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1.  Pioglitazone Enhances Mitochondrial Biogenesis and Ribosomal Protein Biosynthesis in Skeletal Muscle in Polycystic Ovary Syndrome 
PLoS ONE  2008;3(6):e2466.
Insulin resistance is a common metabolic abnormality in women with PCOS and leads to an elevated risk of type 2 diabetes. Studies have shown that thiazolidinediones (TZDs) improve metabolic disturbances in PCOS patients. We hypothesized that the effect of TZDs in PCOS is, in part, mediated by changes in the transcriptional profile of muscle favoring insulin sensitivity. Using Affymetrix microarrays, we examined the effect of pioglitazone (30 mg/day for 16 weeks) on gene expression in skeletal muscle of 10 obese women with PCOS metabolically characterized by a euglycemic-hyperinsulinemic clamp. Moreover, we explored gene expression changes between these PCOS patients before treatment and 13 healthy women. Treatment with pioglitazone improved insulin-stimulated glucose metabolism and plasma adiponectin, and reduced fasting serum insulin (all P<0.05). Global pathway analysis using Gene Map Annotator and Pathway Profiler (GenMAPP 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1) revealed a significant upregulation of genes representing mitochondrial oxidative phosphorylation (OXPHOS), ribosomal proteins, mRNA processing reactome, translation factors, and proteasome degradation in PCOS after pioglitazone therapy. Quantitative real-time PCR suggested that upregulation of OXPHOS genes was mediated by an increase in PGC-1α expression (P<0.05). Pretreatment expression of genes representing OXPHOS and ribosomal proteins was down-regulated in PCOS patients compared to healthy women. These data indicate that pioglitazone therapy restores insulin sensitivity, in part, by a coordinated upregulation of genes involved in mitochondrial OXPHOS and ribosomal protein biosynthesis in muscle in PCOS. These transcriptional effects of pioglitazone may contribute to prevent the onset of type 2 diabetes in these women.
PMCID: PMC2413008  PMID: 18560589
2.  Short-Term Overfeeding May Induce Peripheral Insulin Resistance Without Altering Subcutaneous Adipose Tissue Macrophages in Humans 
Diabetes  2010;59(9):2164-2170.
Chronic low-grade inflammation is a feature of obesity and is postulated to be causal in the development of insulin resistance and type 2 diabetes. The aim of this study was to assess whether overfeeding induces peripheral insulin resistance in lean and overweight humans, and, if so, whether it is associated with increased systemic and adipose tissue inflammation.
Thirty-six healthy individuals undertook 28 days of overfeeding by +1,250 kcal/day (45% fat). Weight, body composition, insulin sensitivity (hyperinsulinemic-euglycemic clamp), serum and gene expression of inflammation markers, immune cell activation, fat cell size, macrophage and T-cell numbers in abdominal subcutaneous adipose tissue (flow cytometry and immunohistochemistry) were assessed at baseline and after 28 days.
Subjects gained 2.7 ± 1.6 kg (P < 0.001) and increased fat mass by 1.1 ± 1.6% (P < 0.001). Insulin sensitivity decreased by 11% from 54.6 ± 18.7 to 48.9 ± 15.7 μmol/(kg of FFM)/min (P = 0.01). There was a significant increase in circulating C-reactive protein (P = 0.002) and monocyte chemoattractant protein-1 (P = 0.01), but no change in interleukin-6 and intercellular adhesion molecule-1. There were no changes in fat cell size, the number of adipose tissue macrophages or T-cells, or inflammatory gene expression and no change in circulating immune cell number or expression of their surface activation markers after overfeeding.
Weight gain-induced insulin resistance was observed in the absence of a significant inflammatory state, suggesting that inflammation in subcutaneous adipose tissue occurs subsequent to peripheral insulin resistance in humans.
PMCID: PMC2927938  PMID: 20547978
3.  BMI-independent inflammation in omental adipose tissue associated with insulin resistance in morbid obesity 
Obesity is a strong risk factor for resistance to insulin-mediated glucose disposal, a precursor of type 2 diabetes and other disorders.
To identify molecular pathways that may cause such obesity-associated insulin resistance in human subjects, we exploited the fact that not all obese individuals are prone to insulin resistance. Thus the degree of obesity as a variable was removed by studying morbidly obese human subjects of similar BMI values who are insulin-sensitive versus insulin-resistant.
University Medical Center, United States
Combining gene expression profiling with computational approaches, we determined the global gene expression signatures of omental and subcutaneous adipose tissue samples obtained from similarly obese patients undergoing gastric bypass surgery.
Gene sets related to chemokine activity and chemokine receptor-binding were identified as most highly expressed in the omental tissue from insulin-resistant compared to insulin sensitive subjects, independent of BMI. These upregulated genes included chemokines CCL2, CCL3, CCL4, CCL18 and IL8/CXCL8, and were not differentially expressed in subcutaneous adipose tissues between the two groups of subjects. Strikingly, insulin resistance, but not BMI, was associated with increased macrophage infiltration in the omental adipose tissue, as was adipocyte size, in these morbidly obese subjects.
Our findings demonstrate that inflammation of omental adipose tissue is strongly associated with insulin resistance in human obesity even in subjects with similar BMI values. Increased omental fat mass may contribute to the amplified inflammatory response observed in this population.
PMCID: PMC2980798  PMID: 20678967
abdominal adipose tissue; insulin resistance; chemokines; inflammation; macrophages
4.  Inverse Regulation of Inflammation and Mitochondrial Function in Adipose Tissue Defines Extreme Insulin Sensitivity in Morbidly Obese Patients 
Diabetes  2013;62(3):855-863.
Obesity is associated with insulin resistance, a major risk factor for type 2 diabetes and cardiovascular disease. However, not all obese individuals are insulin resistant, which confounds our understanding of the mechanistic link between these conditions. We conducted transcriptome analyses on 835 obese subjects with mean BMI of 48.8, on which we have previously reported genetic associations of gene expression. Here, we selected ∼320 nondiabetic (HbA1c <7.0) subjects and further stratified the cohort into insulin-resistant versus insulin-sensitive subgroups based on homeostasis model assessment–insulin resistance. An unsupervised informatics analysis revealed that immune response and inflammation-related genes were significantly downregulated in the omental adipose tissue of obese individuals with extreme insulin sensitivity and, to a much lesser extent, in subcutaneous adipose tissue. In contrast, genes related to β-oxidation and the citric acid cycle were relatively overexpressed in adipose of insulin-sensitive patients. These observations were verified by querying an independent cohort of our published dataset of 37 subjects whose subcutaneous adipose tissue was sampled before and after treatment with thiazolidinediones. Whereas the immune response and inflammation pathway genes were downregulated by thiazolidinedione treatment, β-oxidation and citric acid cycle genes were upregulated. This work highlights the critical role that omental adipose inflammatory pathways might play in the pathophysiology of insulin resistance, independent of body weight.
PMCID: PMC3581230  PMID: 23223024
5.  Differential Effects of Insulin on Sympathetic Nerve Activity in Agouti Obese Mice 
Journal of hypertension  2010;28(9):1913-1919.
Hyperinsulinemia, which often coexists with obesity and type 2 diabetes, is a major risk factor for cardiovascular disease and thought to promote hypertension through the sympathetic effects of insulin. Here, we examined the effect of insulin on regional sympathetic nerve activity (SNA) in obesity.
Glucose and insulin tolerance tests were performed to examine insulin sensitivity in agouti obese mice. We used also multifiber recording to compare the regional SNA response to ICV insulin between lean and agouti obese mice.
Agouti obese mice have significantly elevated levels of blood glucose and plasma insulin associated with glucose intolerance and insulin resistance. In lean mice, ICV administration of insulin (20 and 100 μU) caused a dose-dependent increase in SNA subserving hindlimb, kidney and brown adipose tissue (BAT). Of note, the regional SNA responses to insulin were differentially altered in agouti obese mice. While lumbar SNA response to insulin was intact in the obese mice, renal and BAT sympathetic activation to insulin were significantly attenuated in these agouti obese mice. Finally, we assessed the role of phosphoinositol-3 kinase (PI3K) signaling pathway in mediating sympathetic activation to insulin in obesity. Notably, ICV pre-treatment with a PI3K inhibitor (LY294002) blocked the increase in lumbar SNA induced by ICV insulin in lean and agouti obese mice.
Our data suggest a differential regulation by insulin of sympathetic outflow to peripheral tissues in obesity. Our findings also demonstrate the importance of PI3K in lumbar sympathetic activation to insulin in obesity.
PMCID: PMC2920344  PMID: 20577122
Sympathetic nervous system; obesity; insulin resistance; PI3K
6.  Microarray Evidences the Role of Pathologic Adipose Tissue in Insulin Resistance and Their Clinical Implications 
Journal of Obesity  2011;2011:587495.
Clustering of insulin resistance and dysmetabolism with obesity is attributed to pathologic adipose tissue. The morphologic hallmarks of this pathology are adipocye hypertrophy and heightened inflammation. However, it's underlying molecular mechanisms remains unknown. Study of gene function in metabolically active tissues like adipose tissue, skeletal muscle and liver is a promising strategy. Microarray is a powerful technique of assessment of gene function by measuring transcription of large number of genes in an array. This technique has several potential applications in understanding pathologic adipose tissue. They are: (1) transcriptomic differences between various depots of adipose tissue, adipose tissue from obese versus lean individuals, high insulin resistant versus low insulin resistance, brown versus white adipose tissue, (2) transcriptomic profiles of various stages of adipogenesis, (3) effect of diet, cytokines, adipokines, hormones, environmental toxins and drugs on transcriptomic profiles, (4) influence of adipokines on transcriptomic profiles in skeletal muscle, hepatocyte, adipose tissue etc., and (5) genetics of gene expression. The microarray evidences of molecular basis of obesity and insulin resistance are presented here. Despite the limitations, microarray has potential clinical applications in finding new molecular targets for treatment of insulin resistance and classification of adipose tissue based on future risk of insulin resistance syndrome.
PMCID: PMC3092611  PMID: 21603273
7.  Global Gene Expression Profiles of Subcutaneous Adipose and Muscle From Glucose-Tolerant, Insulin-Sensitive, and Insulin-Resistant Individuals Matched for BMI 
Diabetes  2011;60(3):1019-1029.
To determine altered gene expression profiles in subcutaneous adipose and skeletal muscle from nondiabetic, insulin-resistant individuals compared with insulin-sensitive individuals matched for BMI.
A total of 62 nondiabetic individuals were chosen for extremes of insulin sensitivity (31 insulin-resistant and 31 insulin-sensitive subjects; 40 were European American and 22 were African American) and matched for age and obesity measures. Global gene expression profiles were determined and compared between ethnic groups and between insulin-resistant and insulin-sensitive participants individually and using gene-set enrichment analysis.
African American and European American subjects differed in 58 muscle and 140 adipose genes, including many inflammatory and metabolically important genes. Peroxisome proliferator–activated receptor γ cofactor 1A (PPARGC1A) was 1.75-fold reduced with insulin resistance in muscle, and fatty acid and lipid metabolism and oxidoreductase activity also were downregulated. Unexpected categories included ubiquitination, citrullination, and protein degradation. In adipose, highly represented categories included lipid and fatty acid metabolism, insulin action, and cell-cycle regulation. Inflammatory genes were increased in European American subjects and were among the top Kyoto Encyclopedia of Genes and Genomes pathways on gene-set enrichment analysis. FADS1, VEGFA, PTPN3, KLF15, PER3, STEAP4, and AGTR1 were among genes expressed differentially in both adipose and muscle.
Adipose tissue gene expression showed more differences between insulin-resistant versus insulin-sensitive groups than the expression of genes in muscle. We confirm the role of PPARGC1A in muscle and show some support for inflammation in adipose from European American subjects but find prominent roles for lipid metabolism in insulin sensitivity independent of obesity in both tissues.
PMCID: PMC3046820  PMID: 21266331
8.  Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance. 
Journal of Clinical Investigation  1996;97(11):2601-2610.
To test the hypothesis that obesity/insulin resistance impairs both endothelium-dependent vasodilation and insulin-mediated augmentation of endothelium-dependent vasodilation, we studied leg blood flow (LBF) responses to graded intrafemoral artery infusions of methacholine chloride (MCh) or sodium nitroprusside (SNP) during saline infusion and euglycemic hyperinsulinemia in lean insulin-sensitive controls (C), in obese insulin-resistant subjects (OB), and in subjects with non-insulin-dependent diabetes mellitus (NIDDM). MCh induced increments in LBF were approximately 40% and 55% lower in OB and NIDDM, respectively, as compared with C (P < 0.05). Euglycemic hyperinsulinemia augmented the LBF response to MCh by - 50% in C (P < 0.05 vs saline) but not in OB and NIDDM. SNP caused comparable increments in LBF in all groups. Regression analysis revealed a significant inverse correlation between the maximal LBF change in response to MCh and body fat content. Thus, obesity/insulin resistance is associated with (a) blunted endothelium-dependent, but normal endothelium-independent vasodilation and (b) failure of euglycemic hyperinsulinemia to augment endothelium-dependent vasodilation. Therefore, obese/insulin-resistant subjects are characterized by endothelial dysfunction and endothelial resistance to insulin's effect on enhancement of endothelium-dependent vasodilation. This endothelial dysfunction could contribute to the increased risk of atherosclerosis in obese insulin-resistant subjects.
PMCID: PMC507347  PMID: 8647954
9.  Association of adipocyte genes with ASP expression: a microarray analysis of subcutaneous and omental adipose tissue in morbidly obese subjects 
Prevalence of obesity is increasing to pandemic proportions. However, obese subjects differ in insulin resistance, adipokine production and co-morbidities. Based on fasting plasma analysis, obese subjects were grouped as Low Acylation Stimulating protein (ASP) and Triglyceride (TG) (LAT) vs High ASP and TG (HAT). Subcutaneous (SC) and omental (OM) adipose tissues (n = 21) were analysed by microarray, and biologic pathways in lipid metabolism and inflammation were specifically examined.
LAT and HAT groups were matched in age, obesity, insulin, and glucose, and had similar expression of insulin-related genes (InsR, IRS-1). ASP related genes tended to be increased in the HAT group and were correlated (factor B, adipsin, complement C3, p < 0.01 each). Differences between LAT and HAT group were almost exclusively in SC tissue, with little difference in OM tissue. Increased C5L2 (p < 0.01), an ASP receptor, in HAT suggests a compensatory ASP pathway, associated with increased TG storage.
HAT adipose tissue demonstrated increased lipid related genes for storage (CD36, DGAT1, DGAT2, SCD1, FASN, and LPL), lipolysis (HSL, CES1, perilipin), fatty acid binding proteins (FABP1, FABP3) and adipocyte differentiation markers (CEBPα, CEBPβ, PPARγ). By contrast, oxidation related genes were decreased (AMPK, UCP1, CPT1, FABP7). HAT subjects had increased anti-inflammatory genes TGFB1, TIMP1, TIMP3, and TIMP4 while proinflammatory PIG7 and MMP2 were also significantly increased; all genes, p < 0.025.
Taken together, the profile of C5L2 receptor, ASP gene expression and metabolic factors in adipose tissue from morbidly obese HAT subjects suggests a compensatory response associated with the increased plasma ASP and TG.
PMCID: PMC2843642  PMID: 20105310
10.  What distinguishes adipose tissue of severely obese humans who are insulin sensitive and resistant? 
Current opinion in lipidology  2013;24(1):49-56.
Purpose of review
Despite a strong correlation between obesity and insulin resistance, 25% of severely obese (BMI >40) individuals are insulin sensitive. In this review, we will examine the factors in adipose tissue that distinguish the two groups, as well as reasons for believing the insulin-sensitive group will be less disease prone.
Recent findings
Obesity has been linked to the metabolic syndrome with an increase in visceral (intra-abdominal) compared to subcutaneous fat. Recent studies in which adipose tissue of insulin-sensitive and insulin-resistant patients with severe obesity were compared indicate that the insulin-resistant group is also distinguished by increases in oxidative stress and decreases in AMP-activated protein kinase (AMPK) activity. In contrast, changes in the expression of genes for SIRT1, inflammatory cytokines, mitochondrial biogenesis and function, and the two α-isoforms of AMPK showed more depot variation. Studies of how these and other changes in adipose tissue respond to bariatric surgery are still in their infancy.
Available data suggest that increases in oxidative stress, decreases in AMPK activity and SIRT1 gene expression, depot-specific changes in inflammatory, mitochondrial and other genes distinguish adipose tissue of insulin resistant from insulin-sensitive individuals with severe obesity.
PMCID: PMC3575680  PMID: 23298959
AMP-activated protein kinase; inflammation; insulin resistance; oxidative stress; SIRT1
11.  Chemokine Expression in Inflamed Adipose Tissue Is Mainly Mediated by NF-κB 
PLoS ONE  2013;8(6):e66515.
Immune cell infiltration of expanding adipose tissue during obesity and its role in insulin resistance has been described and involves chemokines. However, studies so far have focused on a single chemokine or its receptor (especially CCL2 and CCL5) whereas redundant functions of chemokines have been described. The objective of this work was to explore the expression of chemokines in inflamed adipose tissue in obesity. Human and mouse adipocytes were analyzed for expression of chemokines in response to inflammatory signal (TNF-α) using microarrays and gene set enrichment analysis. Gene expression was verified by qRT-PCR. Chemokine protein was determined in culture medium with ELISA. Chemokine expression was investigated in human subcutaneous adipose tissue biopsies and mechanism of chemokine expression was investigated using chemical inhibitors and cellular and animal transgenic models. Chemokine encoding genes were the most responsive genes in TNF-α treated human and mouse adipocytes. mRNA and protein of 34 chemokine genes were induced in a dose-dependent manner in the culture system. Furthermore, expression of those chemokines was elevated in human obese adipose tissue. Finally, chemokine expression was reduced by NF-κB inactivation and elevated by NF-κB activation. Our data indicate that besides CCL2 and CCL5, numerous other chemokines such as CCL19 are expressed by adipocytes under obesity-associated chronic inflammation. Their expression is regulated predominantly by NF-κB. Those chemokines could be involved in the initiation of infiltration of leukocytes into obese adipose tissue.
PMCID: PMC3688928  PMID: 23824685
12.  Effects of body weight and alcohol consumption on insulin sensitivity 
Nutrition Journal  2010;9:14.
Obesity is a risk factor for the development of insulin resistance, which can eventually lead to type-2 diabetes. Alcohol consumption is a protective factor against insulin resistance, and thus protects against the development of type-2 diabetes. The mechanism by which alcohol protects against the development of type-2 diabetes is not well known. To determine the mechanism by which alcohol improves insulin sensitivity, we fed water or alcohol to lean, control, and obese mice. The aim of this study was to determine whether alcohol consumption and body weights affect overlapping metabolic pathways and to identify specific target genes that are regulated in these pathways.
Adipose tissue dysfunction has been associated with the development of type-2 diabetes. We assessed possible gene expression alterations in epididymal white adipose tissue (WAT). We obtained WAT from mice fed a calorie restricted (CR), low fat (LF Control) or high fat (HF) diets and either water or 20% ethanol in the drinking water. We screened the expression of genes related to the regulation of energy homeostasis and insulin regulation using a gene array composed of 384 genes.
Obesity induced insulin resistance and calorie restriction and alcohol improved insulin sensitivity. The insulin resistance in obese mice was associated with the increased expression of inflammatory markers Cd68, Il-6 and Il-1α; in contrast, most of these genes were down-regulated in CR mice. Anti-inflammatory factors such as Il-10 and adrenergic beta receptor kinase 1 (Adrbk1) were decreased in obese mice and increased by CR and alcohol. Also, we report a direct correlation between body weight and the expression of the following genes: Kcnj11 (potassium inwardly-rectifying channel, subfamily J, member 11), Lpin2 (lipin2), and Dusp9 (dual-specificity MAP kinase phosphatase 9).
We show that alcohol consumption increased insulin sensitivity. Additionally, alterations in insulin sensitivity related with obesity were coupled with alterations in inflammatory genes. We provide evidence that alcohol may improve insulin sensitivity by up-regulating anti-inflammatory genes. Moreover, we have indentified potential gene targets in energy metabolic pathways and signal transducers that may contribute to obesity-related insulin resistance as well as calorie restriction and alcohol-induced insulin sensitivity.
PMCID: PMC2859759  PMID: 20307313
13.  Insulin Resistance in Non-Obese Subjects Is Associated with Activation of the JNK Pathway and Impaired Insulin Signaling in Skeletal Muscle 
PLoS ONE  2011;6(5):e19878.
The pathogenesis of insulin resistance in the absence of obesity is unknown. In obesity, multiple stress kinases have been identified that impair the insulin signaling pathway via serine phosphorylation of key second messenger proteins. These stress kinases are activated through various mechanisms related to lipid oversupply locally in insulin target tissues and in various adipose depots.
Methodology/Principal Findings
To explore whether specific stress kinases that have been implicated in the insulin resistance of obesity are potentially contributing to insulin resistance in non-obese individuals, twenty healthy, non-obese, normoglycemic subjects identified as insulin sensitive or resistant were studied. Vastus lateralis muscle biopsies obtained during euglycemic, hyperinsulinemic clamp were evaluated for insulin signaling and for activation of stress kinase pathways. Total and regional adipose stores and intramyocellular lipids (IMCL) were assessed by DXA, MRI and 1H-MRS. In muscle of resistant subjects, phosphorylation of JNK was increased (1.36±0.23 vs. 0.78±0.10 OD units, P<0.05), while there was no evidence for activation of p38 MAPK or IKKβ. IRS-1 serine phosphorylation was increased (1.30±0.09 vs. 0.22±0.03 OD units, P<0.005) while insulin-stimulated tyrosine phosphorylation decreased (10.97±0.95 vs. 0.89±0.50 OD units, P<0.005). IMCL levels were twice as high in insulin resistant subjects (3.26±0.48 vs. 1.58±0.35% H2O peak, P<0.05), who also displayed increased total fat and abdominal fat when compared to insulin sensitive controls.
This is the first report demonstrating that insulin resistance in non-obese, normoglycemic subjects is associated with activation of the JNK pathway related to increased IMCL and higher total body and abdominal adipose stores. While JNK activation is consistent with a primary impact of muscle lipid accumulation on metabolic stress, further work is necessary to determine the relative contributions of the various mediators of impaired insulin signaling in this population.
PMCID: PMC3092773  PMID: 21589939
14.  Increased abundance of the receptor-type protein-tyrosine phosphatase LAR accounts for the elevated insulin receptor dephosphorylating activity in adipose tissue of obese human subjects. 
Journal of Clinical Investigation  1995;95(6):2806-2812.
Protein-tyrosine phosphatases (PTPases) have an essential role in the regulation of the steady-state phosphorylation of the insulin receptor and other proteins in the insulin signalling pathway. To examine whether increased PTPase activity is associated with adipose tissue insulin resistance in human obesity we measured PTPase enzyme activity towards the insulin receptor in homogenates of subcutaneous adipose tissue from a series of six lean and six nondiabetic, obese (body mass index > 30) subjects. The obese subjects had a mean 1.74-fold increase in PTPase activity (P < 0.0001) with a striking positive correlation by linear regression analysis between PTPase activity and body mass index among all of the samples (R = 0.918; P < 0.0001). The abundance of three candidate insulin receptor PTPases in adipose tissue was also estimated by immunoblot analysis. The most prominent increase was a 2.03-fold rise in the transmembrane PTPase LAR (P < 0.001). Of the three PTPase examined, only immunodepletion of LAR protein from the homogenates with neutralizing antibodies resulted in normalization of the PTPase activity towards the insulin receptor, demonstrating that the increase in LAR was responsible for the enhanced PTPase activity in the adipose tissue from obese subjects. These studies suggest that increased PTPase activity towards the insulin receptor is a pathogenetic factor in the insulin resistance of adipose tissue in human obesity and provide evidence for a potential role of the LAR PTPase in the regulation of insulin signalling in disease states.
PMCID: PMC295966  PMID: 7769120
15.  Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats. 
Journal of Clinical Investigation  1998;101(6):1354-1361.
Troglitazone (CS-045) is one of the thiazolidinediones that activate the peroxisome proliferator-activated receptor gamma (PPARgamma), which is expressed primarily in adipose tissues. To elucidate the mechanism by which troglitazone relieves insulin resistance in vivo, we studied its effects on the white adipose tissues of an obese animal model (obese Zucker rat). Administration of troglitazone for 15 d normalized mild hyperglycemia and marked hyperinsulinemia in these rats. Plasma triglyceride level was decreased by troglitazone in both obese and lean rats. Troglitazone did not change the total weight of white adipose tissues but increased the number of small adipocytes (< 2,500 micron2) approximately fourfold in both retroperitoneal and subcutaneous adipose tissues of obese rats. It also decreased the number of large adipocytes (> 5,000 micron2) by approximately 50%. In fact, the percentage of apoptotic nuclei was approximately 2.5-fold higher in the troglitazone-treated retroperitoneal white adipose tissue than control. Concomitantly, troglitazone normalized the expression levels of TNF-alpha which were elevated by 2- and 1.4-fold in the retroperitoneal and mesenteric white adipose tissues of the obese rats, respectively. Troglitazone also caused a dramatic decrease in the expression levels of leptin, which were increased by 4-10-fold in the white adipose tissues of obese rats. These results suggest that the primary action of troglitazone may be to increase the number of small adipocytes in white adipose tissues, presumably via PPARgamma. The increased number of small adipocytes and the decreased number of large adipocytes in white adipose tissues of troglitazone-treated obese rats appear to be an important mechanism by which increased expression levels of TNF-alpha and higher levels of plasma lipids are normalized, leading to alleviation of insulin resistance.
PMCID: PMC508690  PMID: 9502777
16.  Switch from Stress Response to Homeobox Transcription Factors in Adipose Tissue After Profound Fat Loss 
PLoS ONE  2010;5(6):e11033.
In obesity, impaired adipose tissue function may promote secondary disease through ectopic lipid accumulation and excess release of adipokines, resulting in systemic low-grade inflammation, insulin resistance and organ dysfunction. However, several of the genes regulating adipose tissue function in obesity are yet to be identified.
Methodology/Principal Findings
In order to identify novel candidate genes that may regulate adipose tissue function, we analyzed global gene expression in abdominal subcutaneous adipose tissue before and one year after bariatric surgery (biliopancreatic diversion with duodenal switch, BPD/DS) (n = 16). Adipose tissue from lean healthy individuals was also analyzed (n = 13). Two different microarray platforms (AB 1700 and Illumina) were used to measure the differential gene expression, and the results were further validated by qPCR. Surgery reduced BMI from 53.3 to 33.1 kg/m2. The majority of differentially expressed genes were down-regulated after profound fat loss, including transcription factors involved in stress response, inflammation, and immune cell function (e.g., FOS, JUN, ETS, C/EBPB, C/EBPD). Interestingly, a distinct set of genes was up-regulated after fat loss, including homeobox transcription factors (IRX3, IRX5, HOXA5, HOXA9, HOXB5, HOXC6, EMX2, PRRX1) and extracellular matrix structural proteins (COL1A1, COL1A2, COL3A1, COL5A1, COL6A3).
The data demonstrate a marked switch of transcription factors in adipose tissue after profound fat loss, providing new molecular insight into a dichotomy between stress response and metabolically favorable tissue development. Our findings implicate homeobox transcription factors as important regulators of adipose tissue function.
PMCID: PMC2882947  PMID: 20543949
17.  Munc18c in Adipose Tissue Is Downregulated in Obesity and Is Associated with Insulin 
PLoS ONE  2013;8(5):e63937.
Munc18c is associated with glucose metabolism and could play a relevant role in obesity. However, little is known about the regulation of Munc18c expression. We analyzed Munc18c gene expression in human visceral (VAT) and subcutaneous (SAT) adipose tissue and its relationship with obesity and insulin.
Materials and Methods
We evaluated 70 subjects distributed in 12 non-obese lean subjects, 23 overweight subjects, 12 obese subjects and 23 nondiabetic morbidly obese patients (11 with low insulin resistance and 12 with high insulin resistance).
The lean, overweight and obese persons had a greater Munc18c gene expression in adipose tissue than the morbidly obese patients (p<0.001). VAT Munc18c gene expression was predicted by the body mass index (B = −0.001, p = 0.009). In SAT, no associations were found by different multiple regression analysis models. SAT Munc18c gene expression was the main determinant of the improvement in the HOMA-IR index 15 days after bariatric surgery (B = −2148.4, p = 0.038). SAT explant cultures showed that insulin produced a significant down-regulation of Munc18c gene expression (p = 0.048). This decrease was also obtained when explants were incubated with liver X receptor alpha (LXRα) agonist, either without (p = 0.038) or with insulin (p = 0.050). However, Munc18c gene expression was not affected when explants were incubated with insulin plus a sterol regulatory element-binding protein-1c (SREBP-1c) inhibitor (p = 0.504).
Munc18c gene expression in human adipose tissue is down-regulated in morbid obesity. Insulin may have an effect on the Munc18c expression, probably through LXRα and SREBP-1c.
PMCID: PMC3659121  PMID: 23700440
18.  Bioinformatics for the NuGO proof of principle study: analysis of gene expression in muscle of ApoE3*Leiden mice on a high-fat diet using PathVisio 
Genes & Nutrition  2008;3(3-4):185-191.
Insulin resistance is a characteristic of type-2 diabetes and its development is associated with an increased fat consumption. Muscle is one of the tissues that becomes insulin resistant after high fat (HF) feeding. The aim of the present study is to identify processes involved in the development of HF-induced insulin resistance in muscle of ApOE3*Leiden mice by using microarrays. These mice are known to become insulin resistant on a HF diet. Differential gene expression was measured in muscle using the Affymetrix mouse plus 2.0 array. To get more insight in the processes, affected pathway analysis was performed with a new tool, PathVisio. PathVisio is a pathway editor customized with plug-ins (1) to visualize microarray data on pathways and (2) to perform statistical analysis to select pathways of interest. The present study demonstrated that with pathway analysis, using PathVisio, a large variety of processes can be investigated. The significantly regulated genes in muscle of ApOE3*Leiden mice after 12 weeks of HF feeding were involved in several biological pathways including fatty acid beta oxidation, fatty acid biosynthesis, insulin signaling, oxidative stress and inflammation.
PMCID: PMC2593012  PMID: 19034557
Pathway analysis; Microarray; Insulin resistance; High-fat diet
19.  The expression of ob gene is not acutely regulated by insulin and fasting in human abdominal subcutaneous adipose tissue. 
Journal of Clinical Investigation  1996;98(2):251-255.
The regulation of ob gene expression in abdominal subcutaneous adipose tissue was investigated using a reverse transcription-competitive PCR method to quantify the mRNA level of leptin. Leptin mRNA level was highly correlated with the body mass index of 26 subjects (12 lean, 7 non-insulin-dependent diabetic, and 7 obese patients). The effect of fasting on ob gene expression was investigated in 10 subjects maintained on a hypocaloric diet (1045 KJ/d) for 5 d. While their metabolic parameters significantly changed (decrease in insulinemia, glycemia, and resting metabolic rate and increase in plasma ketone bodies), the caloric restriction did not modify the leptin mRNA level in the adipose tissue. To verify whether insulin regulates ob gene expression, six lean subjects underwent a 3-h euglycemic hyperinsulinemic (846 +/- 138 pmol/liter) clamp. Leptin and Glut 4 mRNA levels were quantified in adipose tissue biopsies taken before and at the end of the clamp. Insulin infusion produced a significant threefold increase in Glut 4 mRNA while leptin mRNA was not affected. It is concluded that ob gene expression is not acutely regulated by insulin or by metabolic factors related to fasting in human abdominal subcutaneous adipose tissue.
PMCID: PMC507424  PMID: 8755631
20.  Macrophages and Adipocytes in Human Obesity 
Diabetes  2009;58(7):1558-1567.
We investigated the regulation of adipose tissue gene expression during different phases of a dietary weight loss program and its relation with insulin sensitivity.
Twenty-two obese women followed a dietary intervention program composed of an energy restriction phase with a 4-week very-low-calorie diet and a weight stabilization period composed of a 2-month low-calorie diet followed by 3–4 months of a weight maintenance diet. At each time point, a euglycemic-hyperinsulinemic clamp and subcutaneous adipose tissue biopsies were performed. Adipose tissue gene expression profiling was performed using a DNA microarray in a subgroup of eight women. RT–quantitative PCR was used for determination of mRNA levels of 31 adipose tissue macrophage markers (n = 22).
Body weight, fat mass, and C-reactive protein level decreased and glucose disposal rate increased during the dietary intervention program. Transcriptome profiling revealed two main patterns of variations. The first involved 464 mostly adipocyte genes involved in metabolism that were downregulated during energy restriction, upregulated during weight stabilization, and unchanged during the dietary intervention. The second comprised 511 mainly macrophage genes involved in inflammatory pathways that were not changed or upregulated during energy restriction and downregulated during weight stabilization and dietary intervention. Accordingly, macrophage markers were upregulated during energy restriction and downregulated during weight stabilization and dietary intervention. The increase in glucose disposal rates in each dietary phase was associated with variation in expression of sets of 80–110 genes that differed among energy restriction, weight stabilization, and dietary intervention.
Adipose tissue macrophages and adipocytes show distinct patterns of gene regulation and association with insulin sensitivity during the various phases of a dietary weight loss program.
PMCID: PMC2699855  PMID: 19401422
21.  Differential co-expression analysis of obesity-associated networks in human subcutaneous adipose tissue 
To use a unique obesity-discordant sib-pair study design to combine differential expression analysis, expression quantitative trait loci (eQTLs) mapping, and a co-expression regulatory network approach in subcutaneous human adipose tissue to identify genes relevant to the obese state.
Study design
Genome-wide transcript expression in subcutaneous human adipose tissue was measured using Affymetrix U133+2.0 microarrays and genomewide genotyping data was obtained using an Applied Biosystems SNPlex linkage panel.
154 Swedish families ascertained through an obese proband (Body Mass Index >30kg/m2) with a discordant sibling (BMI>10kg/m2 less than proband).
Approximately one-third of the transcripts were differentially expressed between lean and obese siblings. The cellular adhesion molecules (CAMs) KEGG grouping contained the largest number of differentially expressed genes under cis-acting genetic control. By using a novel approach to contrast CAMs co-expression networks between lean and obese siblings, a subset of differentially regulated genes was identified, with the previously GWAS obesity-associated NEGR1 as a central hub. Independent analysis using mouse data demonstrated that this finding for NEGR1 is conserved across species.
Our data suggests that, in addition to its reported role in the brain, NEGR1 is also expressed in subcutaneous adipose tissue and acts as a central “hub” in an obesity-related transcript network.
PMCID: PMC3160485  PMID: 21427694
Gene Expression; network; eQTL; sibpair; linkage; adipose tissue
22.  Coordinated Defects in Hepatic Long Chain Fatty Acid Metabolism and Triglyceride Accumulation Contribute to Insulin Resistance in Non-Human Primates 
PLoS ONE  2011;6(11):e27617.
Non-Alcoholic fatty liver disease (NAFLD) is characterized by accumulation of triglycerides (TG) in hepatocytes, which may also trigger cirrhosis. The mechanisms of NAFLD are not fully understood, but insulin resistance has been proposed as a key determinant.
To determine the TG content and long chain fatty acyl CoA composition profile in liver from obese non-diabetic insulin resistant (IR) and lean insulin sensitive (IS) baboons in relation with hepatic and peripheral insulin sensitivity.
Twenty baboons with varying grades of adiposity were studied. Hepatic (liver) and peripheral (mainly muscle) insulin sensitivity was measured with a euglycemic clamp and QUICKI. Liver biopsies were performed at baseline for TG content and LCFA profile by mass spectrometry, and histological analysis. Findings were correlated with clinical and biochemical markers of adiposity and insulin resistance.
Obese IR baboons had elevated liver TG content compared to IS. Furthermore, the concentration of unsaturated (LC-UFA) was greater than saturated (LC-SFA) fatty acyl CoA in the liver. Interestingly, LC-FA UFA and SFA correlated with waist, BMI, insulin, NEFA, TG, QUICKI, but not M/I. Histological findings of NAFLD ranging from focal to diffuse hepatic steatosis were found in obese IR baboons.
Liver TG content is closely related with both hepatic and peripheral IR, whereas liver LC-UFA and LC-SFA are closely related only with hepatic IR in non-human primates. Mechanisms leading to the accumulation of TG, LC-UFA and an altered UFA: LC-SFA ratio may play an important role in the pathophysiology of fatty liver disease in humans.
PMCID: PMC3220682  PMID: 22125617
Obesity Surgery  2012;22(3):472-477.
Background and Aim
Circulating adiponectin is known to correlate negatively with insulin resistance in patients with obesity and diabetes. The aim of this study was to assess the effect of gastric bypass (GB) surgery on adiponectin gene expression in subcutaneous and omental adipose tissues.
Adipose tissues and plasma were obtained from 25 subjects undergoing GB surgery, 15 non-obese subjects, and 12 subjects after GB surgery. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was used for analysis of the adipose tissues. Adiponectin expression was normalized for GAPDH and expressed as percentage of subject-matched subcutaneous expression which was given an arbitrary value of 100%. Insulin resistance was assessed by the homeostatic model assessment (HOMA). Circulating adiponectin was assayed by ELISA.
Omental adiponectin gene expression was 5-fold higher in subjects after GB when compared with age matched morbidly obese subjects before GB (P<0.01). There were not statistical differences in omental adiponectin gene expression observed in subjects after GB and age matched non-obese subjects. For the entire cohort of subjects, there was a significant negative correlation between omental adiponectin expression and insulin resistance expressed by HOMA values (r=−0.62, P<0.001). Circulating adiponectin was significantly lower (p<0.05) in the obese group than in the non-obese and post-GB groups.
Omental adiponectin gene expression significantly increase after GB surgery reaching levels equal to age matched non-obese subjects. Omental adiponectin expression has a significant negative correlation with the insulin resistance status.
PMCID: PMC3381935  PMID: 22161113
adiponectin; gastric bypass surgery; bariatric surgery; omentum; adipose tissue; insulin resistance
24.  Transcriptomic profiles of skeletal muscle tissue following an euglycemic-hyperinsulinemic clamp in insulin-resistant obese subjects 
Genes & Nutrition  2012;8(1):91-98.
Insulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Muscle is mainly responsible for insulin-stimulated glucose clearance from the bloodstream. Thus, regulation of gene expression in muscle tissue may be involved in the pathogenesis of insulin resistance. The objective was to investigate gene expression and metabolic pathways alterations in skeletal muscle tissue following an euglycemic-hyperinsulinemic clamp in obese insulin-resistant subjects. We carried out a transcriptome comparison of skeletal muscle tissue before and after a 3-h euglycemic-hyperinsulinemic clamp following 8-week supplementation with n-3 polyunsaturated fatty acid (PUFA) (1.8 g/day) with or without a supplement of fish gelatin (FG) (25 % of daily protein intake) in 16 obese insulin-resistant subjects. Results indicate that approximately 5 % (1932) of expressed transcripts were significantly changed after the clamp in both n-3 PUFA and n-3 PUFA + FG supplementation periods. Of these differentially expressed transcripts, 1394 genes associated with enzymes, transcription and translation regulators, transporters, G protein-coupled receptors, cytokines, and ligand-dependent nuclear receptors were modified. Metabolic pathways that were significantly modified included liver X receptor/retinoid X receptors (RXR) activation, vitamin D receptor/RXR activation, interleukin (IL)-8, acute phase response, IL10, triggering receptor expressed on myeloid cells 1, peroxisome proliferator-activated receptor, G-beta/gamma and hepatocyte growth factor and IL6 signaling. Taken together, results suggest that mainly inflammatory and transcription factors are modified following clamp in obese insulin-resistant subjects. Overall, understanding the changes in metabolic pathways due to insulin may be a potential target for the management of insulin resistance.
PMCID: PMC3534998  PMID: 22566203
Diabetes; Obesity; Microarray; Gene expression; Metabolic pathways
25.  Endoplasmic Reticulum Stress Is Reduced in Tissues of Obese Subjects After Weight Loss 
Diabetes  2009;58(3):693-700.
OBJECTIVE—Obesity is associated with insulin resistance and type 2 diabetes, although the mechanisms linking these pathologies remain undetermined. Recent studies in rodent models revealed endoplasmic reticulum (ER) stress in adipose and liver tissues and demonstrated that ER stress could cause insulin resistance. Therefore, we tested whether these stress pathways were also present in obese human subjects and/or regulated by weight loss.
RESEARCH DESIGN AND METHODS—Eleven obese men and women (BMI 51.3 ± 3.0 kg/m2) were studied before and 1 year after gastric bypass (GBP) surgery. We examined systemic insulin sensitivity using hyperinsulinemic-euglycemic clamp studies before and after surgery and collected subcutaneous adipose and liver tissues to examine ER stress markers.
RESULTS—Subjects lost 39 ± 9% body wt at 1 year after GBP surgery (P < 0.001), which was associated with a marked improvement in hepatic, skeletal muscle, and adipose tissue insulin sensitivity. Markers of ER stress in adipose tissue significantly decreased with weight loss. Specifically, glucose-regulated protein 78 (Grp78) and spliced X-box binding protein-1 (sXBP-1) mRNA levels were reduced, as were phosphorylated elongation initiation factor 2α (eIF2α) and stress kinase c-Jun NH2-terminal kinase 1 (JNK1) (all P values <0.05). Liver sections from a subset of subjects showed intense staining for Grp78 and phosphorylated eIF2α before surgery, which was reduced in post-GBP sections.
CONCLUSIONS—This study presents important evidence that ER stress pathways are present in selected tissues of obese humans and that these signals are regulated by marked weight loss and metabolic improvement. Hence, this suggests the possibility of a relationship between obesity-related ER stress and metabolic dysfunction in obese humans.
PMCID: PMC2646068  PMID: 19066313

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