A Role of the Inflammasome in the Low Storage Capacity of the Abdominal Subcutaneous Adipose Tissue in Obese Adolescents

The innate immune cell sensor leucine-rich–containing family, pyrin domain containing 3 (NLRP3) inflammasome controls the activation of caspase-1, and the release of proinflammatory cytokines interleukin (IL)-1β and IL-18. The NLRP3 inflammasome is implicated in adipose tissue inflammation and the pathogenesis of insulin resistance. Herein, we tested the hypothesis that adipose tissue inflammation and NLRP3 inflammasome are linked to the downregulation of subcutaneous adipose tissue (SAT) adipogenesis/lipogenesis in obese adolescents with altered abdominal fat partitioning. We performed abdominal SAT biopsies on 58 obese adolescents and grouped them by MRI-derived visceral fat to visceral adipose tissue (VAT) plus SAT (VAT/VAT+SAT) ratio (cutoff 0.11). Adolescents with a high VAT/VAT+SAT ratio showed higher SAT macrophage infiltration and higher expression of the NLRP3 inflammasome–related genes (i.e., TLR4, NLRP3, IL1B, and CASP1). The increase in inflammation markers was paralleled by a decrease in genes related to insulin sensitivity (ADIPOQ, GLUT4, PPARG2, and SIRT1) and lipogenesis (SREBP1c, ACC, LPL, and FASN). Furthermore, SAT ceramide concentrations correlated with the expression of CASP1 and IL1B. Infiltration of macrophages and upregulation of the NLRP3 inflammasome together with the associated high ceramide content in the plasma and SAT of obese adolescents with a high VAT/VAT+SAT may contribute to the limited expansion of the subcutaneous abdominal adipose depot and the development of insulin resistance.     

Complement Factor H Is Expressed in Adipose Tissue in Association With Insulin Resistance

OBJECTIVE

Activation of the alternative pathway of the complement system, in which factor H (fH; complement fH [CFH]) is a key regulatory component, has been suggested as a link between obesity and metabolic disorders. Our objective was to study the associations between circulating and adipose tissue gene expressions of CFH and complement factor B (fB; CFB) with obesity and insulin resistance.

RESEARCH DESIGN AND METHODS

Circulating fH and fB were determined by enzyme-linked immunosorbent assay in 398 subjects. CFH and CFB gene expressions were evaluated in 76 adipose tissue samples, in isolated adipocytes, and in stromovascular cells (SVC) (n = 13). The effects of weight loss and rosiglitazone were investigated in independent cohorts.

RESULTS

Both circulating fH and fB were associated positively with BMI, waist circumference, triglycerides, and inflammatory parameters and negatively with insulin sensitivity and HDL cholesterol. For the first time, CFH gene expression was detected in human adipose tissue (significantly increased in subcutaneous compared with omental fat). CFH gene expression in omental fat was significantly associated with insulin resistance. In contrast, CFB gene expression was significantly increased in omental fat but also in association with fasting glucose and triglycerides. The SVC fraction was responsible for these differences, although isolated adipocytes also expressed fB and fH at low levels. Both weight loss and rosiglitazone led to significantly decreased circulating fB and fH levels.

CONCLUSIONS

Increased circulating fH and fB concentrations in subjects with altered glucose tolerance could reflect increased SVC-induced activation of the alternative pathway of complement in omental adipose tissue linked to insulin resistance and metabolic disturbances.Obesity is closely associated with a cluster of metabolic diseases, such as dyslipidemia, hypertension, insulin resistance, type 2 diabetes, and atherosclerosis (1). Adipose tissue is well known for its essential role as an energy storage depot and for secreting adipokines that influence sites as diverse as brain, liver, muscle, β-cells, gonads, lymphoid organs, and systemic vasculature (2,3). Expression analysis of macrophage and nonmacrophage cell populations isolated from adipose tissue demonstrates that adipose tissue macrophages are responsible for most of the proinflammatory cytokines (4). In recent years, it has become evident that alterations in the function of the innate immune system are intrinsically linked to metabolic pathways in humans (5–8).The complement system is a major component of the innate immune system, defending the host against pathogens, coordinating various events during inflammation, and bridging innate and adaptive immune responses. Complement deficiency and abnormalities in the regulation of the complement system lead to increased susceptibility to infection and chronic inflammatory diseases (9,10,11).Factor H (fH) is a relatively abundant plasma glycoprotein that is essential to maintain complement homeostasis and to restrict the action of complement to activating surfaces. fH acts as a cofactor for factor I–mediated cleavage of C3b (the active fragment of the third component of complement C3), accelerates the dissociation of the alternative pathway C3 convertases (a bimolecular enzymatic complex formed by active fragments of C3 and factor B [fB]), and competes with fB for binding to C3b. fH regulates complement both in fluid phase and on cellular surfaces (12–16).It has been suggested that activation of the alternative pathway of the complement system could be a link between obesity and metabolic disorders (17–21). Moreover, fB and factor D (fD, adipsin) are produced by adipose tissue where they likely influence formation of the alternative pathway component C3 convertase and the production of the anaphylatoxin C3a and its carboxypeptidase B-anaphylatoxic–inactivated derivative C3adesArg (acylation-stimulating protein [ASP]). Both ASP/C3adesArg and C3a interact with the receptor C5L2 to effectively stimulate triglyceride synthesis in cultured adipocytes (22). C3 knockout (C3KO) mice are obligatorily ASP deficient and present lipid abnormalities (23). In humans, ASP levels are increased in obesity, type 2 diabetes, and in individuals at risk of arterial disease, including those with hypertension, type 2 diabetes, dyslipidemia, and coronary artery disease, whereas exercise or weight loss decreases ASP levels (24,25). These data suggest a relationship between these conditions and activation of the alternative pathway of complement. There is also a correlation between increased C3 concentration and decreased insulin action (26,27). Levels of C3 and fB were higher in subjects with insulin resistance and other features of the metabolic syndrome (28,29).Given these interactions among activation of the alternative pathway of complement, metabolic disturbances, and a chronic low-level inflammatory state, we designed experiments to study the associations among circulating fH, fB, insulin resistance, lipid parameters, and inflammatory markers. We found that circulating fH and fB are strongly associated with obesity. For that reason, we also studied whether adipose tissue could constitute a source of circulating fH and fB.     

Anti-CD44 Antibody Treatment Lowers Hyperglycemia and Improves Insulin Resistance,Adipose Inflammation,and Hepatic Steatosis in Diet-Induced Obese Mice

Type 2 diabetes (T2D) is a metabolic disease affecting >370 million people worldwide. It is characterized by obesity-induced insulin resistance, and growing evidence has indicated that this causative link between obesity and insulin resistance is associated with visceral adipose tissue inflammation. However, using anti-inflammatory drugs to treat insulin resistance and T2D is not a common practice. We recently applied a bioinformatics methodology to open public data and found that CD44 plays a critical role in the development of adipose tissue inflammation and insulin resistance. In this report, we examined the role of CD44 in T2D by administering daily injections of anti-CD44 monoclonal antibody (mAb) in a high-fat–diet mouse model. Four weeks of therapy with CD44 mAb suppressed visceral adipose tissue inflammation compared with controls and reduced fasting blood glucose levels, weight gain, liver steatosis, and insulin resistance to levels comparable to or better than therapy with the drugs metformin and pioglitazone. These findings suggest that CD44 mAb may be useful as a prototype drug for therapy of T2D by breaking the links between obesity and insulin resistance.     

SIRT1 Transcription Is Decreased in Visceral Adipose Tissue of Morbidly Obese Patients with Severe Hepatic Steatosis

Background  

Visceral adipose tissue is known to release greater amounts of adipokines and free fatty acids into the portal vein, being one of the most predictive factors of nonalcoholic fatty liver disease (NAFLD). Our study has the purpose to evaluate sirtuin 1 (SIRT1), adiponectin, Forkhead/winged helix (FOXO1), peroxisome proliferator-activated receptor (PPAR)γ1–3, and PPARβ/δ mRNA expression in morbidly obese patients in three different lipid depots: visceral (VAT), subcutaneous (SAT), and retroperitoneal (RAT). Recent studies suggest that SIRT1, a NAD⁺-dependent deacetylase, protects rats from NAFLD.     

Depletion of Liver Kupffer Cells Prevents the Development of Diet-Induced Hepatic Steatosis and Insulin Resistance

OBJECTIVE

Increased activity of the innate immune system has been implicated in the pathogenesis of the dyslipidemia and insulin resistance associated with obesity and type 2 diabetes. In this study, we addressed the potential role of Kupffer cells (liver-specific macrophages, KCs) in these metabolic abnormalities.

RESEARCH DESIGN AND METHODS

Rats were depleted of KCs by administration of gadolinium chloride, after which all animals were exposed to a 2-week high-fat or high-sucrose diet. Subsequently, the effects of these interventions on the development of hepatic insulin resistance and steatosis were assessed. In further studies, the effects of M1-polarized KCs on hepatocyte lipid metabolism and insulin sensitivity were addressed.

RESULTS

As expected, a high-fat or high-sucrose diet induced steatosis and hepatic insulin resistance. However, these metabolic abnormalities were prevented when liver was depleted of KCs. In vitro, KCs recapitulated the in vivo effects of diet by increasing hepatocyte triglyceride accumulation and fatty acid esterification, and decreasing fatty acid oxidation and insulin responsiveness. To address the mechanisms(s) of KC action, we inhibited a panel of cytokines using neutralizing antibodies. Only neutralizing antibodies against tumor necrosis factor-α (TNFα) attenuated KC-induced alterations in hepatocyte fatty acid oxidation, triglyceride accumulation, and insulin responsiveness. Importantly, KC TNFα levels were increased by diet in vivo and in isolated M1-polarized KCs in vitro.

CONCLUSIONS

These data demonstrate a role for liver macrophages in diet-induced alterations in hepatic lipid metabolism and insulin sensitivity, and suggest a role for these cells in the etiology of the metabolic abnormalities of obesity/type 2 diabetes.The physiological purpose of inflammation, which is an adaptive response to infection, injury, or exposure to toxic substances, is to reestablish a homeostatic state that entails removal of the source of infection, tissue repair, or resolution of toxin-induced stress. Upon the reestablishment of homeostasis, the necessity for the inflammatory response is removed, allowing immune system function to return to the basal state. However, under pathological conditions, a state of chronic inflammation is established, and the consequences of this inappropriate condition are the development of diseases of autoimmunity, sepsis, fibrosis, and cellular stress. Most recently, it has become apparent that the major metabolic diseases of this generation, namely obesity, type 2 diabetes, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and atherosclerosis, are states of chronic inflammation, and a series of studies have demonstrated a role for inflammation in the pathophysiology of the metabolic abnormalities associated with a number of these conditions (1–5).Macrophages are a heterogeneous population of myeloid-derived mononuclear cells that are a critical component of the innate immune response (6,7). They are resident in practically all tissues of the body, are recruited to tissues in response to infection or tissue damage, and are particularly enriched in tissues that are frequently exposed to exogenous and endogenous antigens and toxins, such as the lungs and liver. In all tissues, they act as the first responders to pathogens, toxins, and tissue damage by producing a panel of M1 (Th-1) proinflammatory cytokines, the prototypical ones being tumor necrosis factor-α (TNFα), γ-interferon (IFN-γ), and interleukin (IL)-1β. In states of overnutrition such as obesity, the number and activity of macrophages in adipose tissue are increased in rodents (8,9) and humans (10,11). Furthermore, interventions that inhibit macrophage recruitment to adipose tissue (12,13) or decrease proinflammatory activity (14–16) of macrophages improve the insulin resistance associated with obesity, whereas interventions that induce macrophage recruitment exacerbate insulin resistance (12,17). However, although these studies demonstrate a pathophysiological role for adipose macrophages in the metabolic abnormalities of overnutrition, the role of macrophages in other tissues and the mechanisms of their effects are largely unknown. In this regard, the potential role of liver macrophages (Kupffer cells [KCs]) in the development of hepatic dyslipidemia (steatosis) and insulin resistance is largely unknown. Furthermore, recent studies (15,16) demonstrate that blocking the anti-inflammatory or alternative (M2 or Th-2) activation program of KCs exacerbates obesity-induced insulin resistance and decreases hepatocyte fatty acid oxidation. However, although suggestive, these studies do not directly address the contribution of KCs to the development of diet-induced steatosis and insulin resistance, or the mechanisms of these effects. The current study addressed these issues. The data demonstrate that the depletion of KCs protects against the development of diet-induced steatosis and insulin resistance, and that M1 activation of KCs induces changes in hepatocyte lipid metabolic pathways and insulin action that are consistent with the effects of diet in vivo. Finally, data are presented suggesting that KC–derived TNFα plays a role in mediating the detrimental effects of KCs on hepatocyte lipid metabolism and insulin action.     

Insulin Resistance Is Associated With Diminished Endoplasmic Reticulum Stress Responses in Adipose Tissue of Healthy and Diabetic Subjects

We recently showed that insulin increased ER stress in human adipose tissue. The effect of insulin resistance on ER stress is not known. It could be decreased, unchanged, or increased, depending on whether insulin regulates ER stress via the metabolic/phosphoinositide 3-kinase (PI3K) or alternate signaling pathways. To address this question, we examined effects of lipid-induced insulin resistance on insulin stimulation of ER stress. mRNAs of several ER stress markers were determined in fat biopsies obtained before and after 8-h hyperglycemic-hyperinsulinemic clamping in 13 normal subjects and in 6 chronically insulin-resistant patients with type 2 diabetes mellitus (T2DM). In normal subjects, hyperglycemia-hyperinsulinemia increased after/before mRNA ratios of several ER stress markers (determined by ER stress pathway array and by individual RT-PCR). Lipid infusion was associated with inhibition of the PI3K insulin-signaling pathway and with a decrease of hyperinsulinemia-induced ER stress responses. In chronically insulin-resistant patients with T2DM, hyperglycemic-hyperinsulinemia did not increase ER stress response marker mRNAs. In summary, insulin resistance, either produced by lipid infusions in normal subjects or chronically present in T2DM patients, was associated with decreased hyperinsulinemia-induced ER stress responses. This suggests, but does not prove, that these two phenomena were causally related.     

Skeletal Muscle Insulin Resistance Promotes Increased Hepatic De Novo Lipogenesis,Hyperlipidemia, and Hepatic Steatosis in the Elderly

Aging is closely associated with muscle insulin resistance, hyperlipidemia, nonalcoholic fatty liver disease (NAFLD), and type 2 diabetes. We examined the hypothesis that muscle insulin resistance in healthy aging promotes increased hepatic de novo lipogenesis (DNL) and hyperlipidemia by altering the distribution pattern of postprandial energy storage. Healthy, normal weight, sedentary elderly subjects pair-matched to young subjects were given two high-carbohydrate meals followed by ¹³C/¹H magnetic resonance spectroscopy measurements of postprandial changes in muscle and liver glycogen and lipid content, and assessment of DNL using ²H₂O. Net muscle glycogen synthesis was reduced by 45% (P < 0.007) in the elderly subjects compared with the young, reflecting severe muscle insulin resistance. Net liver glycogen synthesis was similar between groups (elderly, 143 ± 23 mmol/L vs. young, 138 ± 13 mmol/L; P = NS). Hepatic DNL was more than twofold higher in the elderly than in the young subjects (elderly, 14.5 ± 1.4% vs. young, 6.9 ± 0.7%; P = 0.00015) and was associated with approximately threefold higher postprandial hepatic triglyceride (TG) content (P < 0.005) and increased fasting plasma TGs (elderly, 1.19 ± 0.18 mmol/L vs. young, 0.74 ± 0.11 mmol/L; P = 0.02). These results strongly support the hypothesis that muscle insulin resistance in aging promotes hyperlipidemia and NAFLD by altering the pattern of postprandial carbohydrate storage away from muscle glycogen and into hepatic DNL.The prevalence of hyperlipidemia and nonalcoholic fatty liver disease (NAFLD), as part of the metabolic syndrome, increases significantly with age (1,2). However, age is also generally associated with increased weight and a sedentary lifestyle, so it is unclear whether the higher prevalence of hyperlipidemia and NAFLD in older people is a function of age per se, excess weight, and/or inactivity. In young, normal weight, healthy individuals, muscle insulin resistance has been proposed to be an important predisposing factor for atherogenic dyslipidemia and NAFLD by changing the pattern of energy storage from ingested carbohydrate away from skeletal muscle glycogen synthesis into hepatic de novo lipogenesis (DNL), resulting in an increase in plasma triglyceride (TG) concentrations and increased hepatic TG synthesis (3). This hypothesis was further supported by a recent study demonstrating a marked improvement in postprandial muscle glycogen synthesis and a decrease in hepatic DNL after reversal of muscle insulin resistance with a single bout of exercise in young, insulin-resistant individuals (4).We have previously shown that even healthy, normal weight, older individuals (65–80 years) have severe muscle insulin resistance, which is associated with increased intramyocellular lipid (IMCL) content and reduced basal rates of mitochondrial activity in muscle and brain (5). We therefore examined the hypothesis that aging-related hyperlipidemia and hepatic steatosis result from skeletal muscle insulin resistance, causing a redistribution of ingested carbohydrate away from muscle glycogen synthesis to the liver, resulting in increased hepatic DNL. In order to examine this hypothesis, we measured muscle and liver glycogen synthesis by ¹³C magnetic resonance spectroscopy (MRS) (3) and muscle and liver lipid synthesis by ¹H MRS (3) along with hepatic DNL measuring deuterium-labeled water (²H₂O) incorporation into plasma VLDL (6,7) in elderly and young volunteers following ingestion of two high-carbohydrate meals. Healthy, normal weight, sedentary elderly subjects were pair-matched by sex, body weight, height, BMI, lean body mass, fat mass, and physical activity with healthy, young subjects in order to determine the effect of age-related primary muscle insulin resistance on postprandial energy distribution, independent of these potentially confounding factors.     

Improved Insulin Sensitivity 3 Months After RYGB Surgery Is Associated With Increased Subcutaneous Adipose Tissue AMPK Activity and Decreased Oxidative Stress

Morbidly obese individuals are predisposed to a wide range of disorders, including type 2 diabetes, atherosclerotic cardiovascular disease, fatty liver disease, and certain cancers. Remarkably, all of these disorders can be improved or prevented by Roux-en-Y gastric bypass (RYGB) surgery. We have reported that decreased AMPK activity, together with increased oxidative stress and inflammation in adipose tissue, is associated with insulin resistance in morbidly obese bariatric surgery patients. In the current study, we assessed how these parameters are affected by RYGB surgery. Eleven patients (average age of 46 ± 4 years) were studied immediately prior to surgery and 3 months postoperatively. We measured subcutaneous adipose tissue AMPK phosphorylation (threonine 172, an index of its activation), malonyl-CoA content, protein carbonylation (a marker of oxidative stress), plasma adiponectin, and mRNA expression of several inflammatory cytokines. After surgery, AMPK activity increased 3.5-fold and oxidative stress decreased by 50% in subcutaneous adipose tissue. In addition, malonyl-CoA levels were reduced by 80%. Furthermore, patients had improvements in their BMI and insulin sensitivity (HOMA) and had increased circulating high–molecular weight adiponectin and decreased fasting plasma insulin levels. In contrast, the expression of inflammatory markers in subcutaneous adipose tissue was unchanged postoperatively, although plasma CRP was diminished by 50%.     

Macrophage Content in Subcutaneous Adipose Tissue: Associations With Adiposity, Age, Inflammatory Markers, and Whole-Body Insulin Action in Healthy Pima Indians

OBJECTIVE— In severely obese individuals and patients with diabetes, accumulation and activation of macrophages in adipose tissue has been implicated in the development of obesity-associated complications, including insulin resistance. We sought to determine whether in a healthy population, adiposity, sex, age, or insulin action is associated with adipose tissue macrophage content (ATMc) and/or markers of macrophage activation.RESEARCH DESIGN AND METHODS— Subcutaneous ATMc from young adult Pima Indians with a wide range of adiposity (13–46% body fat, by whole-body dual-energy X-ray absorptiometry) and insulin action (glucose disposal rate 1.6–9 mg/kg estimated metabolic body size/min, by glucose clamp) were measured. We also measured expression in adipose tissue of factors implicated in macrophage recruitment and activation to determine any association with ATMc and insulin action.RESULTS— ATMc, as assessed by immunohistochemistry (Mphi) and by macrophage-specific gene expression (CD68, CD11b, and CSF1R), were correlated with percent body fat, age, and female sex. Gene expression of CD68, CD11b, and CSF1R but not Mphi was correlated negatively with glucose disposal rate but not after adjustment for percent body fat, age, and sex. However, adipose tissue expression of plasminogen activator inhibitor type-1 (PAI-1) and CD11 antigen-like family member C (CD11c), markers produced by macrophages, were negatively correlated with adjusted glucose disposal rate (r = −0.28, P = 0.05 and r = −0.31, P = 0.03).CONCLUSIONS— ATMc is correlated with age and adiposity but not with insulin action independent of adiposity in healthy human subjects. However, PAI-1 and CD11c expression are independent predictors of insulin action, indicating a possible role for adipose tissue macrophage activation.Obesity is an inflammatory condition leading to chronic activation of an innate immune response (1). This inflammatory response has been implicated in the pathogenesis of obesity-associated complications, including atherosclerosis (2), nonalcoholic fatty liver disease (3), and insulin resistance (4). Adipose tissue is a primary site of obesity-induced inflammation and a complex organ containing adipocytes as well as connective tissue matrix, nerve tissue, stromal vascular cells, and immune cells. A cardinal feature of obesity-induced inflammation in adipose tissue is the recruitment of immune cells, specifically macrophages (5,6). Although the adipocyte is the defining cell of adipose tissue and does contribute to the production of inflammatory molecules (7), it appears that macrophages contribute substantially to the inflammatory signals that are induced by obesity (5,8–11).Among the inflammatory factors whose expression is upregulated in adipose tissue with the onset of obesity, some have been implicated in recruitment of macrophages to adipose tissue, including chemokines, while others appear to be derived primarily from adipose tissue macrophages (ATMs). Studies in rodents indicate that ATMs are bone marrow–derived cells recruited to adipose tissue during periods of positive energy balance and increasing adiposity (5). However, the physiology of macrophage recruitment remains largely unknown. It has been hypothesized that a metabolic signal(s) or stress(es) leads to activation of endothelial cells, production of chemoattractants with subsequent transendothelial migration of monocytes (12), monocyte differentiation into mature macrophages, and ultimately macrophage activation. A few studies have also suggested that differentiation of adipocyte precursors into macrophage-like cells (6,13) can occur, although this remains controversial. Studies have implicated monocyte chemoattractant proteins, hypoxia, and angiogenesis in ATM recruitment. In particular, the adhesion molecule intercellular adhesion molecule 1 (ICAM1) is important in the recruitment of monocytes to sites of inflammation (14), its soluble plasma concentrations have been found to be positively associated with adiposity (15,16), and in previous microarray studies in mice adipose tissue Icam1 expression was correlated with body mass (5).Animal and human studies of obese and diabetic subjects indicate that adipose tissue macrophage content (ATMc) correlates with degree of adiposity (5,6,8,9,12). In a small, interventional study, the subcutaneous expression of CD68, a macrophage marker, correlated with insulin resistance (10). In obese individuals, the degree of hepatic fibroinflammatory lesions or fat liver content is associated with omental or subcutaneous ATM infiltration (17–19). The association of ATMc with insulin resistance and nonalcoholic fatty liver disease indicates a role for ATMc in obesity-related complications. However, it is not clear yet whether ATMc or activation in healthy adults affects insulin action beyond their association with adiposity. In rodents, genetic manipulation of the activation of myeloid cells, including macrophages, alters insulin sensitivity (20–22).In the present study, we examined in healthy nondiabetic individuals the association of subcutaneous ATMc and activation with direct measurements of both adiposity and whole-body insulin sensitivity. In addition, we investigated the relationship of ATMc and subcutaneous adipose tissue expression of genes potentially involved in attraction of macrophages into adipose tissue.     

Gene Expression of Leptin, Resistin, and Adiponectin in the White Adipose Tissue of Obese Patients with Non-Alcoholic Fatty Liver Disease and Insulin Resistance

Background: Adipose tissue is an active endocrine organ that secretes a variety of metabolically important substances including adipokines. These factors affect insulin sensitivity and may represent a link between obesity, insulin resistance, type 2 diabetes (DM), and nonalcoholic fatty liver disease (NAFLD). This study uses real-time polymerase chain reaction (PCR) quantification of mRNAs encoding adiponectin, leptin, and resistin on snap-frozen samples of intra-abdominal adipose tissue of morbidly obese patients undergoing bariatric surgery. Methods: Morbidly obese patients undergoing bariatric surgery were studied. Patients were classified into two groups: Group A (with insulin resistance) (N=11; glucose 149.84 ± 40.56 mg/dL; serum insulin 8.28 ± 3.52 μU/mL), and Group B (without insulin resistance) (N=10; glucose 102.2 ± 8.43 mg/dL; serum insulin 3.431 ± 1.162 μU/mL). Results: Adiponectin mRNA in intra-abdominal adipose tissue and serum adiponectin levels were significantly lower in Group A compared to Group B patients (P<0.016 and P<0.03, respectively). Although serum resistin was higher in Group A than in Group B patients (P<0.005), resistin gene expression was not different between the two groups. Finally, for leptin, neither serum level nor gene expression was different between the two groups. Serum adiponectin level was the only predictor of nonalcoholic steatohepatitis (NASH) in this study (P=0.024). Conclusions: Obese patients with insulin resistance have decreased serum adiponectin and increased serum resistin. Additionally, adiponectin gene expression is also decreased in the adipose tissue of these patients. This low level of adiponectin expression may predispose patients to the progressive form of NAFLD or NASH.     

Plasma Ceramides Are Elevated in Obese Subjects With Type 2 Diabetes and Correlate With the Severity of Insulin Resistance

OBJECTIVE—To quantitate plasma ceramide subspecies concentrations in obese subjects with type 2 diabetes and relate these plasma levels to the severity of insulin resistance. Ceramides are a putative mediator of insulin resistance and lipotoxicity, and accumulation of ceramides within tissues in obese and diabetic subjects has been well described.RESEARCH DESIGN AND METHODS—We analyzed fasting plasma ceramide subspecies by quantitative tandem mass spectrometry in 13 obese type 2 diabetic patients and 14 lean healthy control subjects. Results were related to insulin sensitivity measured with the hyperinsulinemic-euglycemic clamp technique and with plasma tumor necrosis factor-α (TNF-α) levels, a marker of inflammation. Ceramide species (C18:1, 18:0, 20:0, 24:1, and 24:0) were quantified using electrospray ionization tandem mass spectrometry after separation with high-performance liquid chromatography.RESULTS—Insulin sensitivity (mg · kg⁻¹ · min⁻¹) was lower in type 2 diabetic patients (4.90 ± 0.3) versus control subjects (9.6 ± 0.4) (P < 0.0001). Type 2 diabetic subjects had higher (P < 0.05) concentrations of C18:0, C20:0, C24:1, and total ceramide. Insulin sensitivity was inversely correlated with C18:0, C20:0, C24:1, C24:0, and total ceramide (all P < 0.01). Plasma TNF-α concentration was increased (P < 0.05) in type 2 diabetic subjects and correlated with increased C18:1 and C18:0 ceramide subspecies.CONCLUSIONS—Plasma ceramide levels are elevated in type 2 diabetic subjects and may contribute to insulin resistance through activation of inflammatory mediators, such as TNF-α.Type 2 diabetes is an insulin-resistant state characterized by impaired glucose tolerance (1) and inflammation (2). Much evidence has demonstrated the role of increased circulating free fatty acids and tissue fat accumulation in the development of muscle and liver insulin resistance (1,3,4). The disturbances in plasma and tissue lipid metabolism result from an oversupply of lipid substrates, both exogenously and endogenously (increased lipolysis secondary to adipocyte insulin resistance), and perturbations in fat oxidation and utilization by muscle and liver, resulting in the accumulation of ectopic fat (4). Ectopic fat is “lipotoxic” and has been linked to the severity of insulin resistance and pancreatic β-cell dysfunction, i.e., the core defects in type 2 diabetes (1,4). Ectopic fat comprises various lipid species, including long-chain fatty acyl CoAs, diacylglycerol, and ceramide. It is well documented that ceramide accumulates within insulin-resistant tissues of animals (5–7) and humans (8–10) and inhibits insulin action and subsequent glucose uptake through inactivation of Akt. Ceramide also induces inflammation through activation of the nuclear factor-κB–tumor necrosis factor-α (TNF-α) axis (5–7).TNF-α is released from adipocytes and circulating mononuclear cells (MNCs) in response to stimuli, such as lipid infusion, lipopolysaccharide, reactive oxygen species, and hyperglycemia, and elevated TNF-α concentrations have been shown to induce insulin resistance (11–14). TNF-α also activates the plasma membrane enzyme sphingomyelinase (SMase) that hydrolyzes sphingomyelin to ceramide, allowing ceramides to accumulate within the cell (5,6,15–17). This accumulation of ceramide within tissues is thought to initiate a positive feedback mechanism, leading to enhanced production of proinflammatory cytokines (5), resulting in further inhibition of insulin-stimulated glucose uptake. Both plasma TNF-α concentrations and intracellular lipid intermediates, such as ceramides, are elevated in subjects with type 2 diabetes (8,18). Thus, ceramide is a bioactive lipid and putative mediator of insulin resistance that could link nutrient (fat) oversupply and cytokine-induced inflammation in tissues (5–7).Plasma ceramide levels also have been shown to correlate with coronary artery disease, independent of the plasma cholesterol concentration (19,20). However, the role of circulating ceramides has received little attention with respect to the development of insulin resistance and type 2 diabetes. Conflicting reports exist as to whether total circulating ceramides are elevated in obese (21) and type 2 diabetic subjects (22). Subspecies of plasma ceramides have been demonstrated to be increased in patients with sepsis and atherosclerosis (23–25), but the relationship between plasma ceramide subspecies levels and insulin resistance has not been investigated in patients with type 2 diabetes.Given their central role in the induction of insulin resistance and inflammation, elevated plasma ceramide levels may serve as a biomarker or direct perpetuator of insulin resistance and lipid-induced inflammation. Elevated plasma ceramide concentrations also may serve to identify individuals who are at risk to develop type 2 diabetes. The objective of this study was to quantify the concentration of individual ceramide subspecies in the circulation of patients with type 2 diabetes and healthy control subjects and to examine the correlation between plasma levels of ceramide subspecies and insulin sensitivity, measured with the euglycemic-hyperinsulinemic clamp, and plasma TNF-α concentration, a marker of inflammation.     

Early Hepatic Insulin Resistance Precedes the Onset of Diabetes in Obese C57BLKS-db/db Mice

OBJECTIVE

To identify metabolic derangements contributing to diabetes susceptibility in the leptin receptor–deficient obese C57BLKS/J-db/db (BKS-db) mouse strain.

RESEARCH DESIGN AND METHODS

Young BKS-db mice were used to identify metabolic pathways contributing to the development of diabetes. Using the diabetes-resistant B6-db strain as a comparison, in vivo and in vitro approaches were applied to identify metabolic and molecular differences between the two strains.

RESULTS

Despite higher plasma insulin levels, BKS-db mice exhibit lower lipogenic gene expression, rate of lipogenesis, hepatic triglyceride and glycogen content, and impaired insulin suppression of gluconeogenic genes. Hepatic insulin receptor substrate (IRS)-1 and IRS-2 expression and insulin-stimulated Akt-phosphorylation are decreased in BKS-db primary hepatocytes. Hyperinsulinemic-euglycemic clamp studies indicate that in contrast to hepatic insulin resistance, skeletal muscle is more insulin sensitive in BKS-db than in B6-db mice. We also demonstrate that elevated plasma triglyceride levels in BKS-db mice are associated with reduced triglyceride clearance due to lower lipase activities.

CONCLUSIONS

Our study demonstrates the presence of metabolic derangements in BKS-db before the onset of β-cell failure and identifies early hepatic insulin resistance as a component of the BKS-db phenotype. We propose that defects in hepatic insulin signaling contribute to the development of diabetes in the BKS-db mouse strain.Established in the 1940s, the C57BLKS (BKS) inbred mouse strain represents one of the first animal models of type 2 diabetes (1). Development of diabetes in these mice captures several aspects of the human disease (2,3). First, diabetes in this model is associated with obesity. Whereas lean BKS mice are normoglycemic throughout their life, obese leptin-deficient (BKS-ob) or leptin receptor–deficient (BKS-db) mice develop severe hyperglycemia. Second, the natural history of diabetes in BKS-ob or BKS-db is reminiscent of the human disease. These mice initially compensate for obesity-associated insulin resistance by increasing plasma insulin levels, but exhibit β-cell failure and insulin deficiency later in life. Finally, similarly to humans, diabetes in BKS-db is determined by multiple genetic factors (4,5). Despite extensive genetic analysis, the genes responsible for diabetes susceptibility in the BKS strain remain to be identified (6–8).Early studies on BKS-db mice indicated that the development of diabetes is associated with progressive β-cell degranulation and a precipitous decrease in β-cell mass and plasma insulin levels (2). In vivo radio-labeling studies revealed that after an initial phase of hyperproliferation at 4–6 weeks of age, the replication of β-cells gradually decreases despite increasing glucose levels (9). In contrast to BKS, introduction of the db mutation into the C57BL/6J (B6) genetic background produces a dramatically different β-cell phenotype (2,4). Although similarly obese as BKS-db, B6-db mice compensate for insulin resistance by β-cell hyperplasia, increased islet mass, and hyperinsulinemia and maintain only mildly elevated blood glucose levels throughout their life. The markedly different β-cell responses to obesity in BKS-db and B6-db mice suggest that genetically determined variation in β-cell viability/survival in the face of chronic glycemic stress is responsible for differences in diabetes susceptibility between the two strains. Consistent with this hypothesis, BKS β-cells are more sensitive than B6 to cell death triggered by β-cell toxins, such as alloxan and streptozotocin (10–12), and glucose-stimulated islet cell replication is diminished in BKS (13). In conclusion, previous studies suggest that variant β-cell functions underlie the differences in diabetes susceptibility between BKS-db and B6-db mice.In the current study, we refine the current β-cell–centric model of diabetes susceptibility in BKS-db by demonstrating metabolic defects preceding the onset of β-cell failure. In particular, BKS-db mice exhibit elevated hepatic insulin resistance associated with altered lipogenic and gluconeogenic pathways relative to B6-db. We propose that early hepatic insulin resistance contributes to the development of diabetes in the BKS-db strain.     

Protein Kinase A Regulatory Subunits in Human Adipose Tissue: Decreased R2B Expression and Activity in Adipocytes From Obese Subjects

OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects.RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively.RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects.CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity.Cyclic AMP is implicated in the regulation of a variety of cell functions that are, at least in part, related to protein phosphorylation through the activation of protein kinase A (PKA). In addition to the control of differentiated functions, such as motility, secretion, metabolism, differentiation, synaptic transmission, and ion channel activities, cAMP inhibits or stimulates cell proliferation depending on the cell type. In human adipocytes, the cAMP-dependent pathway mediates signals originating from the activation of β-adrenergic receptors, thus playing a key role in the regulation of important metabolic processes, such as lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by PKA, a tetrameric enzyme composed of two catalytic subunits associated with two regulatory subunits. There are four different regulatory subunit genes and proteins (R1A, R1B, R2A, and R2B) expressed with a tissue-specific pattern and exerting distinct roles in cell differentiation and growth control (1). Dramatic changes in the proportion of the two PKA regulatory subunits, R1 and R2, occur during ontogenic development, differentiation processes, and neoplastic transformation, indicating distinct roles for these isoenzymes in cell homeostasis and growth control (2,3). In the past few years, many studies seem to indicate that signaling via PKA plays an important role in regulating metabolism and body weight (4). In particular, the R2B isoform has been demonstrated to be, in mice, the most expressed in three tissues known to regulate energy homeostasis, i.e., brown adipose tissue, white adipose tissue, and brain (4,5). In general, the activation of the holoenzyme PKA in fat is now thought to decrease obesity, as demonstrated in both genetically obese (ob/ob) (6,7) and diet-induced obese mice (8). As far as the R2B subunit is concerned, studies in mice lacking this specific PKA subunit have revealed an unexpected role for this protein in regulating energy balance (5). R2B knockout mice (RIIß^−/−) remain remarkably lean, even when challenged with a high-fat diet (5). These animals have increased metabolic activity, manifested by increases in body temperature, uncoupling protein 1 concentration, and lipid hydrolysis. Biochemical studies have shown that loss of R2B is compensated by the increased R1A regulatory subunit, which is more sensitive to cAMP activation and results in a net increase in basal PKA activity (9).In contrast to this increasing knowledge in mice, little is known about the differential role played by the different PKA regulatory subunits in humans. Two studies from the same group have described lower R2B levels in adipose tissues from 10 normal-weight women affected with polycystic ovary syndrome (PCOS) when compared with 13 matched control women and associated this event with lipolytic catecholamine resistance and insulin resistance in these patients (10,11). We present the first study evaluating the relative expression of the different PKA isoforms in a large series of human adipose tissues. Our results indicate important BMI-related differences in R2B expression and PKA activation, which might play a role in the different lipolytic response to β-adrenergic activation in obese and nonobese subjects.