Hepatic stearoyl-CoA desaturase (SCD)-1 activity and diacylglycerol but not ceramide concentrations are increased in the nonalcoholic human fatty liver

OBJECTIVE—To determine whether 1) hepatic ceramide and diacylglycerol concentrations, 2) SCD1 activity, and 3) hepatic lipogenic index are increased in the human nonalcoholic fatty liver.RESEARCH DESIGN AND METHODS—We studied 16 subjects with (n = 8) and without (n = 8) histologically determined nonalcoholic fatty liver (NAFL⁺ and NAFL⁻) matched for age, sex, and BMI. Hepatic concentrations of lipids and fatty acids were quantitated using ultra-performance liquid chromatography coupled to mass spectrometry and gas chromatography.RESULTS—The absolute (nmol/mg) hepatic concentrations of diacylglycerols but not ceramides were increased in the NAFL⁺ group compared with the NAFL⁻ group. The livers of the NAFL⁺ group contained proportionally less long-chain polyunsaturated fatty acids as compared with the NAFL⁻ group. Liver fat percent was positively related to hepatic stearoyl-CoA desaturase 1 (SCD1) activity index (r = 0.70, P = 0.003) and the hepatic lipogenic index (r = 0.54, P = 0.030). Hepatic SCD1 activity index was positively related to the concentrations of diacylglycerols (r = 0.71, P = 0.002) but not ceramides (r = 0.07, NS).CONCLUSIONS—We conclude that diacylglycerols but not ceramides are increased in NAFL. The human fatty liver is also characterized by depletion of long polyunsaturated fatty acids in the liver and increases in hepatic SCD1 and lipogenic activities.Nonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver (≥10% of liver weight), which cannot be attributed to alcohol consumption or any other liver disease (1). NAFLD covers a range from simple nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) and fibrosis (1). The fatty liver is resistant to the action of insulin to inhibit hepatic glucose (2,3) and VLDL (4) production, resulting in hyperglycemia and hypertriglyceridemia. The mechanisms underlying insulin resistance in human NAFLD are unclear. While triacylglycerols themselves are inert, lipid intermediates may act as important regulators of both oxidative stress (5) and insulin signaling (6). In vitro studies as well as studies in animals suggest that diacylglycerols, which are immediate precursors of triacylglycerols (7), can induce insulin resistance by activating specific isoforms of protein kinase C (PKC) (8,9). The concentrations of diacylglycerols have recently been shown to be increased in human NAFLD compared with subjects with normal liver histology (10). Ceramides are another class of reactive lipids that mediate saturated fat–induced insulin resistance (6). There are no data comparing ceramide and diacylglycerol concentrations in the human liver or relating them to hepatic fat content.Sources of hepatic lipids include dietary chylomicron remnants, free fatty acids released from either adipose tissue triacylglycerols or chylomicrons hydrolyzed at a rate in excess of what can be taken up by tissues (spillover), and de novo lipogenesis (11). Increased lipolysis is a major contributor to hepatic fat accumulation (12–14). In addition, when estimated using tracer techniques, de novo lipogenesis has been found to be significantly increased in subjects with NAFLD compared with normal subjects (12,15,16). De novo lipogenesis produces saturated fatty acids (17,18). Stearoyl-CoA desaturase 1 (SCD1) converts saturated fatty acids to monounsaturated fatty acids, which are major substrates for synthesis of triacylglycerols and other lipids (19). SCD1 knockout mice are resistant to the development of obesity and hepatic steatosis (20,21), whereas the activity of SCD1 is significantly increased in the fatty livers of ob/ob mice (20,22). These data thus suggest that hepatic SCD1 activity may contribute to lipid accumulation in NAFLD. There are, however, no data on hepatic SCD1 activity in human NAFLD.To address the above questions, we quantified the full range of lipids and fatty acids using ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry (MS) and gas chromatography in the human liver. These analyses were performed in two groups of subjects matched for age, sex, and BMI but with either a normal liver fat content (≤10% macrovesicular steatosis) or a nonalcoholic fatty liver (NAFL) (≥20% macrovesicular steatosis [1]).     

Relationship of abdominal visceral and subcutaneous adipose tissue with lipoprotein particle number and size in type 2 diabetes

OBJECTIVE—Insulin resistance and type 2 diabetes are associated with an atherogenic lipoprotein profile. We examined the role of visceral and subcutaneous fat depots, independent of BMI, on the dyslipidemia associated with type 2 diabetes.RESEARCH DESIGN AND METHODS— A total of 382 subjects with type 2 diabetes underwent abdominal computed tomography to evaluate subcutaneous (SAT) and visceral adipose tissue (VAT) distribution and had anthropometric measurements to determine BMI and waist and hip circumference. Fasting blood was obtained for lipoprotein particle number and size using nuclear magnetic resonance spectroscopy. The relationship of lipoprotein particle number and size with BMI, SAT, and VAT was examined using multivariable regression models adjusted for age, sex, diabetes therapy, duration of diabetes, smoking, statin use, and A1C levels. The relation of VAT to lipoprotein particle number and size was further evaluated after the addition of BMI, BMI plus SAT, or BMI plus homeostatis is model assessment of insulin resistance (HOMA-IR) to the model.RESULTS—VAT was positively related to VLDL particle number (P < 0.0001), LDL particle number (P < 0.01), and VLDL size (P < 0.0001) and negatively related to LDL size (P < 0.0001) and HDL size (P < 0.0001). These relationships remained unchanged after addition of BMI and SAT to the model. After addition of HOMA-IR, VAT remained positively related to VLDL particle number (P < 0.0001) and size (P < 0.01) and negatively related to LDL and HDL particle size (P < 0.0001 for both comparisons). Neither BMI nor SAT was independently related to lipoprotein parameters.CONCLUSIONS—In patients with type 2 diabetes, higher VAT independent of BMI was associated with higher VLDL and LDL particle number, larger VLDL particles, and smaller LDL and HDL particles. This lipoprotein pattern has been associated with increased risk for atherosclerosis and cardiovascular disease.Dyslipidemia and increased adiposity, especially of abdominal type, are common metabolic features of type 2 diabetes. The dyslipidemia associated with type 2 diabetes is characterized by changes in lipoprotein particle number and size and has been attributed to insulin resistance (1,2). Studies using nuclear magnetic resonance (NMR) spectroscopy to analyze lipoprotein subclass profile along with euglycemic-hyperinsulinemic clamps (1) or frequently sampled intravenous glucose tolerance tests (2) to assess insulin sensitivity have clearly demonstrated that all three major human lipoproteins are affected by insulin resistance. The alterations in lipoprotein particle number and size in type 2 diabetes and insulin resistance have been linked to increased risk for cardiovascular disease (CVD) in both cross-sectional (3–9) and prospective studies (10,11).Obesity has been clearly demonstrated to be associated with insulin resistance and its metabolic consequences, including type 2 diabetes, dyslipidemia, and CVD (12–14). Recently, studies have suggested that fat tissue distribution may be more important than overall fat mass for these associations (15–17). Epidemiologic and physiologic studies have suggested that abdominal fat is more strongly associated with metabolic risk factors and CVD than total amount of body fat (15,16,18). Whether specific abdominal fat compartments—for example, visceral abdominal fat (VAT) compared with subcutaneous abdominal fat (SAT)—carry greater metabolic and cardiovascular risks remains more controversial (16,17), especially in subjects with type 2 diabetes (17). Even though many studies have pointed to a greater cardiovascular and metabolic risk associated with VAT (18–27), SAT has also been associated with insulin resistance and metabolic disorders in other studies (27–30). For this report, we examined the association between abdominal fat compartments measured by computed tomography (CT) and lipoprotein particle number and size using NMR spectroscopy in 382 subjects with type 2 diabetes who participated in the CHICAGO study (31). We further analyzed how the relationship of abdominal fat depots to lipoprotein parameters was impacted by BMI as a measure of overall adiposity or by hip circumference as an index of peripheral subcutaneous fat mass.     

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.     

Prevalence of melanocortin-4 receptor deficiency in Europeans and their age-dependent penetrance in multigenerational pedigrees

OBJECTIVE— Melanocortin-4 receptor (MC4R) deficiency is the most frequent genetic cause of obesity. However, there is uncertainty regarding the degree of penetrance of this condition, and the putative impact of the environment on the development of obesity in MC4R mutation carriers is unknown.RESEARCH DESIGN AND METHODS— We determined the MC4R sequence in 2,257 obese individuals and 2,677 nonobese control subjects of European origin and established the likely functional impact of all variants detected. We then included relatives of probands carriers and studied 25 pedigrees, including 97 carriers and 94 noncarriers from three generations.RESULTS— Of the MC4R nonsynonymous mutations found in obese subjects, 68% resulted in a loss of function in vitro. They were found in 1.72% of obese versus 0.15% of nonobesed subjects (P = 6.9 × 10⁻¹⁰). Among the families, abnormal eating behavior was more frequent in both MC4R-deficient children and adults than in noncarriers. Although BMI was inversely associated with educational status in noncarrier adults, no such relationship was seen in MC4R mutation carriers. We observed a generational effect, with a penetrance of 40% in MC4R-deficient adults aged >52 years, 60% in 18- to 52-year-old adults, and 79% in children. The longitudinal study of adult carriers showed an increasing age-dependent penetrance (37% at 20 years versus 60% at >40 years).CONCLUSIONS— We have established a robust estimate of age-related penetrance for MC4R deficiency and demonstrated a generational effect on penetrance, which may relate to the development of an “obesogenic” environment. It remains to be seen whether appropriate manipulation of environmental factors may contribute to preventing the development of obesity even in those strongly genetically predisposed to it.The leptin-melanocortin axis controls human energy homeostasis, and the melanocortin-4 receptor (MC4R) is a key player in its central regulation (1). Loss-of-function mutations in MC4R cause severe familial forms of obesity (2,3), and infrequent gain-of-function polymorphisms have been associated with protection against obesity (4,5). At least 72 nonsynonymous mutations have been discovered so far, but some have no obvious functional consequences (6,7), highlighting the importance of functional characterization of MC4R mutations in the determination of potential pathogenicity. MC4R is a membrane-bound G-protein–coupled receptor that activates adenylate cyclase. Loss-of-function mutations result in a partial or complete loss of function as measured by cAMP production. The majority of missense mutations in MC4R result in intracellular retention of the mutated protein, whereas some primarily affect ligand binding or ligand/receptor activation (8,9). In some cases, the alteration of the basal activity of the receptor (8,10) has been reported.The prevalence of loss-of-function MC4R mutations ranges from 0.5 to 5.8% in childhood-onset obesity (11–14). The contribution of MC4R mutations to late-onset obesity is still debated (13,15–18). Obesity due to MC4R mutations has been extensively studied, and although heterozygous loss-of-function mutations can clearly cause familial obesity, they can be found in individuals who are not obese (19). There is a need for reliable estimates of penetrance. Furthermore, no study has thoroughly assessed the effect of loss-of-function MC4R mutations in elderly subjects. Previous studies using part of our French cohort evidenced the first mutation in MC4R and demonstrated that most of them lead to an intracellular retention of the receptor (2,13,18).Although hyperphagia is a key feature of MC4R deficiency, with increased food intake at an ad libitum test meal reported in severely obese MC4R-deficient children (10), an apparent amelioration of obesity and food intake disturbances has been suggested in adulthood in some studies (6,11). Obesity is a complex trait, and MC4R mutations offer a unique opportunity to analyze the effects of both aging and shared environment on the evolution of body mass in this paradigm. In this extensive study of 2,257 unrelated obese subjects, 2,677 control subjects of European descent, and 25 multigenerational pedigrees with several MC4R mutations carriers, we provide a comprehensive picture of the prevalence of this condition and of factors that determine the expression of the obesity phenotype and support previous observations reported in a German familial study (20).     

m.3243A>G Mutation in Mitochondrial DNA Leads to Decreased Insulin Sensitivity in Skeletal Muscle and to Progressive ��-Cell Dysfunction

OBJECTIVE—To study insulin sensitivity and perfusion in skeletal muscle together with the β-cell function in subjects with the m.3243A>G mutation in mitochondrial DNA, the most common cause of mitochondrial diabetes.RESEARCH DESIGN AND METHODS—We measured skeletal muscle glucose uptake and perfusion using positron emission tomography and 2-[¹⁸F]fluoro-2-deoxyglucose and [¹⁵O]H₂O during euglycemic hyperinsulinemia in 15 patients with m.3243A>G. These patients included five subjects with no diabetes as defined by the oral glucose tolerance test (OGTT) (group 1), three with GHb <6.1% and newly found diabetes by OGTT (group 2), and seven with a previously diagnosed diabetes (group 3). Control subjects consisted of 13 healthy individuals who were similar to the carriers of m.3243A>G with respect to age and physical activity. β-Cell function was assessed using the OGTT and subsequent mathematical modeling.RESULTS—Skeletal muscle glucose uptake was significantly lower in groups 1, 2, and 3 than in the control subjects. The glucose sensitivity of β-cells in group 1 patients was similar to that of the control subjects, whereas in group 2 and 3 patients, the glucose sensitivity was significantly lower. The insulin secretion parameters correlated strongly with the proportion of m.3243A>G mutation in muscle.CONCLUSIONS—Our findings show that subjects with m.3243A>G are insulin resistant in skeletal muscle even when β-cell function is not markedly impaired or glucose control compromised. We suggest that both the skeletal muscle insulin sensitivity and the β-cell function are affected before the onset of the mitochondrial diabetes caused by the m.3243A>G mutation.Impaired insulin sensitivity characterizes adult-onset diabetes and has been attributed to decreased insulin-stimulated glucose uptake in major metabolic tissues such as skeletal muscle, liver, and adipose tissue (1,2). It predicts diabetes strongly in subjects with high hereditary risk (3). Decreased glucose uptake in skeletal muscle is the major determinant of impaired insulin sensitivity, because skeletal muscle is the tissue that accounts for the majority of insulin-stimulated glucose uptake in diabetes and in nondiabetic subjects (4). Impaired insulin sensitivity has been correlated with decreased mitochondrial function and with decreased expression of genes involved in mitochondrial oxidative phosphorylation in skeletal muscle (5,6). Interestingly, similar findings in oxidative phosphorylation have recently been made in healthy insulin-resistant subjects with high hereditary predisposition for diabetes (7).In addition to the impaired insulin sensitivity, a gradual β-cell failure is pivotal to the onset of diabetes in adulthood (8,9). It is noteworthy that most gene variants associated with adult-onset diabetes influence the β-cell insulin secretion (10). Genes that contribute to mitochondrial oxidative phosphorylation are located both in the nuclear DNA and in the maternally inherited mitochondrial DNA (mtDNA) (11). Intrinsic or acquired causes that could impair oxidative phosphorylation in the mitochondrion have been proposed to impair both skeletal muscle insulin sensitivity and β-cell function (12,13).The involvement of mtDNA mutations in the hereditary forms of diabetes is evident (14,15). The mtDNA m.3243A>G mutation accounts for 1–2% of adult-onset diabetes, and it has been estimated that most carriers of this mutation develop diabetes during their adulthood (16). This renders the m.3243A>G mutation an interesting pathogenic model for decreased mitochondrial function in adult-onset diabetes. The m.3243A>G mutation is heteroplasmic, i.e., the mutant allele and the wild-type allele co-occur in mitochondria, and the proportion of mutated mtDNA varies across patients and tissues (11). The hetero-plasmy is known to modify the phenotype in patients with m.3243A>G (17), but previous studies on glucose metabolism have not included mutation heteroplasmy as a variable. Furthermore, such studies have been small, so that more than four subjects with m.3243A>G have been examined in only a few of them (18–24). Most of these studies have revealed defects in insulin secretion (18–21,24). Hyperinsulinemic clamp technique has been applied in only two previous studies on m.3243A>G subjects, but these studies could not identify peripheral insulin resistance as the primary pathogenic factor (18,19).The aims of this study were 1) to characterize insulin secretion and sensitivity in patients with a substantial m.3243A>G mutation load and in age-matched healthy subjects, and 2) by using this disease model, to further enlighten the role of mitochondria in the pathogenesis of diabetes. We assessed whole-body glucose uptake by using the hyperinsulinemic clamp technique together with regional measurements of muscle perfusion and glucose uptake by positron emission tomography (PET). Model-based analysis of β-cell function was carried out, and measurements were correlated with mutation heteroplasmy.     

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.     

Spontaneous diabetes in hemizygous human amylin transgenic mice that developed neither islet amyloid nor peripheral insulin resistance

OBJECTIVES—We sought to 1) Determine whether soluble-misfolded amylin or insoluble-fibrillar amylin may cause or result from diabetes in human amylin transgenic mice and 2) determine the role, if any, that insulin resistance might play in these processes.RESEARCH DESIGN AND METHODS—We characterized the phenotypes of independent transgenic mouse lines that display pancreas-specific expression of human amylin or a nonaggregating homolog, [^25,28,29Pro]human amylin, in an FVB/n background.RESULTS—Diabetes occurred in hemizygous human amylin transgenic mice from 6 weeks after birth. Glucose tolerance was impaired during the mid- and end-diabetic phases, in which progressive β-cell loss paralleled decreasing pancreatic and plasma insulin and amylin. Peripheral insulin resistance was absent because glucose uptake rates were equivalent in isolated soleus muscles from transgenic and control animals. Even in advanced diabetes, islets lacked amyloid deposits. In islets from nontransgenic mice, glucagon and somatostatin cells were present mainly at the periphery and insulin cells were mainly in the core; in contrast, all three cell types were distributed throughout the islet in transgenic animals. [^25,28,29Pro]human amylin transgenic mice developed neither β-cell degeneration nor glucose intolerance.CONCLUSIONS—Overexpression of fibrillogenic human amylin in these human amylin transgenic mice caused β-cell degeneration and diabetes through mechanisms independent from both peripheral insulin resistance and islet amyloid. These findings are consistent with β-cell death evoked by misfolded but soluble cytotoxic species, such as those formed by human amylin in vitro.Increasing evidence indicates that decreased β-cell mass contributes to the impaired insulin secretion characteristic of type 2 diabetes (1–3). Amylin, also referred to as islet amyloid polypeptide, is a 37-amino acid polypeptide (4,5) secreted from pancreatic islet β-cells whose aggregation results in islet amyloid formation in type 2 diabetes (6). Islet amyloid has been reported in 40–90% of pancreases from type 2 diabetic subjects studied post mortem (7–11) and has been linked to both decreased β-cell mass and β-cell dysfunction (12,13). In vitro, human amylin causes apoptosis of islet β-cells, and there is growing evidence that this pathogenic process may contribute to the β-cell deficit in type 2 diabetes (1,2,14,15). However, it remains unresolved whether islet amyloid contributes to the etiopathogenesis of type 2 diabetes or, by contrast, occurs only as a consequence of the disease.Several independent lines of human amylin transgenic mice have been developed to investigate the role of amylin and islet amyloid in the pathogenesis of type 2 diabetes (16–19). The findings and conclusions from phenotypic characterization studies are wide ranging and sometimes at variance. Transgenic animals developed by several research groups did not develop spontaneous diabetes or insulin resistance or exhibit evidence of islet amyloid formation, suggesting that overexpression of human amylin alone was not sufficient to contribute to diabetes development and islet amyloid formation in those models (16–18). In contrast, Janson et al. (19) showed development of spontaneous diabetes in the absence of islet amyloid in homozygous individuals from a further transgenic mouse model, consistent with the view that overexpression of human amylin is sufficient for diabetes development but not islet amyloid formation in that model. It was previously thought that overexpression of human amylin might be sufficient for islet amyloid formation, but some studies have suggested that insulin resistance might also be necessary (20–22).Evidence concerning the role of human amylin in the processes that lead to or cause diabetes remains conflicting, and a clear role for human amylin–mediated β-cell death has not been established at this time, at least in part due to conflicting evidence from the different lines of human amylin transgenic mice. Previous reports have described the noticeable lack of correlation between amyloid deposition and hyperglycemia in other transgenic models of amylin-induced diabetes (21,23). Islets from homozygous individuals from the FVB/n-based line reported by Janson et al. (19) demonstrated a pattern of β-cell loss that closely reflects that in islets from human type 2 diabetic patients (1,3,9), but hemizygous animals from that line reportedly do not develop diabetes.Here, we report a transgenic human amylin mouse model (L13) in which hemizygous individuals developed early-onset diabetes without peripheral insulin resistance and islet amyloid formation. We demonstrate that the disappearance of functional β-cells during the progression of diabetes in this model contributes to the pathogenesis of diabetes. The absence of islet amyloid in the pancreas of transgenic mice before diabetes onset and during its progression, despite the high secretion rates of human amylin, shows that islet amyloid is not required for islet β-cell degeneration and loss of physiological insulin secretion. These findings are consistent with the reports of Janson et al. (19) and provide strong support for continuing exploration of the mechanism by which human amylin evokes β-cell death and contributes to the failure of insulin secretion in type 2 diabetes.     

Plasma YKL-40: a BMI-independent marker of type 2 diabetes

OBJECTIVE—YKL-40 is produced by macrophages, and plasma YKL-40 is elevated in patients with diseases characterized by inflammation. In the present study, YKL-40 was examined in relation to obesity, inflammation, and type 2 diabetes.RESEARCH DESIGN AND METHODS—Plasma YKL-40 and adipose tissue YKL-40 mRNA levels were investigated in 199 subjects who were divided into four groups depending on the presence or absence of type 2 diabetes and obesity. In addition, plasma YKL-40 was examined in healthy subjects during a hyperglycemic clamp, in which the plasma glucose level was kept at 15 mmol/l for 3 h, and during a hyperinsulinemic-euglycemic clamp.RESULTS—Patients with type 2 diabetes had higher plasma YKL-40 (76.7 vs. 45.1 ng/ml, P = 0.0001) but not higher expression in adipose tissue YKL-40 mRNA (1.20 vs. 0.98, P = 0.2) compared with subjects with a normal glucose tolerance. Within the groups with normal glucose tolerance and type 2 diabetes, obesity subgroups showed no difference with respect to either plasma YKL-40 or adipose tissue YKL-40 mRNA levels. Multivariate regression analysis showed that plasma YKL-40 was associated with fasting plasma glucose (β = 0.5, P = 0.0014) and plasma interleukin (IL)-6 (β = 0.2, P = 0.0303). Plasma YKL-40 was not related to parameters of obesity. There were no changes in plasma YKL-40 in healthy subjects during either hyperglycemic or hyperinsulinemic-euglycemic clamps.CONCLUSIONS—Plasma YKL-40 was identified as an obesity-independent marker of type 2 diabetes related to fasting plasma glucose and plasma IL-6 levels.YKL-40 (chitinase-3-like-1 [CHI3L1], human cartilage glycoprotein-39), is a heparin-, chitin-, and collagen-binding lectin produced by immunologically active cells such as macrophages (1) and neutrophils (2). YKL-40 is a member of the mammalian chitinase-like proteins and is a phylogenetically highly conserved serum protein (1,3–5). Other cells shown to produce YKL-40 are vascular smooth muscle and endothelia cells (6–8), arthritic chondrocytes (3), cancer cells (9), and embryonic and fetal cells (10). The exact functions of YKL-40 are unknown. Currently, YKL-40 is known to stimulate growth of fibroblast cells (11), activate the AKT and phosphoinositide-3 kinase signaling pathway, exert antiapoptosis (12), and function in angiogenesis (7) and may take part in the innate immune response (13). High plasma concentrations of YKL-40 are found in patients with diseases characterized by inflammation or increased tissue remodeling or with cancer (1,9).Adipose tissue is recognized as a source of inflammation (14–16). A high BMI is associated with increased levels of proinflammatory cytokines, and obesity is characterized as a state of chronic systemic low-grade inflammation (17). Studies demonstrate an accumulation of activated macrophages and other immune active cells in adipose tissue from obese subjects (17,18) as possible sources of inflammatory cytokines, determining a link between obesity, low-grade inflammation, and insulin resistance, and both obesity and low-grade inflammation have been linked with the development of insulin resistance and type 2 diabetes (19).One previous study (20) has shown an elevation of serum YKL-40 in type 2 diabetes. In the present study, using plasma and adipose tissue biopsy material from 103 healthy control subjects and 96 patients with type 2 diabetes with a wide range of BMI, we studied the possible relationship between plasma YKL-40 and adipose tissue expression of YKL-40 on the one hand and obesity, insulin resistance, and inflammation on the other.We further measured the macrophage marker CD68 in adipose tissue. We hypothesized that macrophages in the adipose tissue might secrete YKL-40 and that plasma YKL-40 would represent macrophage infiltration in adipose tissue and serve as a marker of insulin resistance. In order to obtain further information about the regulation of systemic YKL-40, we examined plasma YKL-40 during hyperglycemic and hyperinsulinemic-euglycemic conditions.     

Small interfering RNA-mediated suppression of proislet amyloid polypeptide expression inhibits islet amyloid formation and enhances survival of human islets in culture

OBJECTIVE—Islet amyloid, formed by aggregation of the β-cell peptide islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of pancreatic islets in type 2 diabetes. Toxic IAPP aggregates likely contribute to the progressive loss of β-cells in this disease. We used cultured human islets as an ex vivo model of amyloid formation to investigate whether suppression of proIAPP expression would inhibit islet amyloid formation and enhance β-cell survival and function.RESEARCH DESIGN AND METHODS—Islets from cadaveric organ donors were transduced with a recombinant adenovirus expressing a short interfering RNA (siRNA) designed to suppress human proIAPP (Ad-hProIAPP-siRNA), cultured for 10 days, and then assessed for the presence of islet amyloid, β-cell apoptosis, and β-cell function.RESULTS—Thioflavine S–positive amyloid deposits were clearly present after 10 days of culture. Transduction with Ad-hProIAPP-siRNA reduced proIAPP expression by 75% compared with nontransduced islets as assessed by Western blot analysis of islet lysates 4 days after transduction. siRNA-mediated inhibition of IAPP expression decreased islet amyloid area by 63% compared with nontransduced cultured islets. Cell death assessed by transferase-mediated dUTP nick-end labeling staining was decreased by 50% in transduced cultured human islets, associated with a significant increase in islet insulin content (control, 100 ± 4 vs. +Ad-siRNA, 153 ± 22%, P < 0.01) and glucose-stimulated insulin secretion (control, 222 ± 33 vs. +Ad-siRNA, 285 ± 21 percent basal, P < 0.05).CONCLUSIONS—These findings demonstrate that inhibition of IAPP synthesis prevents amyloid formation and β-cell death in cultured human islets. Inhibitors of IAPP synthesis may have therapeutic value in type 2 diabetes.Islet amyloid deposits are a pathological lesion of the pancreas in type 2 diabetes (1–3) formed by aggregation of islet amyloid polypeptide (IAPP; amylin) (4,5), a 37–amino acid hormone that is colocalized with insulin in β-cell granules and secreted along with insulin in response to β-cell secretagogues (6–8). Aggregates of IAPP, including small oligomeric species, are toxic to β-cells (9–14) and likely play an important role in the progressive loss of β-cells in this disease. Despite considerable study during the past decade, it is still not well understood why soluble IAPP molecules form toxic IAPP aggregates in type 2 diabetes. The presence of an amyloidogenic amino acid sequence in the human IAPP molecule (Gly-Ala-Iso-Leu-Ser [GAILS]) appears to be important but not sufficient for amyloid formation (2,3,15,16). Because production and secretion of IAPP closely mimic that of insulin, IAPP levels are elevated in conditions associated with insulin resistance and hyperinsulinemia, such as the early stages of type 2 diabetes (17). Elevated IAPP production and secretion because of increased demand for insulin along with defective trafficking and/or processing of proIAPP associated with β-cell dysfunction have been implicated as two possible factors contributing to aggregation of (pro)IAPP (i.e., proIAPP and its intermediate forms) in type 2 diabetes (2,3,16,18–23).Studies performed using transgenic rodent models have demonstrated a strong association between β-cell expression of human IAPP and development of hyperglycemia associated with loss of β-cells (24–29). β-Cell loss in these models was attributed to the formation of toxic IAPP aggregates as either small oligomeric species or amyloid fibrils. Studies on human and nonhuman primates have also demonstrated development of islet amyloid associated with β-cell loss in type 2 diabetes (30,31). It is not clear from these studies, however, whether IAPP aggregation and amyloid formation is the cause or a consequence of β-cell dysfunction and death. In the present study, we tested the hypothesis that suppression of human IAPP expression would inhibit amyloid formation and enhance survival and/or function of human islets. We and others have found that islet amyloid forms rapidly in islets from transgenic mice with β-cell expression of human IAPP, and its formation is potentiated by elevated glucose concentrations (23,32–35). We used cultured human islets as an ex vivo model of amyloid formation to investigate whether short interfering RNA (siRNA)-mediated suppression of endogenously expressed human proIAPP will enhance β-cell survival in human islets.     

Decreased fetal size is associated with beta-cell hyperfunction in early life and failure with age

OBJECTIVE—Low birth weight is associated with diabetes in adult life. Accelerated or “catch-up” postnatal growth in response to small birth size is thought to presage disease years later. Whether adult disease is caused by intrauterine β-cell–specific programming or by altered metabolism associated with catch-up growth is unknown.RESEARCH DESIGN AND METHODS—We generated a new model of intrauterine growth restriction due to fatty acid synthase (FAS) haploinsufficiency (FAS deletion [FASDEL]). Developmental programming of diabetes in these mice was assessed from in utero to 1 year of age.RESULTS—FASDEL mice did not manifest catch-up growth or insulin resistance. β-Cell mass and insulin secretion were strikingly increased in young FASDEL mice, but β-cell failure and diabetes occurred with age. FASDEL β-cells had altered proliferative and apoptotic responses to the common stress of a high-fat diet. This sequence appeared to be developmentally entrained because β-cell mass was increased in utero in FASDEL mice and in another model of intrauterine growth restriction caused by ectopic expression of uncoupling protein-1. Increasing intrauterine growth in FASDEL mice by supplementing caloric intake of pregnant dams normalized β-cell mass in utero.CONCLUSIONS—Decreased intrauterine body size, independent of postnatal growth and insulin resistance, appears to regulate β-cell mass, suggesting that developing body size might represent a physiological signal that is integrated through the pancreatic β-cell to establish a template for hyperfunction in early life and β-cell failure with age.Low birth weight predisposes to type 2 diabetes, cardiovascular disease, and premature death (1,2), prompting the hypothesis (3) that impairing growth in early life programs metabolic disease in adulthood. Much of this programming is attributed to postnatal catch-up growth, which is linked to insulin resistance and cardiovascular disease later in life (4). Modeling impaired growth in utero using calorie restriction (5), protein malnutrition (6), prenatal glucocorticoid administration (7), or ligation of the uterine arteries (8) produces catch-up growth and glucose intolerance. Catch-up growth is associated with changes in food intake, metabolism, and insulin resistance that confound the search for mechanisms linking low birth weight and adult disease. In particular, insulin resistance increases β-cell mass (9) and makes it difficult to determine whether adult disease is caused by in utero β-cell–specific programming instead of altered body composition and feeding behavior associated with accelerated postnatal growth.Insulin and its downstream signals are critical for growth and development in species ranging from worms and insects to mammals (10–15). In Drosophila, body size is sensed in the fat body (equivalent to the vertebrate liver) to antagonize insulin-induced growth by ecdysone (16). If an analogous process occurs in mammals, β-cells are likely involved because they are affected by mediators of body size, including insulin, amino acids, and other signals (17). These factors also regulate fatty acid synthase (FAS), which catalyzes the first committed step in fatty acid biosynthesis (18). FAS is regulated by nutrients independent of insulin, suggesting that it could be important for nutrient-dependent growth. Its global loss results in early embryonic lethality (19). Tissue-specific inactivation of FAS is possible (20), and, surprisingly, the loss of FAS in pancreatic β-cells has no effect on β-cell mass or the capacity to secrete insulin (21). Thus, FAS, unlike glucokinase (22) and insulin receptor substrate (IRS)-2 (23), is not required for normal β-cell function.Here, we report that FAS heterozygous mice are born small yet have expanded β-cell mass and increased insulin secretion without insulin resistance. This hyperplastic β-cell phenotype was reversed by promoting growth in utero, and increased β-cell mass was confirmed in another model of intrauterine growth restriction (IUGR). Despite the absence of catch-up postnatal growth, FAS-deficient mice develop diet-induced diabetes and β-cell failure with age.     

Association of Variants in RETN With Plasma Resistin Levels and Diabetes-Related Traits in the Framingham Offspring Study

OBJECTIVE— The RETN gene encodes the adipokine resistin. Associations of RETN with plasma resistin levels, type 2 diabetes, and related metabolic traits have been inconsistent. Using comprehensive linkage disequilibrium mapping, we genotyped tag single nucleotide polymorphisms (SNPs) in RETN and tested associations with plasma resistin levels, risk of diabetes, and glycemic traits.RESEARCH DESIGN AND METHODS— We examined 2,531 Framingham Offspring Study participants for resistin levels, glycemic phenotypes, and incident diabetes over 28 years of follow-up. We genotyped 21 tag SNPs that capture common (minor allele frequency >0.05) or previously reported SNPs at r² > 0.8 across RETN and its flanking regions. We used sex- and age-adjusted linear mixed-effects models (with/without BMI adjustment) to test additive associations of SNPs with traits, adjusted Cox proportional hazards models accounting for relatedness for incident diabetes, and generated empirical P values (P_e) to control for type 1 error.RESULTS— Four tag SNPs (rs1477341, rs4804765, rs1423096, and rs10401670) on the 3′ side of RETN were strongly associated with resistin levels (all minor alleles associated with higher levels, P_e<0.05 after multiple testing correction). rs10401670 was also associated with fasting plasma glucose (P_e = 0.02, BMI adjusted) and mean glucose over follow-up (P_e = 0.01; BMI adjusted). No significant association was observed for adiposity traits. On meta-analysis, the previously reported association of SNP −420C/G (rs1862513) with resistin levels remained significant (P = 0.0009) but with high heterogeneity across studies (P < 0.0001).CONCLUSIONS— SNPs in the 3′ region of RETN are associated with resistin levels, and one of them is also associated with glucose levels, although replication is needed.Adipose tissue is now recognized as a prolific endocrine organ. In the past few years, several proteins, called adipokines, produced by adipose tissue have been discovered (1). In the process of unraveling the link between obesity and development of diabetes, various adipokines have been suspected to contribute to the pathogenesis of insulin resistance. Resistin is a 12.5-kDa polypeptide that belongs to the resistin-like molecule family of cysteine-rich proteins (2). In murine models, it is produced mainly by adipocytes, and it has been proposed to link obesity with diabetes (3). In humans, adipocytes seem to contribute to only a small fraction of the resistin production (4), and macrophages are considered the predominant source of circulating resistin (5,6). Adipose tissue of obese individuals is characterized by increased infiltration by macrophages (7), which have been proposed to contribute to the proinflammatory state that is characteristic of insulin resistance. Some population studies (8–10) have shown that resistin levels are indeed associated with metabolic risk factors and insulin resistance, suggesting that resistin may play an important role in the pathophysiology of diabetes.The gene encoding resistin (RETN) is located on chromosome 19p13. Genetic variants in RETN have been examined by many groups, and it is estimated that up to 70% of the variation in circulating resistin levels can be explained by genetic factors (11). Several single nucleotide polymorphisms (SNPs) have been associated with resistin levels (9,12–14). However, associations between RETN and BMI or other measures of adiposity have shown very inconsistent results (13,15–21). Polymorphisms in RETN also have been associated with indexes of insulin resistance in some reports (21,22), but lack of replication and null associations (13,19) have raised questions regarding the robustness of these findings. Moreover, most of the analyses examining common variation in RETN and risk of type 2 diabetes have been negative (9,13,16,23–27), with nominal associations only emerging in subanalyses (28,29). The inconsistencies in those studies might be due to low power afforded by small samples or poor coverage of the gene and of its flanking sequences.To address those limitations, we conducted fine mapping of RETN to test if any of the SNPs in or around the gene are associated with resistin levels, diabetes incidence, or glycemic and obesity traits in the Framingham Offspring Study, a large representative community sample. Our goal was to confirm or refute previous reports of association and possibly uncover novel SNP associations using comprehensive tag SNP linkage disequilibrium (LD) mapping.     

Circulating retinol-binding protein-4 concentration might reflect insulin resistance-associated iron overload

OBJECTIVES—The mechanisms behind the association between retinol-binding protein-4 (RBP4) and insulin resistance are not well understood. An interaction between iron and vitamin A status, of which RBP4 is a surrogate, has long been recognized. We hypothesized that iron-associated insulin resistance could be behind the impaired insulin action caused by RBP4.RESEARCH DESIGN AND METHODS—Serum ferritin and RBP4 concentration and insulin resistance were evaluated in a sample of middle-aged men (n = 132) and in a replication independent study. Serum RBP4 was also studied before and after iron depletion in patients with type 2 diabetes. Finally, the effect of iron on RBP4 release was evaluated in vitro in adipose tissue.RESULTS—A positive correlation between circulating RBP4 and log serum ferritin (r = 0.35 and r = 0.61, respectively; P < 0.0001) was observed in both independent studies. Serum RBP4 concentration was higher in men than women in parallel to increased ferritin levels. On multiple regression analyses to predict serum RBP4, log serum ferritin contributed significantly to RBP4 variance after controlling for BMI, age, and homeostasis model assessment value. Serum RBP4 concentration decreased after iron depletion in type 2 diabetic patients (percent mean difference −13.7 [95% CI −25.4 to −2.04]; P = 0.024). The iron donor lactoferrin led to increased dose-dependent adipose tissue release of RBP4 (2.4-fold, P = 0.005) and increased RBP4 expression, while apotransferrin and deferoxamine led to decreased RBP4 release.CONCLUSIONS—The relationship between circulating RBP4 and iron stores, both cross-sectional and after iron depletion, and in vitro findings suggest that iron could play a role in the RBP4–insulin resistance relationship.Adipose tissue is increasingly viewed as an endocrine organ that secretes many types of adipokines (such as leptin, tumor necrosis factor-α, interleukin 6, and adiponectin) that modulate the action of insulin in other tissues. Retinol-binding protein-4 (RBP4), a new fat-derived adipokine that specifically binds to retinol, has recently been reported to provide a link between obesity and insulin resistance (1,2). Circulating RBP4 levels and adipose tissue RBP4 expression were raised in several different mouse models of obesity and insulin resistance. In these animal models, increasing the circulating levels of RBP4 leads to glucose intolerance, augmented hepatic gluconeogenesis, and attenuated insulin signaling in skeletal muscle, whereas knock-out of the RBP4 gene increases insulin sensitivity.In humans, different authors have reported increased serum RBP4 concentration in subjects with obesity, insulin resistance, or type 2 diabetes compared with lean subjects (2–6), although not all studies are concordant. At least two recent studies (7,8) did not observe a relationship between RBP4 and insulin resistance in women. Although some problems exist with serum RBP4 measurements (9), RBP4 mRNA was in fact downregulated in subcutaneous abdominal adipose tissue in postmenopausal women (7). Furthermore, the authors did not see a relationship between adipose tissue RBP4 expression and serum RBP4 levels (7).The mechanisms by which RBP4 induces insulin resistance are not well understood. Treatment of mice with fenretinide (which facilitates the excretion of RBP4 into urine) decreased insulin resistance induced by a high-fat diet (1). Sex and fasting plasma glucose levels seem to be independent determinants of plasma RBP4 concentration. Many adipokines have been found to be sexually dimorphic. Both leptin and adiponectin are increased in serum of women compared with men. This observation has been explained on the basis of different fat amounts and the influences of sex hormones (10,11). Plasma RBP4 concentrations, however, exhibit an opposite pattern. The median (range) for RBP4 in plasma was 21.0 μg/ml (10.7–48.5) for men and 18.1 μg/ml (9.3–34.6) for women (P = 0.001) (3). Since data regarding menopausal status were not available, the authors arbitrarily subdivided sex groups at 50 years of age. Plasma RBP4 levels in women aged >50 years were found to be significantly higher than those in women aged <50 years. However, no such age-associated difference in RBP4 plasma levels was observed in men. Thus, the pattern of serum RBP4 concentrations in serum resembles that of iron stores: higher in men than women, in whom iron stores increase after menopause. Furthermore, treatment with fenretinide induces a dose-dependent peripheral anemia evidenced by erythrocytopenia and decreased hemoglobin concentration and packed cell volume in addition to excretion of RBP4 into urine (12).On the other hand, RBP4 is an indicator of vitamin A (retinol) intake (13,14). The interaction between vitamin A and iron status is well known. For instance, vitamin A deficiency may impair iron metabolism and aggravate anemia (15–17). In fact, iron deficiency anemia and vitamin A deficiency often coexist (15–17).In the last years, increased iron intake and raised iron stores have been recognized as significant, independent contributors to insulin resistance in the general population and in patients with type 2 diabetes (rev. in 18,19). On the other hand, iron supplementation significantly increased plasma retinol and RBP4 (20). Given the interactions between vitamin A and iron, we hypothesized that raised iron stores could be behind the association between increased serum RBP4 concentration and insulin resistance. We found that serum ferritin concentration was positively associated with serum RBP4 concentration in two independent studies. Given these cross-sectional observations, we further aimed to explore these interactions in vitro and to evaluate serum RBP4 concentration after iron depletion in patients with type 2 diabetes.     

Selective small-molecule agonists of G protein-coupled receptor 40 promote glucose-dependent insulin secretion and reduce blood glucose in mice

OBJECTIVE— Acute activation of G protein–coupled receptor 40 (GPR40) by free fatty acids (FFAs) or synthetic GPR40 agonists enhances insulin secretion. However, it is still a matter of debate whether activation of GPR40 would be beneficial for the treatment of type 2 diabetes, since chronic exposure to FFAs impairs islet function. We sought to evaluate the specific role of GPR40 in islets and its potential as a therapeutic target using compounds that specifically activate GPR40.RESEARCH DESIGN AND METHODS— We developed a series of GPR40-selective small-molecule agonists and studied their acute and chronic effects on glucose-dependent insulin secretion (GDIS) in isolated islets, as well as effects on blood glucose levels during intraperitoneal glucose tolerance tests in wild-type and GPR40 knockout mice (GPR40^−/−).RESULTS— Small-molecule GPR40 agonists significantly enhanced GDIS in isolated islets and improved glucose tolerance in wild-type mice but not in GPR40^−/− mice. While a 72-h exposure to FFAs in tissue culture significantly impaired GDIS in islets from both wild-type and GPR40^−/− mice, similar exposure to the GPR40 agonist did not impair GDIS in islets from wild-type mice. Furthermore, the GPR40 agonist enhanced insulin secretion in perfused pancreata from neonatal streptozotocin-induced diabetic rats and improved glucose levels in mice with high-fat diet–induced obesity acutely and chronically.CONCLUSIONS— GPR40 does not mediate the chronic toxic effects of FFAs on islet function. Pharmacological activation of GPR40 may potentiate GDIS in humans and be beneficial for overall glucose control in patients with type 2 diabetes.Loss of glucose-dependent insulin secretion (GDIS) from the pancreatic β-cell is responsible for the onset and progression of type 2 diabetes (1,2). Oral agents that stimulate insulin secretion, such as sulfonylureas and related ATP-sensitive K⁺ channel blockers, reduce blood glucose and have been used as a first-line type 2 diabetes therapy for nearly 30 years (3,4). However, these agents act to force the β-cell to secrete insulin continuously regardless of prevailing glucose levels, thereby promoting hypoglycemia and accelerating the loss of islet function and, eventually, diminished efficacy (5,6). Despite the availability of a range of agents for type 2 diabetes, many diabetic patients fail to achieve or to maintain glycemic targets (7–9). In addition, stricter glycemic guidelines have been proposed to help define a path toward diabetes prevention through identifying and treating the pre-diabetes state (10). Agents that induce GDIS have great potential to replace sulfonylureas as a first-line therapy for the treatment of type 2 diabetes. In particular, agents that have positive effects on arresting or even reversing β-cell demise would represent a major therapeutic advance toward addressing the lack of durability seen with current therapies and perhaps obviate the need for eventual insulin intervention (11–13). The recent emergence of glucagon-like peptide 1–based GDIS agents (14–16), including inhibitors of dipeptidyl peptidase-4 (17) and peptidase-stable analogs such as exendin-4 (18), is undoubtedly a major advance in such a direction. Nevertheless, it remains to be observed whether glucagon-like peptide 1–related agents truly exert durable beneficial effects on β-cell mass and function.The molecular pharmacology of lipid and lipid-like mediators that signal through G protein–coupled receptors (GPCRs) has expanded significantly over the past few years. To date, several orphan GPCRs have been paired with lysophospholipids, bile acids, arachidonic acid metabolites, dioleoyl phosphatidic acid, and short-, medium-, and long-chain free fatty acids (FFAs) (19–21). From these discoveries, GPCR 40 (GPR40), GPR119, and GPR120 have been reported to play a role in regulating GDIS and therefore have potential as novel targets for the treatment of type 2 diabetes (22–26). GPR40 is a G_q-coupled, family A GPCR that is highly expressed in β-cells of human and rodent islets. Several naturally occurring medium- to long-chain FFAs and some thiazolidinedione peroxisome proliferator–activated receptor-γ agonists specifically activate GPR40 (27,28). Activation of GPR40 by FFAs (29–32) or synthetic compounds (23,33) enhances insulin secretion through the amplification of intracellular calcium signaling.The pleiotropic effects of FFAs on the pancreatic β-cell are well known. The fact that FFAs are in vitro ligands for GPR40 is suggestive of the link to the wealth of existing literature data on the acute, stimulatory effects of FFAs on insulin release (34,35). However, FFAs also exert suppressive or detrimental effects on β-cells. Lipotoxicity of β-cells, a condition observed with chronic exposure to high FFA levels, results in impairment in their function and a resulting diminution in their insulin secretory capacity (36,37). Currently, there is an ongoing debate on whether GPR40 mediates the deleterious effects of FFAs on islet function (lipotoxicity) and whether an antagonist of GPR40 is preferable to an agonist for the treatment of type 2 diabetes (38,39). Since FFAs can both be metabolized within cells to act as intracellular signaling molecules (35) and activate more than one receptor (20), they cannot be used as specific and selective tools to unravel the role that GPR40 plays in the β-cell. It is therefore necessary to identify small molecules that specifically activate GPR40.In the following discussion, we will detail the identification and in vitro pharmacology of a novel series of synthetic GPR40 agonists. Using isolated islets from wild-type and homozygous GPR40 knockout (GPR40^−/−) mice (to confirm the on-target activity of small-molecule activators), we not only extended previous findings that acute activation of GPR40 enhances GDIS in pancreatic β-cells but also showed that long-term exposure to the GPR40 agonist, in contrast to FFAs, did not impair β-cell function, thus dissociating the activation of GPR40 from β-cell lipotoxicity. Finally, acute and subchronic dosing of the GPR40 agonist robustly reduced the blood glucose excursion during an intraperitoneal glucose tolerance test (IPGTT) in wild-type, but not GPR40^−/−, mice.