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OBJECTIVE

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.

RESEARCH DESIGN AND METHODS

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.

RESULTS

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.

CONCLUSIONS

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.

Objective

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.

Methods

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.

Results

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.

Conclusions

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.

Background

Obesity is a strong risk factor for resistance to insulin-mediated glucose disposal, a precursor of type 2 diabetes and other disorders.

Objectives

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.

Setting

University Medical Center, United States

Methods

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.

Results

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.

Conclusions

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.

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.

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.

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.

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.

Summary

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.

Background

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.

Method

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.

Results

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).

Conclusion

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.

Background

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.

Conclusions

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.

Background

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).

Conclusions/Significance

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.

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.

OBJECTIVE

We investigated the regulation of adipose tissue gene expression during different phases of a dietary weight loss program and its relation with insulin sensitivity.

RESEARCH DESIGN AND METHODS

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).

RESULTS

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.

CONCLUSIONS

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.

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.

Objective

Insulin resistance induces hyperinsulinemic compensation, which in turn maintains almost a constant disposition index. However, the signal that gives rise to the hyperinsulinemic compensation for insulin resistance remains unknown.

Methods

In a dog model of obesity we examined the possibility that potential early-week changes in plasma FFA, glucose, or both could be part of a cascade of signals that lead to compensatory hyperinsulinemia induced by insulin resistance.

Results

Hypercaloric high fat feeding in dogs resulted in modest weight gain, and an increase in adipose tissue with no change in the non-adipose tissue size. To compensate for the drop in insulin sensitivity, there was a significant rise in plasma insulin, which can be attributed in part to a decrease in the metabolic clearance rate of insulin and increased insulin secretion. In this study we observed complete compensation for high fat diet induced insulin resistance as measured by the disposition index. The compensatory hyperinsulinemia was coupled with significant changes in plasma FFAs and no change in plasma glucose.

Conclusions

We postulate that early in the development of diet induced insulin resistance, a change in plasma FFAs may directly, through signaling at the level of β-cell, or indirectly, by decreasing hepatic insulin clearance, result in the observed hyperinsulinemic compensation.

OBJECTIVE

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.

RESEARCH DESIGN AND METHODS

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.

RESULTS

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.

CONCLUSIONS

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.

OBJECTIVE—Skeletal muscle–specific LPL knockout mouse (SMLPL−/−) were created to study the systemic impact of reduced lipoprotein lipid delivery in skeletal muscle on insulin sensitivity, body weight, and composition.

RESEARCH DESIGN AND METHODS—Tissue-specific insulin sensitivity was assessed using a hyperinsulinemic-euglycemic clamp and 2-deoxyglucose uptake. Gene expression and insulin-signaling molecules were compared in skeletal muscle and liver of SMLPL−/− and control mice.

RESULTS—Nine-week-old SMLPL−/− mice showed no differences in body weight, fat mass, or whole-body insulin sensitivity, but older SMLPL−/− mice had greater weight gain and whole-body insulin resistance. High-fat diet feeding accelerated the development of obesity. In young SMLPL−/− mice, insulin-stimulated glucose uptake was increased 58% in the skeletal muscle, but was reduced in white adipose tissue (WAT) and heart. Insulin action was also diminished in liver: 40% suppression of hepatic glucose production in SMLPL−/− vs. 90% in control mice. Skeletal muscle triglyceride was 38% lower, and insulin-stimulated phosphorylated Akt (Ser473) was twofold greater in SMLPL−/− mice without changes in IRS-1 tyrosine phosphorylation and phosphatidylinositol 3-kinase activity. Hepatic triglyceride and liver X receptor, carbohydrate response element–binding protein, and PEPCK mRNAs were unaffected in SMLPL−/− mice, but peroxisome proliferator–activated receptor (PPAR)-γ coactivator-1α and interleukin-1β mRNAs were higher, and stearoyl–coenzyme A desaturase-1 and PPARγ mRNAs were reduced.

CONCLUSIONS—LPL deletion in skeletal muscle reduces lipid storage and increases insulin signaling in skeletal muscle without changes in body composition. Moreover, lack of LPL in skeletal muscle results in insulin resistance in other key metabolic tissues and ultimately leads to obesity and systemic insulin resistance.

OBJECTIVE

We examined the relationship between the expression of thrombospondin (TSP)1, an antiangiogenic factor and regulator of transforming growth factor-β activity, obesity, adipose inflammation, and insulin resistance.

RESEARCH DESIGN AND METHODS

TSP1 gene expression was quantified in subcutaneous adipose tissue (SAT) of 86 nondiabetic subjects covering a wide range of BMI and insulin sensitivity, from visceral adipose (VAT) and SAT from 14 surgical patients and from 38 subjects with impaired glucose tolerance randomized to receive either pioglitazone or metformin for 10 weeks. An adipocyte culture system was also used to assess the effects of pioglitazone and coculture with macrophages on TSP1 gene expression.

RESULTS

TSP1 mRNA was significantly associated with obesity (BMI) and insulin resistance (low insulin sensitivity index). Relatively strong positive associations were seen with markers of inflammation, including CD68, macrophage chemoattractant protein-1, and plasminogen activator inhibitor (PAI)-1 mRNA (r ≥ 0.46, P = 0.001 for each), that remained significant after controlling for BMI and Si. However, TSP1 mRNA was preferentially expressed in adipocyte fraction, whereas inflammatory markers predominated in stromal vascular fraction. Coculture of adipocytes and macrophages augmented TSP1 gene expression and secretion from both cell types. Pioglitazone (not metformin) treatment resulted in a 54% decrease (P < 0.04) in adipose TSP gene expression, as did in vitro pioglitazone treatment of adipocytes.

CONCLUSIONS

TSP1 is a true adipokine that is highly expressed in obese, insulin-resistant subjects; is highly correlated with adipose inflammation; and is decreased by pioglitazone. TSP1 is an important link between adipocytes and macrophage-driven adipose tissue inflammation and may mediate the elevation of PAI-1 that promotes a prothrombotic state.

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.

Background

Obesity-related adipose inflammation has been thought to be a causal factor for the development of insulin resistance and type 2 diabetes. Infiltrated macrophages in adipose tissue of obese animals and humans are an important source for inflammatory cytokines. Clodronate liposomes can ablate macrophages by inducing apoptosis. In this study, we aim to determine whether peritoneal injection of clodronate liposomes has any beneficial effect on systemic glucose homeostasis/insulin sensitivity and whether macrophage content in visceral adipose tissue will be reduced in diet-induced obese (DIO) mice.

Methodology/Principal Findings

Clodronate liposomes were used to deplete macrophages in lean and DIO mice. Macrophage content in visceral adipose tissue, metabolic parameters, glucose and insulin tolerance, adipose and liver histology, adipokine and cytokine production were examined. Hyperinsulinemic-euglycemic clamp study was also performed to assess systemic insulin sensitivity. Peritoneal injection of clodronate liposomes significantly reduced blood glucose and insulin levels in DIO mice. Systemic glucose tolerance and insulin sensitivity were mildly improved in both lean and DIO mice treated with clodronate liposomes by intraperitoneal (ip) injection. Hepatosteatosis was dramatically alleviated and suppression of hepatic glucose output was markedly increased in DIO mice treated with clodronate liposomes. Macrophage content in visceral adipose tissue of DIO mice was effectively decreased without affecting subcutaneous adipose tissue. Interestingly, levels of insulin sensitizing hormone adiponectin, including the high molecular weight form, were significantly elevated in circulation.

Conclusions/Significance

Intraperitoneal injection of clodronate liposomes reduces visceral adipose tissue macrophages, improves systemic glucose homeostasis and insulin sensitivity in DIO mice, which can be partially attributable to increased adiponectin levels.

Background

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.

Methods

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.

Results

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.

Conclusion

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.

OBJECTIVE

To investigate the role of the endoplasmic reticulum (ER) chaperone glucose-regulated protein (GRP) 78/BiP in the pathogenesis of obesity, insulin resistance, and type 2 diabetes.

RESEARCH DESIGN AND METHODS

Male Grp78+/− mice and their wild-type littermates were subjected to a high-fat diet (HFD) regimen. Pathogenesis of obesity and type 2 diabetes was examined by multiple approaches of metabolic phenotyping. Tissue-specific insulin sensitivity was analyzed by hyperinsulinemic-euglycemic clamps. Molecular mechanism was explored via immunoblotting and tissue culture manipulation.

RESULTS

Grp78 heterozygosity increases energy expenditure and attenuates HFD-induced obesity. Grp78+/− mice are resistant to diet-induced hyperinsulinemia, liver steatosis, white adipose tissue (WAT) inflammation, and hyperglycemia. Hyperinsulinemic-euglycemic clamp studies revealed that Grp78 heterozygosity improves glucose metabolism independent of adiposity and following an HFD increases insulin sensitivity predominantly in WAT. As mechanistic explanations, Grp78 heterozygosity in WAT under HFD stress promotes adaptive unfolded protein response (UPR), attenuates translational block, and upregulates ER degradation-enhancing α-mannosidase–like protein (EDEM) and ER chaperones, thus improving ER quality control and folding capacity. Further, overexpression of the active form of ATF6 induces protective UPR and improves insulin signaling upon ER stress.

CONCLUSIONS

HFD-induced obesity and type 2 diabetes are improved in Grp78+/− mice. Adaptive UPR in WAT could contribute to this improvement, linking ER homeostasis to energy balance and glucose metabolism.

OBJECTIVE

To assess the potential role of FoxP3-expressing regulatory T cells (Tregs) in reversing obesity-linked insulin resistance and diabetic nephropathy in rodent models and humans.

RESEARCH DESIGN AND METHODS

To characterize the role of Tregs in insulin resistance, human visceral adipose tissue was first evaluated for Treg infiltration and second, the db/db mouse model was evaluated.

RESULTS

Obese patients with insulin resistance displayed significantly decreased natural Tregs but an increase in adaptive Tregs in their visceral adipose tissue as compared with lean control subjects. To further evaluate the pathogenic role of Tregs in insulin resistance, the db/db mouse model was used. Treg depletion using an anti-CD25 monoclonal antibody enhanced insulin resistance as shown by increased fasting blood glucose levels as well as an impaired insulin sensitivity. Moreover, Treg-depleted db/db mice developed increased signs of diabetic nephropathy, such as albuminuria and glomerular hyperfiltration. This was paralleled by a proinflammatory milieu in both murine visceral adipose tissue and the kidney. Conversely, adoptive transfer of CD4+FoxP3+ Tregs significantly improved insulin sensitivity and diabetic nephropathy. Accordingly, there was increased mRNA expression of FoxP3 as well as less abundant proinflammatory CD8+CD69+ T cells in visceral adipose tissue and kidneys of Treg-treated animals.

CONCLUSIONS

Data suggest a potential therapeutic value of Tregs to improve insulin resistance and end organ damage in type 2 diabetes by limiting the proinflammatory milieu.

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.

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.

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Both insulin resistance and hyperinsulinemia have been reported to be independent risk factors for cardiovascular diseases. However, little is known regarding insulin signaling in the vascular tissues in insulin-resistant states. In this report, insulin signaling on the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways were compared in vascular tissues of lean and obese Zucker (fa/fa) rats in both ex vivo and in vivo studies. Ex vivo, insulin-stimulated tyrosine phosphorylation of insulin receptor β subunits (IRβ) in the aorta and microvessels of obese rats was significantly decreased compared with lean rats, although the protein levels of IRβ in the 2 groups were not different. Insulin-induced tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and their protein levels were decreased in the aorta of obese rats compared with lean rats. The association of p85 subunit to the IRS proteins and the IRS-associated PI 3-kinase activities stimulated by insulin in the aorta of obese rats were significantly decreased compared with the lean rats. In addition, insulin-stimulated serine phosphorylation of Akt, a downstream kinase of PI 3-kinase pathway, was also reduced significantly in isolated microvessels from obese rats compared with the lean rats. In euglycemic clamp studies, insulin infusion greatly increased tyrosine phosphorylation of IRβ- and IRS-2–associated PI 3-kinase activity in the aorta of lean rats, but only slight increases were observed in obese rats. In contrast, insulin stimulated tyrosine phosphorylation of MAP kinase (ERK-1/2) equally in isolated microvessels of lean and obese rats, although basal tyrosine phosphorylation of ERK-1/2 was higher in the obese rats. To our knowledge, these data provided the first direct measurements of insulin signaling in the vascular tissues, and documented a selective resistance to PI 3-kinase (but not to MAP kinase pathway) in the vascular tissues of obese Zucker rats.

J. Clin. Invest. 104:447–457 (1999).