Neda Rasouli

Adipocytokines and the Metabolic Complications of Obesity

CONTEXT Adipose tissue is increasingly recognized as an active endocrine organ with many secretory products and part of the innate immune system. With obesity, macrophages infiltrate adipose tissue, and numerous adipocytokines are released by both macrophages and adipocytes. Adipocytokines play important roles in the pathogenesis of insulin resistance and associated metabolic complications such as dyslipidemia, hypertension, and premature heart disease. EVIDENCE ACQUISITION Published literature was analyzed with the intent of addressing the role of the major adipose secretory proteins in human obesity, insulin resistance, and type 2 diabetes. EVIDENCE SYNTHESIS This review analyzes the characteristics of different adipocytokines, including leptin, adiponectin, pro-inflammatory cytokines, resistin, retinol binding protein 4, visfatin, and others, and their roles in the pathogenesis of insulin resistance. CONCLUSIONS Inflamed fat in obesity secretes an array of proteins implicated in the impairment of insulin signaling. Further studies are needed to understand the triggers that initiate inflammation in adipose tissue and the role of each adipokine in the pathogenesis of insulin resistance.

Adipose tissue macrophages in insulin-resistant subjects are associated with collagen VI and fibrosis and demonstrate alternative activation

Adipose tissue macrophages are associated with insulin resistance and are linked to changes in the extracellular matrix. To better characterize adipose macrophages, the extracellular matrix, and adipocyte-macrophage interactions, gene expression from adipose tissue and the stromal vascular fraction was assessed for markers of inflammation and fibrosis, and macrophages from obese and lean subjects were counted and characterized immunohistochemically. Coculture experiments examined the effects of adipocyte-macrophage interaction. Collagen VI gene expression was associated with insulin sensitivity and CD68 (r = -0.56 and 0.60, P < 0.0001) and with other markers of inflammation and fibrosis. Compared with adipose tissue from lean subjects, adipose tissue from obese subjects contained increased areas of fibrosis, which correlated inversely with insulin sensitivity (r = -0.58, P < 0.02) and positively with macrophage number (r = 0.70, P < 0.01). Although macrophages in crownlike structures (CLS) were more abundant in obese adipose tissue, the majority of macrophages were associated with fibrosis and were not organized in CLS. Macrophages in CLS were predominantly M1, but most other macrophages, particularly those in fibrotic areas, were M2 and also expressed CD150, a marker of M2c macrophages. Coculture of THP-1 macrophages with adipocytes promoted the M2 phenotype, with a lower level of IL-1 expression and a higher ratio of IL-10 to IL-12. Transforming growth factor-β (TGF-β) was more abundant in M2 macrophages and was further increased by coculture with adipocytes. Downstream effectors of TGF-β, such as plasminogen activator inhibitor-1, collagen VI, and phosphorylated Smad, were increased in macrophages and adipocytes. Thus adipose tissue of insulin-resistant humans demonstrated increased fibrosis, M2 macrophage abundance, and TGF-β activity.

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.

Endoplasmic Reticulum Stress Markers Are Associated with Obesity in Nondiabetic Subjects

OBJECTIVE Adipocyte and hepatocyte endoplasmic reticulum (ER) stress response is activated in dietary and genetic models of obesity in mice. We hypothesized that ER stress was also activated and associated with reduced insulin sensitivity (SI) in human obesity. RESEARCH DESIGN AND METHODS We recruited 78 healthy, nondiabetic individuals over a spectrum of body mass index (BMI) who underwent oral and iv glucose tolerance tests, and fasting sc adipose and muscle biopsies. We tested expression of 18 genes and levels of total and phosphorylated eukaryotic initiation factor 2alpha, c-jun, and c-Jun N-terminal kinase 1 in adipose tissue. We compared gene expression in stromal vascular and adipocyte fractions in paired samples from 22 individuals, and tested clustering on gene and protein markers. RESULTS Adipocyte expression of most markers of ER stress, including chaperones downstream of activating transcription factor 6, were significantly correlated with BMI and percent fat (r>0.5; P<0.00001). Phosphorylation of eukaryotic initiation factor 2alpha but not of c-Jun N-terminal kinase 1 or c-jun was increased with obesity. ER stress response (as elsewhere) was also increased with obesity in a second set of 86 individuals, and in the combined sample (n=161). The increase was only partially attributable to the stromal vascular fraction and macrophage infiltration. ER stress markers were only modestly correlated with S(I). Clustering algorithms supported ER stress activation with high BMI but not low SI. CONCLUSIONS Multiple markers of ER stress are activated in human adipose with obesity, particularly for protective chaperones downstream of activating transcription factor 6alpha.

Muscle inflammatory response and insulin resistance: synergistic interaction between macrophages and fatty acids leads to impaired insulin action

Obesity is characterized by adipose tissue expansion as well as macrophage infiltration of adipose tissue. This results in an increase in circulating inflammatory cytokines and nonesterified fatty acids, factors that cause skeletal muscle insulin resistance. Whether obesity also results in skeletal muscle inflammation is not known. In this study, we quantified macrophages immunohistochemically in vastus lateralis biopsies from eight obese and eight lean subjects. Our study demonstrates that macrophages infiltrate skeletal muscle in obesity, and we developed an in vitro system to study this mechanistically. Myoblasts were isolated from vastus lateralis biopsies and differentiated in culture. Coculture of differentiated human myotubes with macrophages in the presence of palmitic acid, to mimic an obese environment, revealed that macrophages in the presence of palmitic acid synergistically augment cytokine and chemokine expression in myotubes, decrease IkappaB-alpha protein expression, increase phosphorylated JNK, decrease phosphorylated Akt, and increase markers of muscle atrophy. These results suggest that macrophages alter the inflammatory state of muscle cells in an obese milieu, inhibiting insulin signaling. Thus in obesity both adipose tissue and skeletal muscle inflammation may contribute to insulin resistance.

Adipose tissue extracellular matrix and vascular abnormalities in obesity and insulin resistance.

CONTEXT Insulin resistance is associated with inflammation, fibrosis, and hypoxia in adipose tissue. OBJECTIVE This study was intended to better characterize the extracellular matrix (ECM) and vascularity of insulin-resistant adipose tissue. DESIGN Adipose expression of collagens, elastin, and angiogenic factors was assessed using real-time RT-PCR and immunohistochemistry (IHC) in abdominal sc adipose tissue. Adipocyte-macrophage coculture experiments examined the effects of polarized macrophages on adipose ECM gene expression, and the effects of collagens were measured in an angiogenesis assay. PARTICIPANTS AND SETTING A total of 74 nondiabetic subjects participated at a University Clinical Research Center. INTERVENTIONS Interventions included baseline adipose biopsy and measurement of insulin sensitivity. MAIN OUTCOME MEASURES Outcome measures included characterization of vascularity and ECM in adipose tissue. RESULTS CD31 (an endothelial marker) mRNA showed no significant correlation with body mass index or insulin sensitivity. In a subgroup of 17 subjects (nine obese, eight lean), CD31-positive capillary number in obese was decreased by 58%, whereas larger vessels were increased by 70%, accounting for the lack of change in CD31 expression with obesity. Using IHC, obese (compared with lean) subjects had decreased elastin and increased collagen V expression, and adipocytes cocultured with M2 macrophages had reduced elastin and increased collagen V expression. In obese subjects, collagen V was colocalized with large blood vessels, and the addition of collagen V to an angiogenesis assay inhibited endothelial budding. CONCLUSIONS The adipose tissue from obese/insulin-resistant subjects has fewer capillaries and more large vessels as compared with lean subjects. The ECM of adipose tissue may play an important role in regulating the expandability as well as angiogenesis of adipose tissue.

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 < 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 < 0.05) and FAS expression increased by 63 ± 8% (P < 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.

Thrombospondin-1 Is an Adipokine Associated With Obesity, Adipose Inflammation, and 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.

Ectopic fat accumulation and metabolic syndrome

The recent escalation of obesity from an individual health problem to a major public health issue reaching epidemic proportions has drawn attention to a constellation of abnormalities (abdominal obesity, hypertension and dyslipidaemia) collectively referred to as metabolic syndrome. As an indicator of insulin resistance and a harbinger of diabetes, this syndrome has been associated with major cardiovascular mortality and morbidity. Yet, the exact pathophysiological events leading to the development of metabolic syndrome remain unknown. We review some of the current literature on the pathogenesis of metabolic syndrome with an emphasis on the role of ectopic lipid accumulation.

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 < 0.01), along with an increase in lipin-β (but not lipin-α) expression by 200% (P < 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.