Obesity is highly correlated with insulin resistance, but both insulin-resistant lean individuals and metabolically healthy obese individuals are well recognized. A recent study (16
) also showed a high correlation between expressions of a large number of transcripts in subcutaneous adipose with BMI. Thus, in most published studies the search for insulin sensitivity–mediated changes in adipose tissue gene expression was confounded by BMI. In addition, many published investigations compared subjects with overt type 2 diabetes, where gene expression may have been altered by hyperglycemia, to normal control subjects. Gene expression in some previous studies was influenced by obtaining muscle biopsies during the hyperinsulinemic state at the end of a 2-h euglycemic clamp (12
). To examine the role of adipose and muscle gene expression while minimizing the confounders, we matched nondiabetic individuals who were at extremes of insulin sensitivity for BMI and obesity measures and obtained both adipose and muscle biopsies during a fasting state. Additional unique features of the current study were the inclusion of participants of both European and African ancestry, the inclusion of both adipose and muscle tissue from the same individuals, and the relatively large sample size of our study compared with many earlier studies (12
). These features, along with different arrays and probe sets, likely explain the differences between the current study and other reports.
Several conclusions are evident from our study. First, considerably more transcripts differ with SI
in adipose than in muscle tissue, which may suggest a more important role for altered adipocyte transcription in impaired insulin action. Second, although only a small number of transcripts differed significantly between European American and African American participants, many of these transcripts were in pathways likely to contribute to metabolic differences including phospholipases, immune factors, and fatty acid and ceramide metabolism. Using stringent criteria, only six genes with putative metabolic importance (CLIC6
, and TNMD
) showed ethnic difference as well as differential expression in adipose of insulin-resistant individuals. However, using less stringent criteria (single-point P
< 0.05 for African Americans/European Americans, irrespective of q
value), nearly 20% of adipocyte transcripts that were associated with SI
in European American participants also differed between European American and African American samples. A recent study of lymphocytes suggested that levels of key glucose homeostasis transcripts downstream of SREBP1 differed between European American and African American individuals (37
). These expression differences may reflect phenotypic differences in insulin action, secretion, and clearance between European American and African American individuals (21
). Although the African American subset in our study was of a size comparable to previous studies of global transcript levels, a larger study comparing expression patterns in African American and European American individuals matched for obesity measures is needed to determine whether apparent transcript differences result from a combination of small sample size and additional variability and heterogeneity in the African American subset. For example, we observed more differentially expressed transcripts in the smaller European American population than in the full sample set. This may be a result of a type I statistical error; however, an alternative explanation for this paradox is that African American samples introduced heterogeneity and increased the variance, and, thus, fewer transcripts reached significance. In our analysis, comparison between all insulin-resistant and insulin-sensitive individuals were performed after considering African American and European American individuals as separate permutation groups to minimize that error. Differences reported in European American insulin-resistant groups compared with European American insulin-sensitive groups could also be explained by increased visceral fat (indicated by WHR) in European American insulin-resistant groups.
Previous studies in muscle have argued for a key role of oxidative phosphorylation (OXPHOS) (12
). We also found that PPARGC1A
(PGC1α) was reduced in insulin-resistant muscle, but of 17 OXPHOS genes that were reported previously, we found nominal evidence (P
< 0.05) of differential expression in only four genes (NDUFB5
, and NDUFU2
), and for all of these, gene expression increased ~1.2-fold in insulin-resistant muscle, arguing against decreased mitochondrial oxidation in insulin resistance. This conclusion was supported by GSEAs, which failed to identify either KEGG pathways or gene ontology terms related to oxidative phosphorylation. Instead, in addition to PPAR signaling, we found downregulation of pathways for arginine and proline metabolism, lipid and fatty acid metabolism, and hexose transport. Novel findings included differential expression of genes in categories of ubiquitin-protein ligase and apoptosis.
In addition to inflammatory markers, a large number of genes in adipose are reported to be associated with insulin resistance in humans. In agreement with our previous studies (9
), we found no evidence for clustering with insulin sensitivity for 240 transcripts related to endoplasmic reticulum stress. We reviewed the literature and identified 104 transcripts not related to endoplasmic reticulum stress that were reported as associated with insulin resistance (11
). We searched for evidence of an association in the same direction as previously reported and found support for 50 of these transcripts, many falling into pathways identified in our global analysis. In the analysis of all subjects, genes involved in inflammation were missing from the list of 1.5-fold differentially expressed genes between the adipose of insulin-resistant and insulin-sensitive subjects. However, GSEA of all subject data and analysis of the European American subset indicated the differential expression of genes in inflammatory pathways. Inflammation was supported with upregulation of CCL2
, and TNF
. This result is consistent with a recent finding from Hyatt et al., which showed significantly lower inflammation in African American individuals compared with BMI-matched European American individuals (47
). Thus, our study, along with published studies, indicates an important ethnic difference in the pathophysiology of insulin resistance (34
). The recently described (48
) gene SPARC
(osteonectin) was modestly increased with insulin resistance (1.2-fold, q
value <5%). In contrast to our findings in muscle, we found changes in oxidative phosphorylation, with genes NDUFS1
, and NRF1
modestly (1.2-fold) downregulated with insulin resistance. Transcription factors PPARG
, and CEBPB
were all modestly downregulated, as were insulin signaling genes SLC2A4
, and HK2.
However, as we noted above, alterations in lipid metabolic genes in adipose tissue were most prominent with the downregulation of SCD
, and FASN
with insulin resistance (50
In addition to PPARA
, we found downregulation of LIPIN1
in adipose tissue of insulin-resistant compared with insulin-sensitive subjects (51
). Finally, in contrast to published findings (52
), we found that APP
was 1.2-fold downregulated rather than upregulated with insulin resistance. Thus, we suggest that many proposed associations were the result of obesity rather than primarily associated with insulin resistance.
By testing adipose and muscle in the same individuals at extremes of insulin sensitivity, we were able to identify pathways unique to each tissue but also similar mechanisms in adipose and muscle. We identified 69 genes with concordant expression in insulin-resistant muscle and adipose, of which only 14 genes were increased with insulin resistance. Although many of the 69 concordantly regulated transcripts have no obvious roles in adipose or muscle, genes associated with cell-cycle progression and differentiation are well represented. Concordantly upregulated genes S100A4
encode calcium binding proteins that are involved in cell-cycle progression and cell differentiation, ATF5
interacts with cell cycle genes, and downregulated gene PPFIBP1
interacts with S100A4. Other concordantly downregulated genes with cell-cycle roles include CDKAL1.
Interestingly, both S100A4
reside near the well-replicated 1q21 region of type 2 diabetes linkage, whereas CDKAL1
is a well replicated type 2 diabetes gene based on genome-wide association scans (53
). Several additional genes are involved in translation initiation, including helicase gene DDX42
, initiation factor EIF4B
, and CAPRIN1.
Also among the concordantly regulated genes were four histone members (increased in insulin resistance) and the chromatin binding factor, transcriptional regulator, and part of the histone deacetylase complex CTCF.
These findings suggest a prominent role for chromatin modification, translation initiation, and cell-cycle regulation in insulin resistance.
A number of coordinately regulated genes are involved in cellular stress responses, inflammatory pathways, and unfolded protein response including BCL2A1
It was surprising that these genes are concordantly expressed in muscle, but it is consistent with a recent description (54
) of increased macrophages in insulin resistant muscle. Finally, fatty acid metabolism was prominent in both tissues, with reduced expression in insulin-resistant individuals for ACAD10
, and CYP4V2
. Other interesting concordantly downregulated transcripts include PER3
, a gene associated with circadian rhythm but of unknown function in adipose and muscle; AGTR1
encoding the angiotensin 2 receptor; and genes KLF15
, encoding transcription factors primarily known for adipose function. Like CDKAL1
, genes VEGFA
are near well-replicated SNPs associated with type 2 diabetes or fasting glucose (23
The current study included a larger sample than most previous studies, included a biethnic population, and, as noted above, provided the opportunity to compare adipose and muscle in individuals matched for BMI but were discordant for insulin sensitivity. Nonetheless, this cross-sectional study has some limitations. Although larger than most earlier reports, the design of this study was inevitably of limited size relative to studies focused on mapping genetic etiology. Although inclusion of an African American population provided a unique opportunity to define differences among ethnic groups, this population may have added heterogeneity. A cross-sectional study cannot define cause and effect; thus, in common with previous studies we cannot exclude the possibility that some of the differences in expression were the result of impaired insulin action rather than the cause. Insulin signaling affects many downstream networks. In this study, the observed direction of changes of differentially expressed genes in carbohydrate metabolism, cell-cycle regulation, lipid metabolism, and arginine-proline metabolism are consistent with the known functional effect of reduced insulin signaling in insulin-resistant individuals. However, some other differences between insulin-resistant and insulin-sensitive subjects, including the increase in inflammation and reduction in angiogenesis, are less readily explained by altered insulin signaling.
In summary, by comparing BMI-matched individuals at extremes of insulin sensitivity, we show that expression changes are more prominent in adipose than muscle, confirming earlier and smaller array studies of muscle, but we confirm the role for PPARGC1A (encoding PGC1) in muscle. Although we indeed found the expected role for inflammation in adipose, the most highly differentially expressed genes suggest a prominent role for pathways involved in fatty acid metabolism and cell-cycle regulation in both adipose and muscle. Although significant differences observed between African American and European American individuals did not overlap with the significant differences observed between insulin-resistant and insulin-sensitive individuals, ethnic differences between metabolically important genes were prominent and deserve further study.