding in the present study: that palmitate but not oleate treatment induced markers of acute ER stress
in HepG2 cells.
Similar results showing induction of ER stress with saturated but not unsaturated fatty acids have been
reported in a rat liver hepatoma cell line (36), in mouse 3T3-L1 cells and cultured preadipocytes (11), and
in a mouse pancreatic β-cell line (17). In contrast, Ota et al. (23) recently reported that prolonged exposure
of another hepatocyte line to oleate induced steatosis and ER stress. The reason for this discrepancy is
unclear. The mechanism by which saturated fatty acids induce ER stress is uncertain, although recent data
suggest that ceramide accumulation is not part of the mechanism (35, 36). Borradaile et al. (4) provided
evidence for both intracellular accumulation of reactive oxygen and impaired ER morphology resulting
from saturated lipid accumulation and compromised ER membrane integrity.
Previous studies with GN4 rat liver epithelial cells, RINm5F rat pancreatic β-cells, and HepG2 cells (9, 21,
34) showed induction of ER stress with TZD treatment as evidenced by increased phosphorylation of
PERK and eIF2α, as well as increased CHOP transcript levels. The doses of pioglitazone used in the
present study (10 μM) were well above levels effective for transcriptional activation of human PPARγ
(0.28 μM), but well below levels previously shown to be cytotoxic (10). In contrast, ciglitazone and
troglitazone were cytotoxic at much lower levels (10), and treatment levels that induced ER stress were
much closer to levels demonstrated to have cytotoxicity. Thus we observed no induction ER stress
transcripts or splicing of XBP1 in pioglitazone-treated HepG2 cells in the present study, and neither
Maniratanachote et al. (21) nor Weber et al. (34) observed increased levels of HSPA5 transcripts at
rosiglitazone or troglitazone concentrations below 25–30 μM. Like those previous studies, we did observe
a significant increase in eIF2α phosphorylation, suggesting possible selective induction of ER stress by low
doses of TZDs.
Recent work from Lin et al. (19) suggested that the ER stress responses differ over the time course of the
stress and in different tissues. Such observations suggest the importance of studying multiple tissues and
cell lines. For example, in our experiments, ERN1 was threefold increased with palmitate, whereas that
increase was not reported in rat hepatoma cell lines (36). Similarly, different inducers of ER stress gave
very different patterns of response, both in our studies and in other reports, suggesting that different
stresses activate the three proximal ER response pathways to different degrees (8, 27). Regardless of the
Page 6 of 18Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipo...
mechanism and in both liver and adipose, we found that pioglitazone failed to protect against induction of
Surprisingly, few other studies have addressed the role of TZDs or insulin sensitizers on ER stress despite a
strong rationale for TZD effects on the ER stress response in the improvement of S . Han et al. (12)
recently reported that the relatively weak dual α/γ-agonist macelignan significantly improved glucose
homeostasis and insulin action, reduced inflammation, and reduced both hepatic and adipocyte indicators
of ER stress in db/db mice including JNK phosphorylation. In contrast, the TZD troglitazone had no
significant effect on ER stress markers (12). Additionally, macelignan but not troglitazone was protective
in both the 3T3-L1 mouse adipocyte and SK-HEP1 mouse hepatic cell lines treated with either tunicamycin
or thapsigargin (12). These data suggest that effects of macelignan on ER stress are not acting through
PPARγ and thus are similar to our findings with pioglitazone in human tissues.
If, as suggested by our studies and those of Han et al. (12), TZDs do not act to reduce ER stress, small
molecule chaperones (26) that improve insulin action and glucose homeostasis could be synergistic with
TZDs. Although our study is the first to address this question in human tissues, our study also has
limitations that are inevitable in a human-based study. First, the liver may be a more important source of
ER stress-induced insulin resistance than adipocytes but could not be studied in humans. We have used cell
lines to address the role of TZDs in the liver, although they are an imperfect surrogate for human
hepatocytes. Furthermore, given the ability of palmitate to induce ER stress in HepG2 cells, we cannot
exclude the possibility that TZDs act indirectly by reducing plasma free fatty acids to decrease hepatic ER
stress and thus hepatic insulin resistance (1, 2). Free fatty acid levels before and after pioglitazone were not
available in our study. Likewise, we cannot exclude the possibility that TZDs have an effect on ER stress in
visceral adipose, which to our knowledge has not been addressed in mice either. The individuals studied
before and after pioglitazone were otherwise healthy, obese individuals with impaired glucose tolerance.
Both data presented here for ER stress transcript HSPA5 in 86 individuals and extensive studies of
transcription and phosphorylation in subcutaneous adipose tissue from an independent population strongly
support the activation of ER stress with obesity (S. C. Elbein, unpublished data). Whether individuals with
diabetes or poor glucose control have a different pattern of ER stress is presently under study, and we
cannot exclude the possibility that TZDs might reduce ER stress in the setting of hyperglycemia. Finally,
muscle is thought to be the primary organ involved in postprandial glucose uptake and thus peripheral S .
We did not include this tissue because ER stress is not activated in muscle from obese db/db mice (25) or
in humans (S. C. Elbein, unpublished data).
In summary, despite improved S and reduced inflammatory cytokines and increased adiponectin in obese,
glucose-intolerant subjects treated with pioglitazone, our data suggest no improvement in adipocyte ER
stress. These findings are supported by the failure of pioglitazone to protect either the HepG2 hepatocyte or
SGBS adipocyte cell lines from ER stress induced by a variety of mechanisms. We suggest that, in humans,
one can dissociate reduced inflammation and improved S from reduced ER stress. Thus, although
inflammation, ER stress, and S are interconnected, reduced adipocyte ER stress is not necessary to reduce
inflammation and improve S . Whether reductions in ER stress using small molecule chaperones could also
improve S in humans remains to be demonstrated.
P. A. Kern has received speaking honoraria from Takeda Pharmaceuticals. N. Rasouli has received
investigator-initiated grants in addition to honoraria for speaking engagements from Takeda and Abbott
Page 7 of 18 Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipo...
This work was supported by the Research Service of the Department of Veterans Affairs (merit funds to S.
C. Elbein, P. A. Kern, and N. Rasouli; Research Enhancement Award Program funds) and in part by the
General Clinical Research Center (grant no. M01-RR-14288 from the National Center for Research
Resources, National Institutes of Health, to the University of Arkansas for Medical Sciences).
[Supplemental Figures and Tables]
We thank the nursing and laboratory staff of the General Clinical Research Center at the University of
Arkansas for Medical Sciences and study coordinators Leslie Miles and Regina Dennis for assisting with
subject recruitment and support of the clinical studies. We thank Hua Wang for assistance with
development of real-time PCR assays and Takeda Pharmaceuticals for providing pioglitazone for the in
vitro experiments and partial support of the pioglitazone trial.
The costs of publication of this article were defrayed in part by the payment of page charges. The article
must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1. Basu R, Shah P, Basu A, Norby B, Dicke B, Chandramouli V, Cohen O, Landau BR, Rizza RA.
Comparison of the effects of pioglitazone and metformin on hepatic and extra-hepatic insulin action in
people with type 2 diabetes. Diabetes 57: 24–31, 2008. [PubMed: 17914032]
2. Boden G, Zhang M. Recent findings concerning thiazolidinediones in the treatment of diabetes. Expert
Opin Investig Drugs 15: 243–250, 2006.
3. Bodles AM, Banga A, Rasouli N, Ono F, Kern PA, Owens RJ. Pioglitazone increases secretion of high-
molecular-weight adiponectin from adipocytes. Am J Physiol Endocrinol Metab 291: E1100–E1105, 2006.
4. Borradaile NM, Han X, Harp JD, Gale SE, Ory DS, Schaffer JE. Disruption of endoplasmic reticulum
structure and integrity in lipotoxic cell death. J Lipid Res 47: 2726–2737, 2006. [PubMed: 16960261]
5. Bouskila M, Pajvani UB, Scherer PE. Adiponectin: a relevant player in PPARgamma-agonist-mediated
improvements in hepatic insulin sensitivity? Int J Obes (Lond) 29, Suppl 1: S17–S23, 2005.
6. Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976. [PubMed: 942051]
7. DeFronzo RA Pathogenesis of type 2 diabetes mellitus. Med Clin North Am 88: 787–835, ix, 2004.
8. DuRose JB, Tam AB, Niwa M. Intrinsic capacities of molecular sensors of the unfolded protein response
to sense alternate forms of endoplasmic reticulum stress. Mol Biol Cell 17: 3095–3107, 2006.
[PMCID: PMC1483043] [PubMed: 16672378]
9. Gardner OS, Shiau CW, Chen CS, Graves LM. Peroxisome proliferator-activated receptor gamma-
independent activation of p38 MAPK by thiazolidinediones involves calcium/calmodulin-dependent
Page 8 of 18 Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipo...
protein kinase II and protein kinase R: correlation with endoplasmic reticulum stress. J Biol Chem 280:
10109–10118, 2005. [PubMed: 15649892]
10. Guo L, Zhang L, Sun Y, Muskhelishvili L, Blann E, Dial S, Shi L, Schroth G, Dragan YP. Differences
in hepatotoxicity and gene expression profiles by anti-diabetic PPAR gamma agonists on rat primary
hepatocytes and human HepG2 cells. Mol Divers 10: 349–360, 2006. [PubMed: 17031537]
11. Guo W, Wong S, Xie W, Lei T, Luo Z. Palmitate modulates intracellular signaling, induces
endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes. Am J
Physiol Endocrinol Metab 293: E576–E586, 2007. [PubMed: 17519282]
12. Han KL, Choi JS, Lee JY, Song J, Joe MK, Jung MH, Hwang JK. Therapeutic potential of peroxisome
proliferators–activated receptor-alpha/gamma dual agonist with alleviation of endoplasmic reticulum stress
for the treatment of diabetes. Diabetes 57: 737–745, 2008. [PubMed: 18065517]
13. Hotamisligil GS Role of endoplasmic reticulum stress and c-Jun NH -terminal kinase pathways in
inflammation and origin of obesity and diabetes. Diabetes 54, Suppl 2: S73–S78, 2005.
14. Kaneto H, Nakatani Y, Kawamori D, Miyatsuka T, Matsuoka TA, Matsuhisa M, Yamasaki Y. Role of
oxidative stress, endoplasmic reticulum stress, and c-Jun N-terminal kinase in pancreatic beta-cell
dysfunction and insulin resistance. Int J Biochem Cell Biol 37: 1595–1608, 2005. [PubMed: 15878838]
15. Kaneto H, Matsuoka TA, Nakatani Y, Kawamori D, Miyatsuka T, Matsuhisa M, Yamasaki Y.
Oxidative stress, ER stress, and the JNK pathway in type 2 diabetes. J Mol Med 83: 429–439, 2005.
16. Kankaanpaa M, Lehto HR, Parkka JP, Komu M, Viljanen A, Ferrannini E, Knuuti J, Nuutila P,
Parkkola R, Iozzo P. Myocardial triglyceride content and epicardial fat mass in human obesity: relationship
to left ventricular function and serum free fatty acid levels. J Clin Endocrinol Metab 91: 4689–4695, 2006.
17. Karaskov E, Scott C, Zhang L, Teodoro T, Ravazzola M, Volchuk A. Chronic palmitate but not oleate
exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic beta-cell
apoptosis. Endocrinology 147: 3398–3407, 2006. [PubMed: 16601139]
18. Li H, Ruan XZ, Powis SH, Fernando R, Mon WY, Wheeler DC, Moorhead JF, Varghese Z. EPA and
DHA reduce LPS-induced inflammation responses in HK-2 cells: evidence for a PPAR-gamma-dependent
mechanism. Kidney Int 67: 867–874, 2005. [PubMed: 15698426]
19. Lin JH, Li H, Yasumura D, Cohen HR, Zhang C, Panning B, Shokat KM, Lavail MM, Walter P. IRE1
signaling affects cell fate during the unfolded protein response. Science 318: 944–949, 2007.
[PMCID: PMC3670588] [PubMed: 17991856]
20. Loffler M, Bilban M, Reimers M, Waldhausl W, Stulnig TM. Blood glucose-lowering nuclear receptor
agonists only partially normalize hepatic gene expression in db/db mice. J Pharmacol Exp Ther 316: 797
–804, 2006. [PubMed: 16260581]
21. Maniratanachote R, Minami K, Katoh M, Nakajima M, Yokoi T. Chaperone proteins involved in
troglitazone-induced toxicity in human hepatoma cell lines. Toxicol Sci 83: 293–302, 2005.
22. Nakatani Y, Kaneto H, Kawamori D, Yoshiuchi K, Hatazaki M, Matsuoka TA, Ozawa K, Ogawa S,
Hori M, Yamasaki Y, Matsuhisa M. Involvement of endoplasmic reticulum stress in insulin resistance and
diabetes. J Biol Chem 280: 847–851, 2005. [PubMed: 15509553]
Page 9 of 18 Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipo...
23. Ota T, Gayet C, Ginsberg HN. Inhibition of apolipoprotein B100 secretion by lipid-induced hepatic
endoplasmic reticulum stress in rodents. J Clin Invest 118: 316–332, 2008. [PMCID: PMC2104481]
24. Ozawa K, Miyazaki M, Matsuhisa M, Takano K, Nakatani Y, Hatazaki M, Tamatani T, Yamagata K,
Miyagawa J, Kitao Y, Hori O, Yamasaki Y, Ogawa S. The endoplasmic reticulum chaperone improves
insulin resistance in type 2 diabetes. Diabetes 54: 657–663, 2005. [PubMed: 15734840]
25. Ozcan U, Cao Q, Yilmaz E, Lee AH, Iwakoshi NN, Ozdelen E, Tuncman G, Gorgun C, Glimcher LH,
Hotamisligil GS. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science
306: 457–461, 2004. [PubMed: 15486293]
26. Ozcan U, Yilmaz E, Ozcan L, Furuhashi M, Vaillancourt E, Smith RO, Gorgun CZ, Hotamisligil GS.
Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2
diabetes. Science 313: 1137–1140, 2006. [PubMed: 16931765]
27. Pirot P, Ortis F, Cnop M, Ma Y, Hendershot LM, Eizirik DL, Cardozo AK. Transcriptional regulation
of the endoplasmic reticulum stress gene chop in pancreatic insulin-producing cells. Diabetes 56: 1069
–1077, 2007. [PubMed: 17395747]
28. Qin S, Liu T, Kamanna VS, Kashyap ML. Pioglitazone stimulates apolipoprotein A-I production
without affecting HDL removal in HepG2 cells: involvement of PPAR-alpha. Arterioscler Thromb Vasc
Biol 27: 2428–2434, 2007. [PubMed: 17872455]
29. Rasouli N, Kern PA, Reece EA, Elbein SC. Effects of pioglitazone and metformin on beta-cell function
in nondiabetic subjects at high risk for type 2 diabetes. Am J Physiol Endocrinol Metab 292: E359–E365,
2007. [PubMed: 16968813]
30. Rasouli N, Raue U, Miles LM, Lu T, Di Gregorio GB, Elbein SC, Kern PA. Pioglitazone improves
insulin sensitivity through reduction in muscle lipid and redistribution of lipid into adipose tissue. Am J
Physiol Endocrinol Metab 288: E930–E934, 2005. [PubMed: 15632102]
31. Rasouli N, Yao-Borengasser A, Miles LM, Elbein SC, Kern PA. Increased plasma adiponectin in
response to pioglitazone does not result from increased gene expression. Am J Physiol Endocrinol Metab
290: E42–E46, 2006. [PubMed: 16118250]
32. Seufert J, Lubben G, Dietrich K, Bates PC. A comparison of the effects of thiazolidinediones and
metformin on metabolic control in patients with type 2 diabetes mellitus. Clin Ther 26: 805–818, 2004.
33. Wang H, Kouri G, Wollheim CB. ER stress and SREBP-1 activation are implicated in beta-cell
glucolipotoxicity. J Cell Sci 118: 3905–3915, 2005. [PubMed: 16091421]
34. Weber SM, Chambers KT, Bensch KG, Scarim AL, Corbett JA. PPARγ ligands induce ER stress in
pancreatic β-cells: ER stress activation results in attenuation of cytokine signaling. Am J Physiol
Endocrinol Metab 287: E1171–E1177, 2004. [PubMed: 15315910]
35. Wei Y, Wang D, Pagliassotti MJ. Saturated fatty acid-mediated endoplasmic reticulum stress and
apoptosis are augmented by trans-10, cis-12-conjugated linoleic acid in liver cells. Mol Cell Biochem 303:
105–113, 2007. [PubMed: 17426927]
36. Wei Y, Wang D, Topczewski F, Pagliassotti MJ. Saturated fatty acids induce endoplasmic reticulum
stress and apoptosis independently of ceramide in liver cells. Am J Physiol Endocrinol Metab 291: E275
–E281, 2006. [PubMed: 16492686]
Page 10 of 18 Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adi...
37. Yang X, Smith U. Adipose tissue distribution and risk of metabolic disease: does thiazolidinedione-
induced adipose tissue redistribution provide a clue to the answer? Diabetologia 50: 1127–1139, 2007.
38. Yao-Borengasser A, Varma V, Bodles AM, Rasouli N, Phanavanh B, Lee MJ, Starks T, Kern LM,
Spencer HJ 3rd, Rashidi AA, McGehee RE Jr, Fried SK, Kern PA. Retinol binding protein 4 expression in
humans: relationship to insulin resistance, inflammation, and response to pioglitazone. J Clin Endocrinol
Metab 92: 2590–2597, 2007. [PMCID: PMC2893415] [PubMed: 17595259]
39. Yoshiuchi K, Kaneto H, Matsuoka TA, Kohno K, Iwawaki T, Nakatani Y, Yamasaki Y, Hori M,
Matsuhisa M. Direct monitoring of in vivo ER stress during the development of insulin resistance with ER
stress-activated indicator transgenic mice. Biochem Biophys Res Commun 366: 545–550, 2008.
Figures and Tables
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Endoplasmic reticulum (ER) stress is activated in subcutaneous adipose tissue of obese human subjects. Subcutaneous
adipose tissue expression of the ER chaperone HSPA5 from 86 individuals over a range of body mass indexes (BMIs) from
19 to 40 kg/m shows significant increase from lean to overweight and obese individuals. Raw expression values of HSPA5
were normalized to 18S RNA. Data are presented as means ± SD of normalized HSPA5 expression for 3 BMI classes: lean
(BMI <25 kg/m ), overweight (BMI ≥25 kg/m and BMI ≤30 kg/m ), or obese (BMI >30 kg/m ). P = statistical
significance determined from Mann-Whitney U-test.
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Adipocyte ER stress markers in response to pioglitazone. Subcutaneous adipose tissue expression of 5 ER stress pathway
genes before and after pioglitazone treatment in 20 human volunteers. Raw expression values of ER stress response
(ERSR) genes were normalized to 18S RNA. Data are presented as means ± SD of 18S-normalized gene expression.
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ER stress response to tunicamycin or thapsigargin with pioglitazone pretreatment. A: expression of ER stress transcripts
after 16 h of pretreatment with pioglitazone or DMSO, followed by 12 h of treatment with tunicamycin (1 μg/ml) or control
(DMSO). B: ER stress transcripts after 16 h of pretreatment of pioglitazone or DMSO, followed by 12 h of treatment with
thapsigargin (25 nM) or control (ethanol). Raw expression values were normalized to 18S RNA. All data were normalized
to the lowest value of the control condition as 1 and are presented as means ± SD for 3 independent experiments with 2
biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control (pioglitazone alone). C: representative gels
showing spliced and unspliced XBP1 transcripts for the pioglitazone/tunicamycin experiment described in A. D:
representative gel showing XBP1 splicing for the pioglitazone/thapsigargin experiment described in B.
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Palmitate induces ER stress. A: expression of ER stress transcripts after 12 h of treatment with 1 mM palmitate, 1 mM
oleate, or BSA control. Raw expression values were normalized to 18S RNA. Values were expressed relative to the lowest
control value as 1 and are presented as means ± SD for 3 independent experiments with 2 biological replicates. *P < 0.05
and **P < 0.01 vs. control (BSA). B: XBP1 splicing for the experiment in A from a representative gel.
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