Global Gene Expression Profiles of Subcutaneous Adipose and Muscle from Glucose-Tolerant, Insulin-Sensitive, and Insulin-Resistant Individuals Matched for BMI

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 down-regulated. 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. Diabetes 60:1019–1029, 201

The Lipogenic Enzymes DGAT1, FAS, and LPL in Adipose Tissue: Effects of Obesity, Insulin Resistance, and TZD Treatment

Acyl-coenzyme A:diacylglycerol transferase (DGAT), fatty acid synthetase (FAS), and LPL are three enzymes important in adipose tissue triglyceride accumulation. To study the relationship of DGAT1, FAS, and LPL with insulin, we examined adipose mRNA expression of these genes in subjects with a wide range of insulin sensitivity (SI). DGAT1 and FAS (but not LPL) expression were strongly correlated with SI. In addition, the expression of DGAT1 and FAS (but not LPL) were higher in normal glucose-tolerant subjects compared with subjects with impaired glucose tolerance (IGT) (P \u3c 0.005). To study the effects of insulin sensitizers, subjects with IGT were treated with pioglitazone or metformin for 10 weeks, and lipogenic enzymes were measured in adipose tissue. After pioglitazone treatment, DGAT1 expression was increased by 33 ± 10% (P \u3c 0.05) and FAS expression increased by 63 ± 8% (P \u3c 0.05); however, LPL expression was not altered. DGAT1, FAS, and LPL mRNA expression were not significantly changed after metformin treatment. The treatment of mice with rosiglitazone also resulted in an increase in adipose expression of DGAT1 by 2- to 3-fold, as did the treatment of 3T3 F442A adipocytes in vitro with thiazolidinediones. These data support a more global concept suggesting that adipose lipid storage functions to prevent peripheral lipotoxicity

Pioglitazone Induces Apoptosis of Macrophages in Human Adipose Tissue

Metabolic syndrome and type 2 diabetes mellitus are associated with an increased number of macrophage cells that infiltrate white adipose tissue (WAT). Previously, we demonstrated that the treatment of subjects with impaired glucose tolerance (IGT) with the peroxisome proliferator-activated receptor γ (PPARγ) agonist pioglitazone resulted in a decrease in macrophage number in adipose tissue. Here, adipose tissue samples from IGT subjects treated with pioglitazone were examined for apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. TUNEL-positive cells were identified, and there was a significant 42% increase in TUNEL-positive cells following pioglitazone treatment. Overlay experiments with anti-CD68 antibody demonstrated that most of theTUNEL-positive cellsweremacrophages.To determine whether macrophage apoptosis was a direct or indirect effect of pioglitazone treatment, human THP1 cells were treated with pioglitazone in vitro, demonstrating increased TUNEL staining in a dose- and time-dependent manner. Furthermore, the appearance of the active proteolytic subunits of caspase-3 and caspase-9 were detected in cell lysate from THP1 cells and also increased in a dose- and time-dependent manner following pioglitazone treatment. Pretreatment with a PPARγ inhibitor, GW9662, prevented pioglitazone induction of the apoptotic pathway in THP1 cells. Differentiated human adipocytes did not show any significant increase in apoptosis after treatment in vitro with piolgitazone. These findings indicate that PPARγ has distinct functions in different cell types in WAT, such that pioglitazone reduces macrophage infiltration by inducing apoptotic cell death specifically in macrophages through PPARγ activation

Expression of CD68 and Macrophage Chemoattractant Protein-1 Genes in Human Adipose and Muscle Tissues: Association with Cytokine Expression, Insulin Resistance, and Reduction by Pioglitazone

To examine the role of adipose-resident macrophages in insulin resistance, we examined the gene expression of CD68, a macrophage marker, along with macrophage chemoattractant protein-1 (MCP-1) in human subcutaneous adipose tissue using real-time RT-PCR. Both CD68 and MCP-1 mRNAs were expressed in human adipose tissue, primarily in the stromal vascular fraction. When measured in the adipose tissue from subjects with normal glucose tolerance, covering a wide range of BMI (21-51 kg/m2) and insulin sensitivity (SI) (0.6-8.0 × 10-4 min-1 · μU-1 · ml-1), CD68 mRNA abundance, which correlated with the number of CD68-positive cells by immunohistochemistry, tended to increase with BMI but was not statistically significant. However, there was a significant inverse relation between CD68 mRNA and SI (r = -0.55, P = 0.02). In addition, there was a strong positive relationship among adipose tissue CD68 mRNA, tumor necrosis factor-α (TNF-α) secretion in vitro (r = 0.79, P \u3c 0.005), and plasma interleukin-6 (r = 0.67, P \u3c 0.005). To determine whether improving SI in subjects with impaired glucose tolerance (IGT) was associated with decreased CD68 expression, IGT subjects were treated for 10 weeks with pioglitazone or metformin. Pioglitazone increased S1 by 60% and in the same subjects reduced both CD68 and MCP-1 mRNAs by \u3e50%. Furthermore, pioglitazone resulted in a reduction in the number of CD68-positive cells in adipose tissue and reduced plasma TNF-α. Metformin had no effect on any of these measures. Thus, treatment with pioglitazone reduces expression of CD68 and MCP-1 in adipose tissue, apparently by reducing macrophage numbers, resulting in reduced inflammatory cytokine production and improvement in SI

Lipin Expression Is Attenuated in Adipose Tissue of Insulin-Resistant Human Subjects and Increases With Peroxisome Proliferator-Activated Receptor γ Activation

Lipin-α and -β are the alternatively spliced gene products of the Lpin1 gene, whose product lipin is required for adipocyte differentiation. Lipin deficiency causes lipodystrophy, fatty liver, and insulin resistance in mice, whereas adipose tissue lipin overexpression results in increased adiposity but improved insulin sensitivity. To assess lipin expression and its relation to insulin resistance in humans, we examined lipin-α and -β mRNA levels in subjects with normal or impaired glucose tolerance. We found higher expression levels of both lipin isoforms in lean, insulin-sensitive subjects. When compared with normal glucose-tolerant subjects, individuals with impaired glucose tolerance were more insulin resistant, demonstrated higher levels of intramyocellular lipids (IMCLs), and expressed ∼50% lower levels of lipin-α and -β. In addition, there was a strong inverse correlation between adipose tissue lipin expression and muscle IMCLs but no evidence for an increase in muscle lipid oxidation. After treatment of the impaired glucose-tolerant subjects with insulin sensitizers for 10 weeks, pioglitazone (but not metformin) resulted in a 60% increase in the insulin sensitivity index (Si) and a 32% decrease in IMCLs (both P \u3c 0.01), along with an increase in lipin-β (but not lipin-α) expression by 200% (P \u3c 0.005). Lipin expression in skeletal muscle, however, was not related to obesity or insulin resistance. Hence, high adipose tissue lipin expression is found in insulin-sensitive subjects, and lipin-β expression increases following treatment with pioglitazone. These results suggest that increased adipogenesis and/or lipogenesis in subcutaneous fat, mediated by the LPIN1 gene, may prevent lipotoxicity in muscle, leading to improved insulin sensitivity

Human Visfatin Expression: Relationship to Insulin Sensitivity, Intramyocellular Lipids, and Inflammation

Context: Visfatin (VF) is a recently described adipokine preferentially secreted by visceral adipose tissue (VAT) with insulin mimetic properties. Objective: The aim of this study was to examine the association of VF with insulin sensitivity, intramyocellular lipids (IMCL), and inflammation in humans. Design and Patients: VF mRNA was examined in paired samples of VAT and abdominal sc adipose tissue (SAT) obtained from subjects undergoing surgery. Plasma VF and VF mRNA was also examined in SAT and muscle tissue, obtained by biopsy from well-characterized subjects with normal or impaired glucose tolerance, with a wide range in body mass index (BMI) and insulin sensitivity (SI). Setting: The study was conducted at a University Hospital and General Clinical Research Center. Intervention: SI was measured, and fat and muscle biopsies were performed. In impaired glucose tolerance subjects, these procedures were performed before and after treatment with pioglitazone or metformin. Main Outcome Measures: We measured the relationship between VF and obesity, SI, adipose tissue inflammation, IMCL, and response to insulin sensitizers. Results: No significant difference in VF mRNA was seen between SAT and VAT depots. VAT VF mRNA associated positively with BMI, whereas SAT VF mRNA decreased with BMI. SAT VF correlated positively with SI, and the association of SAT VF mRNA with SI was independent of BMI. IMCL and markers of inflammation (adipose CD68 and plasma TNFα) were negatively associated with SAT VF. Impaired glucose tolerance subjects treated with pioglitazone showed no change in SAT VF mRNA despite a significant increase in SI. Plasma VF and muscle VF mRNA did not correlate with BMI or SI or IMCL, and there was no change in muscle VF with either pioglitazone or metformin treatments. Conclusion: SAT VF is highly expressed in lean, more insulinsensitive subjects and is attenuated in subjects with high IMCL, low SI, and high levels of inflammatory markers. VAT VF and SAT VF are regulated oppositely with BMI

OXPAT/PAT-1 is a PPAR-Induced Lipid Droplet Protein that Promotes Fatty Acid Utilization

Lipid droplet proteins of the PAT (perilipin, adipophilin, and TIP47) family regulate cellular neutral lipid stores. We have studied a new member of this family, PAT-1, and found that it is expressed in highly oxidative tissues. We refer to this protein as OXPAT. Physiologic lipid loading of mouse liver by fasting enriches OXPAT in the lipid droplet tissue fraction. OXPAT resides on lipid droplets with the PAT protein adipophilin in primary cardiomyocytes. Ectopic expression of OXPAT promotes fatty acid-induced triacylglycerol accumulation, long-chain fatty acid oxidation, and mRNAs associated with oxidative metabolism. Consistent with these observations, OXPAT is induced in mouse adipose tissue, striated muscle, and liver by physiological (fasting), pathophysiological (insulin deficiency), pharmacological (peroxisome proliferator-activated receptor [PPAR] agonists), and genetic (muscle-specific PPARα overexpression) perturbations that increase fatty acid utilization. In humans with impaired glucose tolerance, PPARγ agonist treatment induces adipose OXPAT mRNA. Further, adipose OXPAT mRNA negatively correlates with BMI in nondiabetic humans. Our collective data in cells, mice, and humans suggest that OXPAT is a marker for PPAR activation and fatty acid oxidation. OXPAT likely contributes to adaptive responses to the fatty acid burden that accompanies fasting, insulin deficiency, and overnutrition, responses that are defective in obesity and type 2 diabetes

Retinol Binding Protein 4 Expression in Humans: Relationship to Insulin Resistance, Inflammation, and Response to Pioglitazone

Context: Retinol binding protein 4 (RBP4) was recently found to be expressed and secreted by adipose tissue, and was strongly associated with insulin resistance. Objective: The aim was to determine the relationship between RBP4 and obesity, insulin resistance, and other markers of insulin resistance in humans. Design and Patients: RBP4 mRNA levels in adipose tissue and muscle of nondiabetic human subjects with either normal or impaired glucose tolerance (IGT) were studied, along with plasma RBP4. RBP4 gene expression was also measured in adipose tissue fractions, and from visceral and sc adipose tissue (SAT) from surgical patients. Setting: The study was conducted at University Hospital and General Clinical Research Center. Intervention: Insulin sensitivity (SI) was measured, and fat and muscle biopsies were performed. In IGT subjects, these procedures were performed before and after treatment with metformin or pioglitazone. Main Outcome Measures: The relationship between RBP4 expression and obesity, SI, adipose tissue inflammation, and intramyocellular lipid level, and response to insulin sensitizers was measured. Results: RBP4 was expressed predominantly from the adipocyte fraction of SAT. Although SAT RBP4 expression and the plasma RBP4 level demonstrated no significant relationship with body mass index or SI, there was a strong positive correlation between RBP4 mRNA and adipose inflammation (monocyte chemoattractant protein-1 and CD68), and glucose transporter 4 mRNA. Treatment of IGT subjects with pioglitazone resulted in an increase in SI and an increase in RBP4 gene expression in both adipose tissue and muscle, but not in plasma RBP4 level, and the in vitro treatment of cultured adipocytes with pioglitazone yielded a similar increase in RBP4 mRNA. Conclusions: RBP4 gene expression in humans is associated with inflammatory markers, but not with insulin resistance. The increase in RBP4 mRNA after pioglitazone treatment is unusual, suggesting a complex regulation of this novel adipokine

Insulin Resistance in African-American and Caucasian Women: Differences in Lipotoxicity, Adipokines, and Gene Expression in Adipose Tissue and Muscle

Objectives: We tested whether African-American (AA) women are different from Caucasian women in regard to lipotoxicity, adipokines, and gene expression in adipose tissue and muscle