Mechanisms of human insulin resistance and
thiazolidinedione-mediated insulin sensitization
D. D. Searsa,1, G. Hsiaob, A. Hsiaob, J. G. Yua, C. H. Courtneya, J. M. Ofrecioa, J. Chapmanc, and S. Subramaniamb,1
Departments ofaMedicine andbBioengineering, University of California at San Diego, La Jolla, CA 92093; andcPfizer, Inc., La Jolla, CA 92121
Edited by Inder M. Verma, The Salk Institute for Biological Studies, La Jolla, CA, and approved August 31, 2009 (received for review April 9, 2009)
Cellular and tissue defects associated with insulin resistance are
coincident with transcriptional abnormalities and are improved after
insulin sensitization with thiazolidinedione (TZD) PPAR? ligands. We
characterized 72 human subjects by relating their clinical phenotypes
with functional pathway alterations. We transcriptionally profiled
identified molecular and functional characteristics of insulin resistant
subjects and distinctions between TZD treatment responder and
in skeletal muscle (e.g., glycolytic flux and intramuscular adipocytes)
and adipose tissue (e.g., mitochondrial metabolism and inflamma-
tion) that improved relative to TZD-induced insulin sensitization.
Pre-TZD treatment expression of MLXIP in muscle and HLA-DRB1 in
adipose tissue from insulin resistant subjects was linearly predictive
of post-TZD insulin sensitization. We have uniquely characterized
coordinated cellular and tissue functional pathways that are charac-
teristic of insulin resistance, TZD-induced insulin sensitization, and
potential TZD responsiveness.
muscle and adipose tissue ? transcriptional mechanisms ? diabetes ?
branched chain amino acid (BCAA) ? inflammation
and the primary defect leading to type 2 diabetes (1, 2). Impaired
insulin-stimulated glucose uptake in skeletal muscle and lipid metab-
olism in adipocytes are central characteristics of insulin-resistance.
Other manifestations of the condition include elevated intramuscular
fat content (3), dysregulation of adipokine secretion, and chronic low-
grade inflammation in adipose tissue (4). Macrophage infiltration in
olism (5). Several studies have shown that decreased mitochondrial
protein and oxidative phosphorylation (OXPHOS) in skeletal muscle
and adipocytes are also underlying factors of insulin resistance (6, 7).
and lipid metabolism, altering adipokine secretion, and reducing adi-
pose tissue inflammation (4, 8). Although TZDs improve insulin
sensitivity and the glycemic, lipid, and inflammatory profiles of most
patients, approximately 30% of diabetic subjects do not respond to
TZD treatment, as gauged by fasting plasma glucose or HbA1c levels
of genes in skeletal muscle, adipocytes, and macrophages. PPAR?-
TZD-induced gene expression changes lead to insulin sensitization or
by which TZD-induced insulin sensitization is prevented are poorly
We have conducted a mechanistic analysis of the gene expression
profiles of adipose tissue (AT) and skeletal muscle (SM) from 72
nsulin resistance is a pathological state in which insulin action is
characterization. We have measured the transcriptional alterations
responded to TZD treatment with varied improvements in insulin
insulin resistant subjects by their degree of TZD response (i.e., in-
creased insulin sensitivity based on rate of glucose disposal during the
hyperinsulinemic-euglycemic clamp) to define responder and
nonresponder subgroups. Based on our analysis of SM and AT
from these subjects, we have defined pathways and key mecha-
nisms of disease that distinguish the responder and nonre-
Characterization of Insulin Sensitive Subjects and Insulin Resistant TZD-
sion profiles from 72 subjects with a broad range of insulin sensitivity,
before and after TZD treatment, to identify transcriptional and bio-
resistance, and pharmacological insulin sensitization. Each subject
underwent a hyperinsulinemic-euglycemic clamp at the start of the
study and a second hyperinsulinemic-euglycemic clamp after 3-month
tazone. Vastus lateralus SM biopsies were harvested immediately
before (basal) and after each clamp (postclamp); abdominal s.c. AT
biopsies were harvested immediately before each clamp (Fig. 1A). AT
biopsies were harvested only from the pioglitazone and rosiglitazone
study groups (44 subjects). Dataset S1 shows selected clinical charac-
gauged the insulin sensitivity of each subject by their rate of glucose
groups, based on their rate of glucose disposal during the first hyper-
insulinemic-euglycemic clamp (Rd1). Subjects with Rd1 values ?8
IRd). From each AT and SM biopsy, we generated gene expression
biochemical pathway analyses. We compared our results from NRd
subjects, IRdsubjects, and IRdsubject subgroups (see Materials and
Fig. 1B shows the TZD-induced fractional Rd change [(Rd2 ?
treatment did not increase the insulin sensitivity (Rd) of most NRd
subjects (see also Dataset S1). As expected, most IRdsubjects re-
(Rd2 ? Rd1). Insulin-sensitizing efficacy was not significantly different
Author contributions: D.D.S. and S.S. designed research; D.D.S., G.H., J.G.Y., C.H.C., and
J.M.O. performed research; G.H., A.H., and J.C. contributed new reagents/analytic tools;
D.D.S., G.H., and S.S. analyzed data; and D.D.S., G.H., and S.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
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between the three TZDs. Approximately 25% of the IRdsubjects did
ment in insulin-sensitivity. To contrast the expression profiles of IRd
TZD-responders (those having the best insulin sensitization response)
with IRdTZD-nonresponders (those having the worst insulin sensiti-
zation response), all of the IRdsubjects (referred to below as IRdall)
were ranked by their fractional Rdchange and divided into quartile
subgroups. IRdsubjects in the top quartile of fractional change in Rd
subjects not only had the largest fractional Rdchange but also had the
in the bottom quartile of fractional Rdchange (?13% increase in Rd)
two quartiles of fractional Rdchange (13–83% increase in Rd) were
of the IRd?, IRd?, IRdmidsubject groups.
Insulin Resistance Is Associated with Impaired Insulin-Induced Metabolic
sion of lipoprotein lipase (LPL), a critical regulator of SM lipid
metabolism (11) and a target gene of PPAR? (12), was 48% lower in
IRdallsubjects in the pre-TZD fasting state (?Bonf? 0.05), was fully
rescued by TZD treatment, and was positively correlated with Rd(r ?
0.53). All genes with baseline expression values that correlate with Rd
genes suppressed or activated in the insulin-stimulated (postclamp)
state in NRdsubjects. One hundred sixteen insulin-target genes were
functionally involved with metabolism (Dataset S2). The insulin-
induced response of 42 genes was significantly altered in the IRdall
hexokinase 2 (HK2) and pyruvate dehydrogenase kinase 4 (PDK4)
compared with NRdsubjects. HK2 expression is normally induced by
insulin, but this induction was blunted in IRdallsubjects (Fig. 2A).
50% lower (?Bonf? 0.05) than in NRdsubjects. Insulin activation of
HK2 commits glucose to the intracellular compartment in muscle,
facilitating glucose metabolism in the fed state. PDK4 expression is
normally suppressed by insulin, but this effect was blunted in IRdall
into acetyl-CoA, effectively disrupting glucose utilization. The sche-
and PDK4 in insulin resistant subjects would reduce glycolytic flux.
Additional genes involved in the glycolytic pathway (PFKFB3 and
also shown in Fig. 2C. We observed insulin-induced, transcriptionally
TZD treatment, IRdallsubjects exhibited improved insulin-induced
HK2 and PDK4 expression changes and postclamp HK2 and PDK4
expression levels compared with NRdsubjects (red and blue bars,
respectively, in Fig. 2 A and B). We contrasted these HK2 and PDK4
expression patterns with those from the responder (IRd?) and nonre-
A and B). Interestingly, post-TZD improvements in the expression of
HK2 and PDK4 were more robust in the IRd?subject subgroup
compared with the IRdallsubject group and absent in the IRd?subject
subgroup (Fig. 2 A and B). Thus, TZD-improved insulin-regulation of
of TZD-improved insulin sensitization.
human insulin-resistance study. (B) Distribution of baseline insulin sensitivity and
TZD-mediated insulin sensitization response of study subjects. NRd(insulin sensitive
subjects with normal Rd, ?8 mg/kg per min, blue square symbols), IRdall(insulin
group of insulin resistant subjects that are TZD responders, in upper quartile of
post-TZD fractional Rdchange, pink triangles), IRd?(subgroup of insulin resistant
subjects that are TZD nonresponders, in bottom quartile of post-TZD fractional Rd
Study design and subject insulin sensitivity distribution. (A) Schematic of
and B) Pre- and postclamp HK2 and PDK4 expression data from the subject groups
NRdsubjects was completely blocked in IRdallsubjects. After TZD-treatment, this
defect was improved in IRdallsubjects, normalized in IRd?subgroup subjects and
improved in IRdallsubjects, normalized in IRd?subgroup subjects and unaffected in
IRd?subgroup subjects. NRd(blue), IRdall(red), IRd?subgroup (pink), IRd?subgroup
in IRdallsubjects compared with NRdsubjects before TZD treatment. Green fill indi-
cates significantly blunted insulin-induced expression in IRdallsubjects, resulting in
low postclamp expression. Red fill indicates significantly blunted insulin-repressed
expression in IRdallsubjects, resulting in high postclamp expression.*, significant
www.pnas.org?cgi?doi?10.1073?pnas.0903032106 Sears et al.
Insulin Resistance Is Associated with Elevated Adipocyte Markers in the
Skeletal Muscle, Which Are Increased After TZD Treatment. Despite the
gross exclusion of perimuscular fat from SM biopsies, SM from IRd
set S2) compared with SM from NRdsubjects. Several of these genes
and retinol-binding protein 4 (RBP4). Leptin was overexpressed ?5-
fold in IRdallsubjects at baseline and in IRd?subjects before and after
insulin-stimulation and/or TZD treatment. Leptin expression in IRd?
IRd?subjects and were significantly further elevated after TZD treat-
ment. ADIPOQ and RBP4 were overexpressed in IRd?subjects only
after TZD treatment and this pattern was significantly blunted com-
pared to the over-expression we observed in IRd?subjects. Leptin,
have paracrine effects on glucose uptake, fatty acid uptake, and fatty
increased adipocyte-myocyte cross-talk.
Genes involved in other aspects of adipocyte biology were differen-
had higher basal and post-TZD expression of genes that regulate lipid
uptake and storage, compared with NRdsubjects. Perilipin (PLIN),
fatty acid binding protein (FABP4), stearoyl-CoA desaturase (SCD),
cell death-inducing DFFA-like effector c (CIDEC) are among these
genes. Overexpression of these genes was increased after TZD treat-
IRd?subjects and weakest in IRd?subjects suggesting that elevated
lipid uptake and storage is related to insulin sensitization. In fact,
fractional CIDEC expression change correlated with fractional Rd
change (r ? 0.57). We observed significant overexpression of genes
involved in adipogenesis in SM of IRdsubjects, including CCAAT/
enhancer binding protein ? (CEBPA), sterol regulatory element bind-
ing transcription factor (SREBF1), and early growth response 2
(EGR2). CEBPA overexpression and SREBF1 expression each in-
creased significantly after insulin infusion in IRdallsubjects. Interest-
ingly, only the IRd?subjects exhibited significant up-regulation of
EGR2 (8.6-fold) after TZD treatment (?Bonf? 0.05). Together, these
findings suggest that TZD-induced insulin-sensitization is mediated, in
part, by stimulation of intramuscular adipocyte differentiation (see
transendothelial migration pathway that highlight genes that are overexpressed in IRdallsubjects before TZD treatment (C) and down-regulated in IRd?subjects after TZD
NRdand IRdall(A and C) or IRd?(B and D) subjects. Pink ovals – genes still significantly overexpressed in IRd?subjects compared with NRdsubjects but are significantly
down-regulated compared with pre-TZD levels. Schematics are adapted from KEGG. (E) CD74 was significantly overexpressed in all IRdsubject groups compared with NRd
subjects. After TZD treatment, CD74 expression was significantly down-regulated in IRd?subgroup subjects (pink) but not in IRdallsubjects (red) or IRd?subgroup subjects
(yellow).*, significant difference between bracketed groups (?bonf? 0.05). (F) Negative correlation between CD74 expression and Rd (r ? ?0.59). Graph includes pre- and
Sears et al. PNAS ?
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Insulin Resistance Is Associated with Increased Inflammatory Markers in
within this gene set (Dataset S6). We identified statistical enrichment
Several immune system pathways were also significantly enriched,
including antigen processing and presentation and leukocyte transen-
dothelial migration. Thirty-four percent of the adipose tissue genes
differentially expressed IRdallsubjects were associated with inflamma-
tion and virtually all of these (192 of 202) were overexpressed. This is
shown schematically in Fig. 3A for genes involved in the antigen
processing and presentation pathway and leukocyte transendothelial
migration pathway (Fig. 3C), two critical pathways regulating tissue
inflammation and immune cell infiltration. AT is a heterogeneous
mixture of cell types. The enriched functional ontology and pathway
annotations, of the differentially expressed genes, associated with the
immune system are evidence of increased antigen-presenting cells in
AT from the IRdallsubjects.
Overexpression of inflammatory markers was universally decreased
in IRd?subjects after TZD treatment. Interestingly, most genes over-
expressed in IRdallsubjects and subsequently down-regulated after
TZD treatment were macrophage-specific (Dataset S4). Several facil-
itate macrophage chemotaxis, including matrix remodeling genes
(MMP7, MMP9) and osteopontin (SPP1). Post-TZD repression of
antigen processing and presentation (compare Fig. 3 A and B) and
leukocyte transendothelial migration pathways (compare Fig. 3 C and
D). A significantly smaller percentage of inflammatory markers were
but not IRd?subjects after TZD treatment are shown in Dataset S4.
Overexpression of CD74, a MHC class II invariant chain gene, was
significantly repressed 46% in IRd?subjects but not in IRd?subjects
after TZD treatment (Fig. 3E). In fact, CD74 expression was inversely
correlated with Rd(r ? ?0.60) (Fig. 3F).
Gene Expression Signatures that Correlate with Insulin Resistance and
TZD-Induced Insulin Sensitization. To further elucidate relationships be-
relations between genome-wide gene expression and Rdvalues (Data-
set S7 shows all correlations with r ? 0.45). As expected, a set of 319
?0.5) was enriched for genes functionally annotated with immune
response (?Bonf? 0.05) (Dataset S6), including ?60% of the genes
shown in Fig. 3 A and C. We used statistical regression test (see
sue is correlated with insulin sensitivity. Schematics of mi-
was positively correlated with Rd. (A) ?-oxidation of fatty
acids, TCA cycle, and branched chain amino acid degrada-
tion. (B) Oxidative phosphorylation. Dark orange ovals –
genes with expression vs. Rdcorrelations of r ? 0.5. Light
orange ovals – genes with expression vs. Rdcorrelations of
r ? 0.45–0.50.
Mitochondrial metabolic function in adipose tis-
www.pnas.org?cgi?doi?10.1073?pnas.0903032106Sears et al.
was inversely correlated with insulin sensitivity (Rd) (Dataset S7).
between NRdand IRdallsubjects before TZD treatment (Dataset S4),
represented in Fig. 3 A and C. These results suggest that insulin resis-
criptional markers of insulin resistance. Negative correlations between
insulin sensitivity and expression levels of these and the other genes
associated with leukocyte transendothelial migration show the associ-
sensitization (observed in IRd?subjects but not IRd?subjects) corre-
as a result of attenuated immune cell residency and recruitment.
We observed robust positive correlations (r ? 0.5) between Rdand
the pre- and post-TZD expression of 315 genes. This positively corre-
lated gene set was statistically enriched for genes annotated with
mitochondria functional pathways (Dataset S6). Expression of the
majority of genes involved in mitochondrial ?-oxidation of fatty acids
TCA cycle was correlated with insulin sensitivity (Fig. 4A), including
delta isomerase (DCI, r ? 0.64), and succinate-CoA ligase (SUCLG2,
in the oxidative phosphorylation pathway was correlated with insulin
sensitivity (Fig. 4B), including NADH dehydrogenase (NDUFB5, r ?
0.62), electron-transfer-flavoprotein alpha (ETFA, r ? 0.69), and
cytochrome c reductase (UQCRC2, r ? 0.66). Expression of genes
involved in branched chain amino acid (BCAA) metabolism (several
also involved in TCA cycle) is also correlated with insulin sensitivity
BCKDHA, and BCKDHB). Overall, these results indicate that im-
are proportional to the degree of insulin resistance.
Several published studies have attempted to identify plasma com-
effect of TZDs on individual patients (9, 20). We have identified gene
subjects, i.e., pre-TZD gene expression that correlates with fractional
Rdchange [(Rd2 ? Rd1)/Rd1] (Dataset S3). MLX interacting protein
1 (HLA-DRB1) in AT had gene expression signatures that predict
insulin sensitization by TZDs. MLXIP is a transcription factor that
activates glycolytic genes (including HK2) in SM (21). Pre-TZD, post-
clamp SM expression of MLXIP correlated with fractional Rdchange
expression before TZD treatment exhibited greater relative insulin-
sensitization after TZD treatment [pre-TZD MLXIP expression in
Adipose tissue HLA-DRB1 expression was negatively correlated with
fractional Rdchange (r ? ?0.48). HLA-DRB1 plays a central role in
(APCs). Expression of HLA-DRB1 was 3.4-fold higher in IRd?sub-
jects compared with IRd?subjects (Fig. 5B). Thus, patients who
expressed less HLA-DRB1 in AT before TZD treatment experienced
greater relative increases in insulin-sensitivity after TZD treatment.
The multidimensionality of our human gene expression dataset allows
us to investigate fundamental questions about insulin resistance and
insulin-resistant subjects and uniquely identify gene expression signa-
tures that characterize TZD-mediated insulin sensitization and predict
TZD responsiveness in insulin resistant subjects. Notably, physiologic
and individual proteomic data in the literature support many of the
statistical data analyses. Our findings demonstrate that insulin resis-
genes in skeletal muscle. The fasting-to-feeding transition altered the
expression of many genes in insulin sensitive subjects including key
regulators of glycolysis and other metabolic pathways. Interestingly,
resistant subjects. Our data support the notion that over-nutrition
insulin resistance is associated with impaired metabolic flexibility in
response to insulin (22). For example, defective insulin-induced regu-
lation of HK2 and PDK4 in insulin resistant subjects (Fig. 2C) would
responder (IRd?) subjects was associated with improved insulin-
expression was ameliorated in TZD-treated responder subjects, con-
comitant with insulin sensitization, but not in TZD-treated nonre-
sponder (IRd?) subjects who did not become more insulin sensitive.
with improved glycolytic flux and overall metabolic flexibility.
Surprisingly, elevated intramuscular adipocyte marker gene expres-
sion in the insulin resistant subjects further increased after TZD
potential to improve myocyte sensitivity through paracrine signaling
and alleviating local hyperlipidemia by storing excess fatty acids (Fig.
S3). For example, TZD-induced elevation of adiponectin secretion
in skeletal muscle of TZD-treated subjects (23). Thus, presence of
adipocytes in skeletal muscle of insulin resistant subjects may not be a
Expression of MLXIP in pre-TZD skeletal muscle was positively correlated with frac-
tional Rdchange in IRdallsubjects (r ? 0.54) and significantly higher in IRd?vs. IRd?
correlated with fractional Rdchange in IRdallsubjects (r ? ?0.47) and significantly
lower in IRd?vs. IRd?subjects. Bars indicate the expression mean and range of
(pink, red, and yellow triangles), IRd?subgroup subjects (pink), IRd?subgroup sub-
Gene expression predictors of TZD-mediated insulin sensitization. (A)
Sears et al.PNAS ?
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causal factor mediating insulin resistance, but an adaptation that
accommodates for excess energy storage.
Chronic low-grade inflammation is associated with and can cause
insulin resistance. Macrophages infiltrate adipose tissue of obese ani-
mals and humans where they secrete cytokines that interfere with
insulin signaling (4, 24). Our pathway analysis results indicate that
transendothelial migration and antigen presentation in resident leuko-
activation of proinflammatory pathways correlated with the degree of
sion of inflammatory genes in adipose tissue was relative to TZD-
induced insulin sensitization of the subjects, i.e., TZD-induced repres-
sion was greater in responder than in nonresponder subjects. Notably,
markers of macrophage infiltration were most robust in nonresponder
subjects before TZD treatment and may play a role in making insulin-
resistance more refractory to TZD treatment.
Decreased mitochondrial capacity, gene expression, and mass
are observed in adipose tissue from insulin resistant humans and
rodents and are improved after TZD treatment (6, 22, 25–28).
Our studies of s.c. adipose tissue extend previous findings and
show that fatty acid and BCAA oxidation, TCA cycle, and
oxidative phosphorylation pathways are down-regulated in pro-
portion to insulin resistance, i.e., TZD-induced insulin sensiti-
zation was positively correlated with expression of genes regu-
lating mitochondrial activity. Our findings are evidence that the
magnitude of insulin resistance in insulin resistant individuals is
mechanistically linked to the magnitude of dysfunctional mito-
chondrial capacity driving pathogenic lipotoxicity, futile triac-
ylglycerol cycling, and generation of reactive oxygen species.
Clinical or transcriptional signatures that differentiate insulin-
resistant TZD responder and nonresponder subjects have not hereto-
fore been characterized.We have identified distinctions between TZD
responders and nonresponders and have identified gene expression
predictors of TZD-mediated insulin sensitization. In skeletal muscle,
insulin resistant responder subjects exhibited greater post-TZD nor-
malization of insulin-induced glycolytic flux than insulin resistant non-
responder subjects, indicating that responders recover metabolic flex-
ibility as they become more insulin sensitive. This may be due to their
higher pre-TZD expression level of MLXIP which was predictive of
post-TZD insulin sensitization. MLXIP is a metabolic sensor that
shuttles between mitochondria and the nucleus where it is a transcrip-
tion factor activating HK2 and other glycolytic target genes (21). In
adipose tissue, pre-TZD HLA-DRB1 expression was correlated with
DRB1, an antigen-presenting cell gene, might be mechanistically in-
volved in determining the dichotomous anti-inflammation and insulin
sensitization responses exhibited by TZD-treated responder and non-
responder subjects in our study.
In conclusion, this study describes system-wide mechanistic dif-
ferences between normal and insulin-resistant subjects and identi-
fies transcriptional signatures that differentiate insulin-resistant
TZD responder from nonresponder subjects.
Materials and Methods
and hyperinsulinemic euglycemic clamp protocols (23, 29, 32).
Microarray Studies. We generated gene expression profiles from tissue biopsy
rays (Affymetrix, Inc., Santa Clara, CA). See SI Text for specific details.
Statistical Analyses. See SI Text (18, 34, 35).
ACKNOWLEDGMENTS. We thank Dr. J. Denis Heck, Sriti Misra, and Lana Bord-
cosh at the DNA & Protein MicroArray Facility, University of California, Irvine for
conducting the RNA microarray analyses and Jerrold Olefsky for support in the
study, interpretation and manuscript preparation. Funding was provided by the
National Institutes of Health (NIH) National Institute of Diabetes and Digestive
and Kidney Diseases (NIDDK) Grant K01-DK62025 (D.D.S.); the Eunice Kennedy
Shriver National Institute of Child Health and Human Development and NIH
cooperative agreement U54-HD012303 as part of the Specialized Cooperative
Centers Programs in Reproduction and Infertility Research (J.M.O., J.G.Y., and
C.H.C.); NIH Grants P01-DK074868 (D.D.S., J.M.O., J.G.Y., and C.H.C.) and R01-
DK033651 (J.M.O., J.G.Y., and C.H.C.); National Heart, Lung and Blood Institute
Grant R33-HL087375 (S.S.); National Institute of General Medical Sciences Grant
U54-GM69338 (S.S.); NIDDK Grant P01-DK074868 (S.S.); and the University of
California Discovery Program Project #bio03–10383 with matching funds from
Pfizer, Inc. (D.D.S., J.G.Y., C.H.C., and S.S.).
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