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The stress protein heat shock protein 60 (Hsp60) induces secretion of proinflammatory mediators from murine adipocytes. This study aimed to study Hsp60 as a mediator of adipose tissue inflammation and skeletal muscle cell (SkMC) insulin sensitivity and to quantify plasma Hsp60 concentrations in lean and obese individuals. Regulation of Hsp60 release and Hsp60-induced cytokine secretion and signaling was measured in human adipocytes and SkMCs. Adipocytes exhibited higher Hsp60 release than preadipocytes and SkMCs, which was further stimulated by cytokines and Toll-like receptor (TLR)-4 activation. Hsp60 activated extracellular signal–related kinase (ERK)-1/2, Jun NH2-terminal kinase (JNK), p38, nuclear factor (NF)-κB, and impaired insulin-stimulated Akt phosphorylation in adipocytes. Furthermore, Hsp60 stimulated adipocytes to secrete tumor necrosis factor-α, interleukin (IL)-6, and IL-8. In SkMCs, Hsp60 activated ERK1/2, JNK, and NF-κB and inhibits insulin signaling and insulin-stimulated glucose uptake. SkMCs released IL-6, IL-8, and monocyte chemoattractant protein-1 on Hsp60 stimulation. Plasma Hsp60 was higher in obese males than in lean males and correlated positively with BMI, blood pressure, leptin, and homeostasis model assessment–insulin resistance. In summary, Hsp60 is released by human adipocytes, increased in plasma of obese humans, and induces insulin resistance. This is accompanied by activation of proinflammatory signaling in human adipocytes and SkMCs. Thus, Hsp60 might be a factor underlying adipose tissue inflammation and obesity-associated metabolic disorders.

Isolated adult ventricular cardiomyocytes have been used to study the effects of insulin-deficient diabetes on the expression of cardiac G protein alpha-subunits. Immunoblot analysis of plasma membranes revealed the presence of three different Gs proteins with molecular masses of 45, 47, and 52 kDa. Furthermore, cardiomyocytes were found to contain Gi-2 (41 kDa) and G(o) (39 kDa). Heart cells from streptozotocin-diabetic rats exhibited an unaltered expression of the Gs proteins, whereas Gi-2 and G(o) were reduced by 58 +/- 2 and 27 +/- 11%, respectively. In cells from diabetic rats, adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in response to isoproterenol decreased by approximately 30% at agonist concentrations of 10(-7) to 10(-5) M, with an unaltered maximum stimulation by forskolin. Treatment of cardiomyocytes with pertussis toxin resulted in an incremental increase of isoproterenol-stimulated cAMP formation, which was significantly lower in cardiac myocytes from streptozotocin-diabetic animals (19.2 +/- 1.7 vs. 11.5 +/- 2.4 pmol cAMP.5 x 10(4) cells-1 times 10 min-1). The inhibition of the isoproterenol-induced cAMP accumulation by carbachol in the intact cell was not altered in streptozotocin-diabetes. In conclusion, our data show that insulin-deficient diabetes is associated with a reduced expression and concomitant functional loss of Gi in ventricular cardiomyocytes. Receptor-mediated inhibition of adenylate cyclase remains unaffected by this process, whereas the beta-adrenergic stimulatory pathway involves an additional defect upstream of the adenylate cyclase/G protein system.

Obesity is closely associated with muscle insulin resistance and is a major risk factor for the pathogenesis of type 2 diabetes. Regular physical activity not only prevents obesity, but also considerably improves insulin sensitivity and skeletal muscle metabolism. We sought to establish and characterise an in vitro model of human skeletal muscle contraction, with a view to directly studying the signalling pathways and mechanisms that are involved in the beneficial effects of muscle activity. Contracting human skeletal muscle cell cultures were established by applying electrical pulse stimulation. To induce insulin resistance, skeletal muscle cells were incubated with human adipocyte-derived conditioned medium, monocyte chemotactic protein (MCP)-1 and chemerin. Similarly to in exercising skeletal muscle in vivo, electrical pulse stimulation induced contractile activity in human skeletal muscle cells, combined with the formation of sarcomeres, activation of AMP-activated protein kinase (AMPK) and increased IL-6 secretion. Insulin-stimulated glucose uptake was substantially elevated in contracting cells compared with control. The incubation of skeletal muscle cells with adipocyte-conditioned media, chemerin and MCP-1 significantly reduced the insulin-stimulated phosphorylation of Akt. This effect was abrogated by concomitant pulse stimulation of the cells. Additionally, pro-inflammatory signalling by adipocyte-derived factors was completely prevented by electrical pulse stimulation of the myotubes. We showed that the effects of electrical pulse stimulation on skeletal muscle cells were similar to the effect of exercise on skeletal muscle in vivo in terms of enhanced AMPK activation and IL-6 secretion. In our model, muscle contractile activity eliminates insulin resistance by blocking pro-inflammatory signalling pathways. This novel model therefore provides a unique tool for investigating the molecular mechanisms that mediate the beneficial effects of muscle contraction.

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic and anti-angiogenic properties. More recently it became evident that PEDF is upregulated in patients with type 2 diabetes and also contributes to insulin resistance in mice. During characterization of the secretome of in vitro differentiated human adipocytes by two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-MS, we found that PEDF is one of the most abundant proteins released by adipocytes. The aim of this study was to investigate the regulation and autocrine function of PEDF in human adipocytes and to determine its paracrine effects on human skeletal muscle cells (hSkMC) and human smooth muscle cells (hSMC). Human primary adipocytes secrete 130 ng ml−1 PEDF over 24 h from 1 million cells, which is extremely high as compared with adiponectin, interleukin-6 (IL-6) or IL-8. This release of PEDF is significantly higher than from other primary cells, such as adipose-tissue located macrophages (50-times), hSkMC and hSMC (5-times). PEDF protein expression significantly increases during adipogenesis, which is paralleled by increased PEDF secretion. Furthermore, tumor necrosis factor-α and hypoxia significantly downregulate PEDF protein levels. PEDF secretion was significantly reduced by troglitazone and hypoxia and significantly increased by insulin. Treatment of adipocytes and hSkMC with PEDF induced insulin resistance in adipocytes, skeletal and smooth muscle cells at the level of insulin-stimulated Akt phosphorylation, which was dose dependent and more prominent in adipocytes. Furthermore, inflammatory nuclear factor-κB (NF-κB) signaling was induced by PEDF. In hSMC, PEDF induced proliferation (1.7-fold) and acutely activated proliferative and inflammatory signaling pathways (NF-κB, p38 mitogen-activated protein kinase and mammalian target of rapamycin). PEDF is one of the most abundant adipokines and its secretion is inversely regulated by insulin and hypoxia. PEDF induces insulin resistance in adipocytes and hSkMC and leads to inflammatory signaling in hSMC. Because of these diverse actions, PEDF is a key adipokine, which could have an important role in diabetes and obesity-related disorders.

Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) has been recognized as an appropriate technology for biomarker discovery. Nevertheless, whenever human blood serum is studied, its sensitivity is restricted due to the wide dynamic range of serum protein concentrations. In this context, sample pre-fractionation targeting the low abundant proteins may help to overcome these limitations. Here, we evaluate the combination of ProteoMiner™ pre-fractionation and SELDI based protein profiling. We introduce a simplified workflow and demonstrate the high sensitivity and reproducibility of this combined profiling approach. Our analyses show that this combination is suitable for large-scale serum proteome profiling studies yielding reliable and reproducible results.

Obesity is associated with low-grade chronic inflammation in adipose tissue. This review presents an update on human and rodent studies analyzing the nature of fat-infiltrating immune cells, the time course of adipose tissue infiltration and underlying mechanisms.Intensive studies in rodents have shown that not only cells of the innate immune system traffic into adipose tissue but also various lymphocytes of the adaptive immunity are involved in inflammatory processes in fat. Several studies also provide insight in the order of appearance of macrophages and lymphocytes during the onset of obesity. Adipocytes and preadipocytes are also active players by their secretion of chemotactic adipokines.This review summarizes strong evidence for a link between the action of innate and adaptive immune systems in adipose tissue in the context of obesity and metabolism in rodents, but more studies in humans are necessary to relate this topic to human physiology. Targeting different immune cells at different stages of obesity may eventually lead to novel therapeutic approaches for the metabolic syndrome.

Chemerin is a new adipokine involved in in vitro adipogenesis and insulin resistance and associates with body mass index (BMI) in vivo.We investigated the role of chemerin in morbid obesity, associated metabolic diseases (insulin resistance, hepatic diseases), and postsurgery-induced weight loss.This was a prospective study performed at a university hospital.Subjects included 60 obese female patients (BMI 50.0 +/- 1.0 kg/m(-2)) being candidates for gastric bypass.Patients were examined before and 3, 6, and 12 months after surgery. In 27 patients, chemerin was measured after 2 yr.Outcomes included chemerin, anthropometric parameters, homeostasis model assessment for insulin resistance index (HOMA-IR), cholesterol, high-density lipoprotein, triglycerides, C-reactive protein, adipokines at all time points; and liver histology and macrophage content in fat at baseline.Chemerin was substantially elevated in obese patients compared with nonobese persons (353.8 +/- 18.0 vs. 191 +/- 14 ng/ml, P0.001). Preoperatively, chemerin concentrations correlated positively with BMI, C-reactive protein, IL-6, HOMA-IR, and the amount of omental macrophages and negatively with high-density lipoprotein levels. Baseline chemerin was elevated in patients with a significant activity score for nonalcoholic fatty liver disease, portal inflammation, fibrosis, and fibroinflammation. After surgery, chemerin decreased significantly to 253.0 +/- 14.9 ng/ml after 1 yr and pursued its decrease in patients studied for 2 yr. After surgery, chemerin concentrations positively correlated with triglycerides. The strong decrease of chemerin in the 3 months after surgery was associated with the decrease in HOMA-IR and blood glucose.Chemerin concentrations are elevated in morbidly obese patients and correlated with insulin resistance and markers of liver pathology. Chemerin plasma concentrations decreased after bariatric surgery. This study suggests that chemerin might mediate metabolic alterations in obesity, drastically improving after gastric bypass.

The present study examined the effect of GTP-gamma-S on the function of insulin receptors partially purified from adult rat cardiomyocytes by WGA chromatography. GTP-gamma-S increased receptor autophosphorylation about two times and fully mimicked the stimulatory action of insulin on poly(Glu:Tyr) phosphorylation with no additional effect of the hormone. The effect of GTP-gamma-S was specific, dose-dependent, and due to an increase in the Vmax of the kinase. In the presence of ATP or AMP-PNP, insulin significantly enhanced the binding of [35S]GTP-gamma-S to the partially purified insulin receptor. The findings suggest coupling of the insulin receptor to a G-protein which may be involved in the regulation of tyrosine kinase activity.

Field stimulation of isolated adult ventricular cardiomyocytes was used to study the effect of contractile activity on 3-O-methylglucose transport and the subcellular distribution of Glut4. Cells contracting at a frequency of 1 Hz for 30 min exhibited unaltered basal and insulin-stimulated rates of glucose transport when compared to resting cells. However, at 5 Hz 3-O-methylglucose transport increased to 224% of control after 5 min. Under these conditions insulin was unable to produce a significant additional stimulation of glucose transport. Immunoblotting with an anti-Glut4 polyclonal antibody showed that both insulin and contraction (5 Hz) increased the amount of Glut4 in a plasma membrane fraction by about 8-fold with a parallel decrease in an intracellular membrane fraction by 60-65%. These data suggest the existence of an identical insulin- and contraction-recruitable Glut4 transporter pool in cardiomyocytes.

A strong causal link between increased adipose tissue mass and insulin resistance in tissues such as liver and skeletal muscle exists in obesity-related disorders such as type 2 diabetes. Increased adipose tissue mass in obese patients and patients with diabetes is associated with altered secretion of adipokines, which also includes chemotactic proteins. Adipose tissue releases a wide range of chemotactic proteins including many chemokines and chemerin, which are interesting targets for adipose tissue biology and for biomedical research in obesity and obesity-related diseases. This class of adipokines may be directly linked to a chronic state of low-grade inflammation and macrophage infiltration in adipose tissue, a concept intensively studied in adipose tissue biology in recent years. The inflammatory state of adipose tissue in obese patients may be the most important factor linking increased adipose tissue mass to insulin resistance. Furthermore, chemoattractant adipokines may play an important role in this situation, as many of these proteins possess biological activity beyond the recruitment of immune cells including effects on adipogenesis and glucose homeostasis in insulin-sensitive tissues. The present review provides a summary of experimental evidence of the role of adipose tissue-derived chemotactic cytokines and their function in insulin resistance in vivo and in vitro.

Blood serum samples are the major source for clinical proteomics approaches, which aim to identify diagnostically relevant or treatment-response related proteins. But, the presence of very high-abundance proteins and the enormous dynamic range of protein distribution hinders whole serum analysis. An innovative tool to overcome these limitations, utilizes combinatorial hexapeptide ligand libraries (ProteoMiner). Here, we demonstrate that ProteoMiner can be used for comparative and quantitative analysis of complex proteomes. We spiked serum samples with increasing amounts (3 microg to 300 microg) of whole E. coli lysate, processed it with ProteoMiner and performed quantitative analyses of 2D-gels. We found, that the concentration of the spiked bacteria proteome, reflected by the maintained proportional spot intensities, was not altered by ProteoMiner treatment. Therefore, we conclude that the ProteoMiner technology can be used for quantitative analysis of low abundant proteins in complex biological samples.

Sleep profoundly affects metabolic pathways. In healthy subjects, experimental sleep restriction caused insulin resistance (IR) and increased evening cortisol and sympathetic activation. Increased obesity in subjects reporting short sleep duration leads to speculation that, during recent decades, decreased sleeping time in the general population may have contributed to the increasing prevalence of obesity. Causal inference is difficult due to lack of control for confounders and inconsistent evidence of temporal sequence. In the general population, obstructive sleep apnoea (OSA) is associated with glucose intolerance. OSA severity is also associated with the degree of IR. However, OSA at baseline does not seem to significantly predict the development of diabetes. Prevalence of the metabolic syndrome is higher in patients with OSA than in obese subjects without OSA. Treatment with continuous positive airway pressure seems to improve glucose metabolism both in diabetic and nondiabetic OSA but mainly in nonobese subjects. The relative role of obesity and OSA in the pathogenesis of metabolic alterations is still unclear and is intensively studied in clinical and experimental models. In the intermittent hypoxia model in rodents, strong interactions are likely to occur between haemodynamic alterations, systemic inflammation and metabolic changes, modulated by genetic background. Molecular and cellular mechanisms are currently being investigated.

Insulin resistance is often associated with obstructive sleep apnoea syndrome (OSAS) and could contribute to cardiovascular risk in OSAS. Sleep loss and intermittent hypoxia could contribute to the pathogenesis of the metabolic alterations associated with obesity, a common feature of OSAS. The biology of the adipocyte is being increasingly studied, and it has been found that hypoxia negatively affects adipocyte function. In November 2007, the European Respiratory Society and two EU COST Actions (Cardiovascular risk in OSAS (B26) and Adipose tissue and the metabolic syndrome (BM0602)), held a Research Seminar in Du¨sseldorf, Germany, to discuss the following: 1) the effects of hypoxia on glucose metabolism and adipocyte function; 2) the role of inflammatory activation in OSAS and obesity; 3) the alarming rates of obesity and OSAS in children; 4) the harmful effects of the metabolic syndrome in OSAS; 5) the effects of OSAS treatment on metabolic variables; and 6) the relationship between daytime sleepiness and hormonal and inflammatory responses. Insulin resistance in skeletal muscle, the role of the endocannabinoid system and novel pharmacological approaches to treat insulin resistance were also discussed. As obesity and hypoxia could be the basic links between OSAS and adipocyte dysfunction, further research is needed to translate these new data into clinical practice.

The effect of alpha-adrenoceptor stimulation on the concentration of cytosolic free calcium ( Ca i 2+ ) was determined by measuring indo-1 fluorescence in isolated ventricular cardiomyocytes from normal and streptozotocin-diabetic rat; 1.3 × 105 alpha1-adrenoceptors per normal myocyte and an unaltered number of these receptors in cells from diabetic rats were detected using the alpha1-selective ligand WB-4101. Under basal conditions, Ca i 2+ was found to be 154 ± 4 n m (n = 34) reaching a value of 192 ± 10 n m (n = 15) after stimulation of myocytes with a maximal dose of methoxamine for 5 min. Under the same conditions the leakage of dye produced a significantly smaller increase of basal values to 169 ± 5 n m (n = 17). Indo-1 loaded cells did not respond to beta-stimulation unless in the presence of KCl (50 m m ), demonstrating the specificity of methoxamine action. Treatment of cells with nifedipine or chelation of extracellular calcium by EGTA did not modify the alpha-adrenergic response. Experiments with cardiomyocytes from streptozotocin-diabetic rats showed an unaltered modulation of Ca i 2+ by both alpha- and beta-receptor stimulation.It is concluded that signalling by alpha1-adrenoceptors in ventricular cardiomyocytes results in mobilization of intracellular calcium stores.

Glucagon like peptide-1 (Glp-1) exhibits beneficial effects on beta cell mass by both enhancing proliferation and inhibiting apoptosis. The precise mechanism of the anti-apoptotic effect of Glp-1 and Glp-1 mimetics like exendin-4 has remained elusive. Here, we studied cytokine-induced apoptosis in the pancreatic beta cell line INS-1 and performed a comparative mitochondrial protein pattern analysis using two-dimensional difference gel electrophoresis (2D-DIGE). Cytokine incubation of INS-1 cells increased caspase-3 activity about 3-fold, which was reduced by 60% in the presence of exendin-4. Production of reactive oxygen species in response to cytokines was completely prevented after preincubation with exendin-4. Highly purified mitochondria were obtained and mitochondrial proteins were labeled with Cy-dyes and separated on overlapping zoom 2D gels spanning a pH-range of 4-9. Protein spots with significant changes after cytokine and exendin-4 treatment were identified by MALDI mass spectrometry. Comparing all treatment conditions, comparative mitochondrial proteome analysis allowed to identify 33 different proteins, which were significantly altered between comparison groups. Changes in protein patterns revealed involvement of cytokine-induced electron transport chain damage. Thus, cytochrome bc1 complex subunit I and ATP synthase subunit beta were downregulated by 30-40%. This was abrogated by the presence of exendin-4. In conclusion, this study provides further insights into the role of mitochondria in cytokine-induced apoptosis. We show here that exendin-4 significantly counter-regulates the reduced abundance of electron transport chain proteins, leading to a reduction of oxidative stress and most likely contributing to the anti-apoptotic action of this drug.

A variety of studies have documented significant improvements in the treatment of type 1 and 2 diabetes after the introduction of artificial insulins. This review gives an overview of insulin analogues which are currently approved for therapeutical use. Clinical data regarding the efficiency to control blood glucose level as well as improving HbA1c level in comparison to conventional insulin preparations in type 1 and 2 diabetic patients are summarized. Furthermore, special features of insulin analogues regarding their signalling properties are discussed with focus on the proliferative effects of insulin glargine as well as some recent data of insulin detemir.

We recently showed that insulin analogues exhibit a beta-cell protective function. The aim of this study was to test if the anti-apoptotic activity of GLP-1 agonists and insulin analogues is mediated by different pathways and if combined treatment may provide augmented protection against beta-cell death. Incubation of INS-1 cells with cytokines or fatty acids increased the number of apoptotic cells and caspase 3 activity, which was reduced by pretreatment with GLP-1 and its receptor agonists exendin-4 and AVE0010 by 50-60%. Similar effects (about 40% reduction) were observed after pretreatment with several insulin analogues. Combined treatment revealed additive activity and resulted in prevention of both cytokine- and fatty acid-induced apoptosis by up to 80%. No acute Akt-phosphorylation in response to GLP-1 receptor agonists could be observed, however, it became detectable after 24-hour stimulation. Gene silencing of Akt2 increased cytokine-induced apoptosis 2-fold. Under these conditions the beta-cell protective activity of AVE0010 remained completely unaltered. We show here that the anti-apoptotic activity of GLP-1 and its receptor agonists AVE0010 and exendin-4 is enhanced by addition of insulin analogues and that the anti-apoptotic action of GLP-1 mimetics is mostly unrelated to Akt2 signaling. It is suggested that combination of GLP-1 receptor agonists and insulin analogues, specifically insulin glargine, may represent a new therapeutic option for preservation of beta-cell mass in type 2 diabetic patients.

Objective: Many adipokines, inflammatory cytokines, and other proteins produced by adipose tissue have been shown to be involved in the development of obesity-related insulin resistance. Nevertheless, new factors that play an important role in these processes are still emerging. Therefore, we screened the level of 120 different proteins in biopsies of subcutaneous adipose tissue (ScAT) of lean and obese subjects. Research Methods and Procedures: All studied volunteers (12 obese with BMI >30 and 6 lean with BMI

Tobago and Trinidad are two Caribbean islands with distinct genetic background and lifestyles; while Tobago is serene and a tourist centre, Trinidad is characterized by a hustling and bustling lifestyle. The study was aimed at determining and comparing the prevalence of the metabolic syndrome (MetS) and its critical components in type 2 diabetic patients using the new International Diabetes Federation (IDF) definition.Four hundred and thirteen (166 Tobago, 247 Trinidad) type 2 diabetic patients visiting 10 lifestyle disease clinics were studied. Blood pressure, anthropometric parameters (height, weight, body mass index and waist circumference) and overnight fasting blood samples were taken. Plasma glucose and serum triglycerides, total cholesterol, LDL- and HDL-cholesterol, insulin, and adiponectin were determined. Insulin resistance (IR) was determined using the HOMA method.The patients in Tobago were significantly older than patients in Trinidad (p0.001) but the duration of diabetes (9.4 +/- 0.5 vs. 11.1 +/- 0.7 yr), medications, generalized (31.7 vs. 38.8%) and central (78.5 vs. 83.7%) obesity were similar (p0.05). In comparison with patients in Tobago, diabetic patients in Trinidad, irrespective of gender, had significantly higher prevalence of IDF critical components such as raised BP, raised triglycerides and reduced HDL-cholesterol (all, p0.001). Thus, while more patients in Trinidad were diagnosed with MetS based on three or four components, more patients in Tobago were diagnosed based on two components (p0.001).There were high prevalence rates of the components of the MetS in both the islands of Tobago and Trinidad. Quantitatively, the aggregation of the components is higher in patients in Trinidad, which constitute greater risk for adverse cardiovascular outcome. Controlling central obesity should be the target in preventing MetS in the two islands.

Mitogenic activity of insulin and insulin analogues and the involvement of the IGF-1 receptor (IGF-1R) is still a controversial issue. We compared levels of the proteins IGF-1R and insulin receptor (InsR) in fibroblasts and smooth muscle cells from healthy donors and assessed the downstream signalling and growth-promoting activity of insulin and insulin analogues.DNA synthesis was monitored in human fibroblasts and coronary artery smooth muscle cells. Using small interfering RNAs, the levels of IGF-1 and InsR were reduced by 95 and 75%, respectively.Enhanced mitogenic potency of insulin and insulin analogues was observed which correlated with increased levels of IGF-1R and/or IRS-1. A reduction in the IGF-1R level significantly blunted stimulation of Akt phosphorylation by IGF-1, AspB10 and glargine by 72, 58 and 40%, respectively. Akt phosphorylation in response to insulin remained unaffected. Silencing of InsR did not significantly alter Akt phosphorylation in response to IGF-1, AspB10 and glargine. IGF-1R knockdown reduced the stimulation of DNA synthesis in response to IGF-1 and glargine to a level identical to that produced by insulin.These data show a prominent role of IGF-1R/Akt signalling in mediating the mitogenic effects of insulin analogues. Regular insulin stimulates DNA synthesis by exclusively activating InsR, whereas insulin analogues mainly signal through IGF-1R. It is suggested that inter-individual differences in the levels of proteins of the IGF-1R system may function as a critical determinant of the mitogenic potency of insulin analogues.

Adipocyte-derived factors might play a role in the development of hepatic insulin resistance. Resistin was identified as an adipokine linking obesity and insulin resistance. Resistin is secreted from adipocytes in rodents but in humans it was proposed to originate from macrophages and its impact for insulin resistance has remained elusive. To analyze the role of adipokines in general and resistin as a special adipokine, we cultured the human liver cell line HepG2 with adipocyte-conditioned medium (CM) containing various adipokines such as IL-6 and MCP-1, and resistin. CM and resistin both induce insulin resistance with a robust decrease in insulin-stimulated phosphorylation of Akt and GSK3. Insulin resistance could be prevented by co-treatment with troglitazone but not by co-stimulation with adiponectin. As human adipocytes do not secrete resistin, HepG2 cells were also treated with resistin added into CM. CM with resistin addition induced stronger insulin resistance than CM alone pointing to a specific role of resistin in the initiation of hepatic insulin resistance in humans.

In obesity, there is a strong link between increased adipose tissue mass and development of insulin resistance in tissues such as liver and muscle. Under these conditions, adipose tissue synthesizes various pro-inflammatory chemokines such as monocyte chemotactic protein-1. This review provides a summary of recent knowledge on the role of monocyte chemotactic protein-1 in adipose tissue inflammation and insulin resistance.Monocyte chemotactic protein-1 is a proinflammatory adipokine that is believed to play a role in the pathogenesis of obesity and diabetes. New in-vitro data demonstrate that monocyte chemotactic protein-1 has the ability to induce insulin resistance in adipocytes and skeletal muscle cells. By using mice that either overexpress monocyte chemotactic protein-1 or are deficient in monocyte chemotactic protein-1 or its receptor, exciting new insights have been obtained into the role of monocyte chemotactic protein-1 in adipose tissue inflammation and insulin resistance.Monocyte chemotactic protein-1 is an adipokine with insulin-resistance-inducing capacity that is related to increased adipose tissue mass in obesity and insulin resistance. It plays an important role in adipose tissue inflammation by recruiting macrophages into fat. Monocyte chemotactic protein-1 is thus a therapeutic target, and may represent an important factor linking adipose tissue inflammation, obesity and type 2 diabetes.

Primary prevention of Coronary Heart Disease (CHD) in diabetic patients should be based on absolute CHD risk calculation. This study was aimed to determine the levels of 10-year CHD risk in Caribbean type 2 diabetic patients using the diabetes specific United Kingdom Prospective Diabetes Study (UKPDS) risk engine calculator.Three hundred and twenty-five (106 males, 219 females) type 2 diabetic patients resident in two Caribbean Islands of Tobago and Trinidad met the UKPDS risk engine inclusion criteria. Records of their sex, age, ethnicity, smoking habit, diabetes duration, systolic blood pressure, total cholesterol, HDL-cholesterol and glycated haemoglobin were entered into the UKPDS risk engine calculator programme and the absolute 10-year CHD and stroke risk levels were computed. The 10-year CHD and stroke risks were statistically stratified into15%, 15-30% and30% CHD risk levels and differences between patients of African and Asian-Indian origin were compared.In comparison with patients in Tobago, type 2 diabetic patients in Trinidad, irrespective of gender, had higher proportion of 10-year CHD risk (10.4 vs. 23.6%, P0.001) whereas the overall 10-year stroke risk prediction was higher in patients resident in Tobago (16.9 vs. 11.4%, P0.001). Ethnicity-based analysis revealed that irrespective of gender, higher proportion of patients of Indian origin scored30% of absolute 10-year CHD risk compared with patients of African descent (3.2 vs. 28.2%, P0.001).The results of the study identified diabetic patients resident in Trinidad and patients of Indian origin as the most vulnerable groups for CHD. These groups of diabetic patients should have priority in primary or secondary prevention of coronary heart disease.

Es gibt Hinweise darauf, dass epikardiales Fett (Epi) ahnliche Eigenschaften wie abdominales Fett aufweist und in der Lage ist, pro-inflammatorische Zytokine und Fettsauren in die koronare Strombahn freizusetzen. Damit ware eine ahnlich direkte Interaktion zwischen Epi und dem Herz als Zielorgan moglich, wie sie fur das abdominale Fett und die Leber diskutiert wird. Unsere Hypothese geht deshalb davon aus, dass Diabetes Typ 2 und andere Insulin-resistenten Zustande das Muster und die Menge der Sekretionsprodukte (Zytokine, Fettsauren) des Epi modifizieren und dadurch zur Entwicklung von Insulinresistenz im Herzen, zur kardialen Dysfunktion und zum im Diabetes erhohten kardialen Risiko beitragen. Ein geeignetes experimentelles Modell um die spezifischen Wechselwirkungen zwischen Epi und dem Herzen zu untersuchen, wurde bisher nicht beschrieben. Bekannt ist aber, dass es im Meerschweinchen ahnlich wie bei Mensch und Affe zur Anreicherung von Epi in Abhangigkeit von Ernahrung und Alter kommt. Um die Eignung des Meerschweinchenherzens als Model zu untersuchen, wurden in einem ersten Ansatz die Freisetzung und Synthese von Zytokinen durch Epi im Vergleich zu subkutanem Fett (Sc) mit einer Array Technik untersucht. Beide Gewebe synthetisieren ein breites Spektrum an pro- und anti-inflammatorischen Zytokinen (>50). Im Epi uberwiegen Leptin, Eotaxin 3, Fraktalkalin, BDNF, IL1a, MDC, IGFBP1, 2 und 4, FAS, TIMP 2, Adiponektin, Angiopoietin 2, GCSF und bFGF. Abhangig vom Alter und der Menge an EPi wird nicht nur eine vermehrte Freisetzung von Zytokinen beobachtet, sondern das Spektrum der sezernierten Zytokine verschiebt sich pro-inflammatorisch. Morphologisch liegt ein hoher Anteil von Epi dem Myokard auf; ein wesentlicher Anteil durchzieht aber auch die epikardialen Muskelschichten, die dadurch eine aufgelockerte Schichtung erfahren. Zusatzlich akkumuliert ein erheblicher Teil entlang der grosen Gefase und an der Herzspitze. Mit der Zunahme an Epi sind das flachenartige Auftreten von perivaskularem und interstitiellem Kollagen und die vermehrte Ablagerung von Grundsubstanz (Vorstufe zur Kollagenablagerung) assoziiert. Die Muskelzelle und die Zellen des parasympathischen Nervensystems erscheinen demgegenuber unauffallig. Die morphologischen Veranderungen legen nahe, dass es in Abhangigkeit von der Menge an Epi zur Beeintrachtigung der myokardialen Struktur kommt und Defekte der diastolischen Dysfunktion und beim venosen Abfluss auftreten. Trotz der morphologischen Unauffalligkeit weist der Herzmuskel biochemisch charakteristische Auffalligkeiten auf: mit zunehmendem Epi und Alter werden die 12-Lipoxygenase, Calreticulin, der Glucosetransporter 4 sowie PPARa down-reguliert. Diese Veranderungen stellen erste Hinweise auf die Entwicklung von Storungen des Glucosestoffwechsels und der Calcium-Homoostase dar.

Clinical insulin resistance is associated with decreased activation of phosphatidylinositol 3′-kinase (PI3K) and its downstream substrate protein kinase B (PKB)/Akt. However, its physiological protein substrates remain poorly characterized. In the present study, the effect of in vivo insulin action on phosphorylation of the PKB/Akt substrate 40 (PRAS40) was examined. In rat and mice, insulin stimulated PRAS40-Thr246 phosphorylation in skeletal and cardiac muscle, the liver, and adipose tissue in vivo. Physiological hyperinsulinemia increased PRAS40-Thr246 phosphorylation in human skeletal muscle biopsies. In cultured cell lines, insulin-mediated PRAS40 phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002. Immunohistochemical and immunofluorescence studies showed that phosphorylated PRAS40 is predominantly localized to the nucleus. Finally, in rats fed a high-fat diet (HFD), phosphorylation of PRAS40 was markedly reduced compared with low-fat diet–fed animals in all tissues examined. In conclusion, the current study identifies PRAS40 as a physiological target of in vivo insulin action. Phosphorylation of PRAS40 is increased by insulin in human, rat, and mouse insulin target tissues. In rats, this response is reduced under conditions of HFD-induced insulin resistance.

The expression and activity of the arachidonic acid-metabolizing enzyme leukocyte-type 12-lipoxygenase (12-LO) are augmented in cultured vascular endothelial and smooth muscle cells exposed to high glucose concentrations and in blood vessels of diabetic animals. The product of this enzyme, 12-hydroxyeicosatetraenoic acid (12-HETE), evokes two types of interactions in these cells: on one hand it acts as a pro-inflammatory factor that contributes to the initiation and progression of atherosclerotic lesions. Yet on the other, it protects the same cells against deleterious effects of high levels of intracellular glucose by downregulating the glucose transport system in the cells. In addition, it has been shown that 12-LO and 12-HETE support insulin-dependent glucose transporter-4 translocation to the plasma membrane by maintaining intact actin fiber network in the cardiomyocytes. Here we focus on the disparate cellular interactions by which 12-LO and 12-HETE affect the glucose transport system in vascular endothelial and smooth muscle cells and in cardiomyocytes.

The adipocyte hormone adiponectin is negatively correlated with obesity and insulin resistance and may exert an important antidiabetes function. In this study, primary human skeletal muscle cells were cocultured with human fat cells or incubated with adipocyte-conditioned medium in the presence or absence of the globular domain of adiponectin (gAcrp30) to analyze its capacity to restore normal insulin signaling in the muscle cells. Human skeletal muscle cells cocultured with adipocytes or treated with adipocyte-conditioned medium showed an impaired Akt and glycogen synthase kinase 3 serine phosphorylation in response to insulin. Furthermore, insulin-stimulated GLUT4 translocation was reduced by adipocyte-conditioned medium. Impaired insulin signaling was normalized upon addition of gAcrp30 to the coculture. Further, adipocyte-conditioned medium generated in the presence of gAcrp30 was unable to perturb insulin-stimulated Akt phosphorylation. Concomitant addition of gAcrp30 and adipocyte-conditioned medium to the myocytes failed to restore normal insulin action. Protein array analysis of adipocyte-conditioned medium indicated that the secretion of at least eight different cytokines was diminished in response to gAcrp30. We therefore suggest that adiponectin operates as a key regulator of adipocyte secretory function. This autocrine action may prevent the induction of skeletal muscle insulin resistance and may partly explain the antidiabetes action of this hormone.

Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449)(-)(664)) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1(449)(-)(664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449)(-)(664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449)(-)(664) was then phosphorylated by PKC-zeta using [(32)P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rIRS-1(449)(-)(664). Ser(570) was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.

To examine the relationship between adiponectin and metabolic variables in the offspring of patients with type 2 diabetes mellitus.Fasting blood samples and anthropometric indices were taken from 34 subjects, offspring of patients with type 2 diabetes, and 24 healthy control subjects without any immediate family history of diabetes. Plasma glucose and serum adiponectin, insulin, triglycerides, total cholesterol, HDL and LDL cholesterol levels were measured, and insulin resistance (IR) was calculated based on the homeostasis model assessment (HOMA) method.Offspring and control subjects were sex-matched, but the offspring were older and had higher body mass index and waist circumference than the control subjects (p0.05). The offspring had significantly higher mean fasting plasma glucose concentrations; however, their mean serum insulin, adiponectin, triglyceride, total cholesterol, HDL and LDL cholesterol and HOMA-derived IR levels did not significantly differ from those of the control subjects (p0.05). While the negative correlation between serum adiponectin and HDL cholesterol levels in the offspring remained statistically significant after adjusting for the effect of age, sex and BMI (r = -0.37, p0.05), the negative correlation between adiponectin and serum triglyceride, LDL cholesterol or IR levels became non-significant after controlling for the above variables (p0.05 in all cases).The correlation between adiponectin and some known biochemical risk factors for developing diabetes and cardiovascular disease in the offspring of patients with diabetes warrants further study to evaluate its potential in assessing the risk of developing these disorders.

We aimed to characterise the development of autoregulation of glucose transport in vascular endothelial cells and its relationship to 12-lipoxygenase (12-LO) expression.Bovine aortic endothelial cells were exposed to 5.5 and 23.0 mmol/l glucose for up to 48 h. The rates of glucose transport, GLUT-1 and 12-LO expression and of 12-hydroxyeicosatetraenoic acid (12-HETE) production were determined.We showed high glucose-dependent downregulation of glucose transport and transporter in vascular endothelial cells within 36-48 h. A similar time-dependent increase in the expression of 12-LO and the generation of its product 12-HETE was also observed. This downregulatory process was prevented when lipoxygenase activity was inhibited.Vascular endothelial cells, which were previously thought to be "glucose-blind", do in fact downregulate GLUT-1 expression and the rate of glucose transport in response to extended exposure to high glucose concentrations. This slow development of glucose-induced downregulation in vascular endothelial cells is related to the slower basal rate of glucose transport in these cells and the slow induction of 12-LO. These data are interesting in view of current hypotheses that attribute vascular endothelial cell dysfunction in diabetes to the lack of a glucose-induced autoregulatory response.

The potentially enhanced mitogenic activity of insulin analogs represents a safety risk that requires detailed analysis of new analogs considered for therapeutic applications. We assessed the signaling properties and mitogenic potency of two novel rapid-acting insulin analogs, Lys(B3),Glu(B29) insulin (HMR 1964) and Lys(B3),Ile(B28) insulin (HMR 1153) using myoblasts and cardiomyocytes. In myoblasts, both binding and internalization were two- to threefold higher for Asp(B10) insulin and HMR 1153 when compared with HMR 1964 and regular insulin. This finding correlated with a prominent Shc/IGF-I receptor interaction, tyrosine phosphorylation of Shc, activation of extracellular signal-regulated protein kinase (ERK)-1 and -2, and stimulation of DNA synthesis by HMR 1153 and Asp(B10) insulin. In contrast, HMR 1964 produced a marginal activation of the Shc/ERK kinase cascade and was equipotent to insulin in stimulating DNA synthesis in myoblasts. Further, the in vivo growth-promoting activity of this analog was found to be identical to that of regular human insulin. In myoblasts, HMR 1964 produced a minor activation of insulin receptor substrate (IRS)-1 tyrosine phosphorylation, but a prominent activation of IRS-2, with a significantly stronger effect than insulin in human myoblasts. Predominant activation of IRS-2 was also observed in adult cardiomyocytes where HMR 1964 increased 3-O-methylglucose transport and the activation of Akt and glycogen synthase kinase-3 to the same extent as human insulin. We concluded that 1) the mitogenic properties of insulin analogs may result from a series of initial receptor interactions, including internalization and phosphorylation; 2) the mitogenic and metabolic potential of HMR 1964 is identical to that of insulin; and 3) predominant activation of IRS-2 may open new avenues for optimized insulin therapies.

Pancreatic beta-cell apoptosis is a common feature of Type 1 and Type 2 diabetes and leptin exerts an anti-apoptotic function in these cells. The beta-cell line INS-1 was used to test the hypothesis that the adipocyte hormone adiponectin might mediate an anti-apoptotic effect comparable to leptin.Apoptosis was induced by culturing cells with a cytokine combination (interleukin-1beta/interferon-gamma) or palmitic acid in absence or presence of leptin or the globular domain of adiponectin (gAcrp30), respectively.INS-1 cells had a prominent sensitivity towards cytokine- and fatty acid-induced apoptosis, resulting in about three- and six-fold increases in caspase 3 activation and DNA fragmentation, respectively. gAcrp30 strongly (50-60%) inhibited palmitic acid-induced apoptosis, with a weaker effect against cytokine-induced apoptosis (35%). The same result was observed for leptin with both adipokines being non-additive. Reduction of apoptosis by an inhibitor of IkappaB-kinase (IKK) indicated the involvement of the nuclear factor (NF)-kappaB pathway in both cytokine- and fatty acid-induced apoptosis, however, leptin and gAcrp30 were unable to block NF-kappaB activation. Cytokine- and fatty-acid-induced suppression of glucose/forskolin-stimulated insulin secretion was completely prevented through the action of gAcrp30, whereas leptin was only effective against lipotoxicity-mediated beta-cell dysfunction.Our data show that gAcrp30 partially rescues beta cells from cytokine- and fatty-acid-induced apoptosis and completely restores autoimmune- and lipotoxicity-induced dysfunction of insulin-producing cells. We suggest that gAcrp30 exerts its anti-apoptotic function without modulating NF-kappaB activation. This novel beta cell protective function of gAcrp30 might serve to counteract autoimmune- and lipotoxicity-induced beta-cell destruction.

Insulin receptor substrate (IRS) proteins represent key elements of the insulin-signaling cascade. IRS-4 is the most recently characterized member of the IRS family with an undefined in vivo function. In contrast to IRS-1 and IRS-2, IRS-4 exhibits a limited tissue expression, and IRS-4 protein has not been detected in any mouse or primary human tissue so far. The purpose of the present study was to analyze the expression of IRS-4 in rat muscle and human skeletal muscle cells and assess involvement of IRS-4 in initial insulin signaling. Using immunoblotting and immunoprecipitation, the specific expression of IRS-4 protein could be demonstrated in rat soleus and cardiac muscle and human skeletal muscle cells, but it was not significantly detectable in quadriceps and gastrocnemius. A prominent down-regulation of IRS-4 was observed in heart and soleus muscle of WOKW rats, an animal model of the metabolic syndrome. In human skeletal muscle cells, both IRS-1 and IRS-2 are rapidly phosphorylated on tyrosine in response to insulin, whereas essentially no tyrosine phosphorylation of IRS-4 was observed in response to both insulin and IGF-I. Instead, a 2-fold increase in IRS-4 tyrosine phosphorylation was observed in myocytes subjected to osmotic stress. In conclusion, IRS-4 protein is expressed in heart and skeletal muscle in a fiber type specific fashion. Our data suggest that IRS-4 does not function as a substrate of the insulin and the IGF-I receptor in primary muscle cells but may be involved in nonreceptor tyrosine kinase signaling.

Intact actin microfilaments are necessary for insulin-regulated GLUT4 translocation from intracellular pools to the plasma membrane. Products of the lipoxygenase (LO) pathway were shown to be implicated in the regulation of actin cytoskeleton rearrangement. The aim of this study was to examine the role of these LO products for cardiac insulin signaling and glucose uptake, GLUT4 translocation, and actin-based cytoskeleton structure. Exposure of cardiomyocytes to esculetin or NDGA, two structurally different LO inhibitors, induced a complete inhibition of insulin-stimulated glucose uptake, whereas control cells showed a threefold stimulation by insulin. Addition of 12(S)-HETE rendered the NDGA-treated cells insulin-sensitive. Early insulin signaling was not changed in cells exposed to LO inhibitors. Cell surface biotinylation of control cells showed a twofold increase of GLUT4 at the cell surface after insulin stimulation. In contrast, the LO inhibitors induced a complete inhibition of insulin-stimulated GLUT4 translocation. Labeling of the F-actin cytoskeleton revealed a prominent disassembly of actin fibers in cells exposed to the LO inhibitors. In conclusion, we show here that products of the LO reaction participate in the organization of the actin network in ventricular cardiomyocytes. Inhibition of LO blocks GLUT4 translocation without affecting insulin signaling events. These data suggest that products of the LO reaction participate in the regulation of glucose transport by contribution to a rearrangement of actin cytoskeletal elements.

Tamoxifen (TAM) is a well-tolerated compound in the treatment of breast cancer and is primarily considered to act by competition with estrogen receptors (ER). Here we investigated the in vitro efficacy and potentially underlying mechanisms of TAM in established cell lines of squamous cell carcinomas of the head and neck (SCCHN). Using proliferation and apoptosis assays the antitumor activity of TAM in five SCCHN and the breast carcinoma line MCF-7 (positive control) was determined. MCF-7 was more sensitive to low-dose TAM (below 1 microM), whereas SCCHN showed significant growth inhibition at higher TAM concentrations (5-10 microM). Growth curve analysis and apoptosis assays were indicative for a cytostatic effect of low-dose TAM and high-dose TAM led to cell loss by apoptosis in sensitive SCCHN. In order to further characterize the observed antitumor effects we determined the amount of steroid hormone receptors with the dextran-coated charcoal method and immunocytochemistry. In addition, production of transforming growth factor (TGF-)-alpha, -beta1 and -beta2 was measured by ELISA, and protein kinase C (PKC) activity was assessed with a radioligand assay. Except MCF-7, none of the SCCHN lines was positive for ER. TAM caused decreased TGF-alpha and increased TGF-beta levels in MCF-7, but not in SCCHN supernatants. Furthermore, the antiestrogen reduced PKC activity in MCF-7, but not in SCCHN. In the present in vitro system, the observed antitumor activity of high-dose TAM in SCCHN cannot be explained by estrogen antagonism, alterations of TGF-alpha/beta levels or decreased PKC activity.

Aims/hypothesis To identify a GTPase of 24,000 M(r) which we recently found to co-localize with GLUT4 in cardiac muscle. Methods A 24,000 M(r)-GTP-binding fraction was purified from pig heart by a three-step chromatographic procedure, followed by two-dimensional electrophoresis and electrospray ionization-mass spectrometry. Subcellular distribution of the GTPase was assessed by western blotting. Co-localization with GLUT4 was assessed by continuous sucrose density gradient fractionation and immunoadsorption of GLUT4-containing vesicles. Results The Rab11 protein was identified as a major component of the GTP-binding fraction and its expression in rat cardiac muscle was confirmed. In vivo insulin treatment resulted in the recruitment of Rab11 from the microsomal fraction to the plasma membrane. Subcellular fractionation indicated two immunoreactive GLUT4 pools. Most of the intracellular pool of Rab11 overlapped with the high-density GLUT4 pool and most of the transferrin receptor pool. The Rab11 protein also co-sedimented with the low-density, non-endosomal GLUT4 pool and substantially increased in this fraction after insulin treatment. It was specifically present in GLUT4-containing vesicles and insulin increased its abundance in these vesicles 2.2-fold relative to the amount of GLUT4. These vesicles also containend Rab4 and Akt-2, the latter being only associated after insulin stimulation. Insulin was unable to alter the cellular localization of Rab11 in insulin-resistant obese Zucker rats. Conclusion/interpretation These results support the hypothesis that at least two GTPases of the Rab family participate in GLUT4-vesicle trafficking. We suggest that Rab11 is involved in the endosomal recycling, sorting and exocytotic movement of the glucose transporter.

Ventricular cardiomyocytes and cardiac tissue of lean and genetically obese (fa/fa) Zucker rats were used 1) to study the role of the p85 regulatory subunit isoforms p85 alpha and p85 beta for insulin signaling through the phosphatidylinositol (PI) 3-kinase pathway, and 2) to elucidate the implications of these mechanisms for cardiac insulin resistance. Western blot analysis of cardiomyocyte lysates revealed expression of p85 alpha and p85 beta but no detectable amounts of the splice variants of p85 alpha. Essentially no p85 alpha subunit of PI 3-kinase was found to be associated with insulin receptor substrate (IRS)-1 or IRS-2 in basal and insulin-stimulated (5 min) cardiomyocytes. Instead, insulin produced a twofold increase in p85 beta associated with IRS-1, leading to a three- to fourfold increase in p85 beta-associated PI 3-kinase activity. This response was significantly reduced in obese animals. Comparable results were obtained in the intact heart after in vivo stimulation. In GLUT-4-containing vesicles, an increased abundance (3.7 +/- 0.7-fold over basal) of p85 alpha was observed after insulin stimulation of lean animals, with no significant effect in the obese group. No p85 beta could be detected in GLUT-4-containing vesicles. Recruitment of the p110 catalytic subunit of PI 3-kinase and a twofold increase in enzyme activity in GLUT-4-containing vesicles by insulin was observed only in lean rats. We conclude that, in the heart, p85 alpha recruits PI 3-kinase activity to GLUT-4 vesicles, whereas p85 beta represents the main regulator of IRS-1- and IRS-2-mediated PI 3-kinase activation. Furthermore, multiple defects of PI 3-kinase activation, involving both the p85 alpha and the p85 beta adaptor subunits, may contribute to cardiac insulin resistance.

In differentiating human preadipocytes glucose uptake in the presence of insulin is a prerequisite for lipid accumulation. The aim of this study was to characterize the insulin-regulated glucose transport system during and after differentiation.Human adipocyte precursor cells kept in primary culture were allowed to differentiate into fat cells under serum-free hormone-supplemented conditions. 2-Deoxy-glucose uptake was measured as a functional parameter of the glucose transport system, the amount of GLUT1 and GLUT4 protein was determined by Western blotting.In the undifferentiated state, cells did not increase 2-deoxy-glucose uptake in response to insulin. On day 16, when cells have acquired the adipocyte phenotype, there was a 3-4-fold stimulation of glucose transport by insulin compared to basal rates, whereas basal glucose uptake was dramatically diminished. Measurement of GLUT4 protein in cell extracts, showed a marked increase in the amount of this insulin-regulated transporter isoform during the differentiation period. On average, the amount of GLUT4 was 16.7-fold greater after than before differentiation. In contrast, the amount of GLUT1 protein decreased during differentiation to almost undetectable levels on day 16. When newly developed adipocytes were maintained in culture for another 14d, the stimulation of glucose uptake and the amount of GLUT4 remained stable.Differentiating human fat cells in primary culture develops an insulin-responsive glucose transport system which exhibits a high stability, thereby providing a valuable model for long-term studies of glucose transport and GLUT4 expression in human adipocytes.

Freshly isolated rat cardiomyocytes, primary cultured cardiomyocytes and the cardiac cell line H9c2 were used to elucidate acute and chronic effects of the sulfonylurea glimepiride on basal and insulin-stimulated glucose uptake and on the expression of the transporter isoforms GLUT1 and GLUT4. Initial rates of 3-O-methylglucose transport in freshly isolated cardiocytes were not acutely affected by glimepiride (100 microM) both in the absence or presence of insulin (10(-7)M). Cells were then transferred to a serum-free culture for 20 h in the presence of glimepiride and a physiological insulin dose. Under these conditions the sulfonyl urea induced a dose-dependent increase in 2-deoxyglucose uptake reaching 186% of control at 10 microM glimepiride, an effect that could also be demonstrated in the absence of insulin during the culture period. Acute stimulation of glucose transport by insulin was additive to the effect of glimepiride and the insulin responsiveness of glucose transport remained unaltered in sulfonylurea treated cultures. Western blot analysis was then performed on crude membrane fractions obtained from primary cultured cardiocytes and H9c2 myotubes (14 days glimepiride). In the former cells the sulfonylurea increased the expression of both GLUT1 and GLUT4 to 164 +/- 21 and 148 +/- 5% of control, respectively. In the myotubes a large, stable increase of GLUT1 could be detected. It is concluded that glimepiride increases cardiac glucose uptake by an insulin-independent pathway most probably involving an increased protein expression of GLUT1 and GLUT4. The increased expression of glucose transporters may have therapeutic impact for the treatment of insulin resistant states and may be beneficial for the diabetic heart.

Ventricular cardiomyocytes isolated from adult rat heart were used to analyze the effect of insulin on the phosphorylation of DNA-binding nuclear proteins and to elucidate the potential involvement of protein phosphatase-1 (PP-1) and PP-2A in this hormonal action. Cells were labelled with [ 33 P]orthophosphate, stimulated with insulin (1.7 × 10 −7 M) and processed for the isolation of nuclei and extraction of DNA-binding proteins. Insulin was found to induce a rapid and constant increase in the serine/threonine phosphorylation of a 38 kDa DNA-binding protein, reaching 150% of control after 15 min and 180% after 150 min. Immunoprecipitation and Western blotting experiments revealed the presence of phosphorylated numatrin in the nuclear extract, however, insulin did not modify its phosphorylation state. Treatment of cardiomyocytes with okadaic acid (1 μM) resulted in a large increase (246 ± 30%) in the phosphorylation of the 38 kDa protein. Using 32 P-labelled phosphorylase as a substrate, we observed a significant inhibition of nuclear PP-1 activity to 38.5 ± 7% ( n = 3) of control after incubation of cardiomyocytes with insulin for 15 min. PP-2A, which corresponds to about 25% of total phosphatase activity, was also inhibited to the same extent. These data show the presence of an insulin-responsive 38 kDa DNA-binding phosphoprotein in the nucleus of cardiomyocytes, which is at least partly regulated by nuclear phosphatase activity. It is suggested that inhibition of nuclear PP-1 and PP-2A represents a possible mechanism of insulin signalling to the nucleus of target cells.

Freshly isolated and primary cultured adult rat cardiomyocytes were used to elucidate the mechanism of action of the new oral antidiabetic agent (+/-)-5-[4-(6-hydroxy-2, 5, 7, 8-tetramethyl-chroman-2-yl-methoxy)benzyl]-2,4-thiazolidinedione (troglitazone) on the heart. Interaction with protein kinase C (PKC) and regulation of glucose transport were evaluated as possible sites of drug action. Acute treatment (30 min) of cardiomyocytes with troglitazone did not affect the phorbolester-induced membrane association of PKC-delta and PKC-epsilon, which represent the major isoforms present in these cells. However, under these conditions the phorbolester-mediated increase in membrane associated PKC activity was inhibited by 43 +/- 4% (n = 4) without affecting the basal distribution of PKC activity. In contrast to these findings, troglitazone had no acute effect on basal or insulin-stimulated glucose transport in freshly isolated cardiomyocytes; even after 120 min treatment an unaltered release of lactate was determine in the presence of the drug. After 20 h in serum-free culture troglitazone induced a dose-dependent increase in 2-deoxyglucose uptake reaching a 40-fold stimulation at 5 mumol/l. This was paralleled by a dose-dependent increase of glucose transporter-1 (GLUT1) and GLUT4 protein expression to 320 +/- 80 and 156 +/- 15% of control, respectively. In addition, chronic exposure to troglitazone increased the GLUT4 abundance in a plasma membrane fraction about twofold. These data show that troglitazone exerts multiple effects on cardiomyocytes involving inhibition of PKC and regulation of glucose transporter expression and distribution. We suggest that an increased glucose supply may be beneficial for the diabetic heart and that modulation of PKC-activity could be relevant for improving insulin action in muscle tissue.

The failure of insulin-regulated recruitment of the GLUT4 glucose transporter in cardiac muscle of obese Zucker rats is associated with alterations of the subcellular distribution of the small-molecular-mass GTP-binding protein rab4A. Here, we show by subcellular fractionation and Western blotting a translocation of the small-molecular-mass GTP-binding protein rab3C from microsomal membranes to plasma membranes in lean control rats following in vivo insulin stimulation. However, in cardiac muscle of obese animals no significant effect of the hormone on the subcellular distribution of rab3C was observed. In GLUT4-enriched membrane vesicles, obtained from cardiac microsomes of the obese group as well as of lean controls, rab3C was not detectable. It is suggested that the altered behaviour of rab3C may contribute to an impaired trafficking of GLUT4 in the insulin-resistant state.

Cardiac ventricular tissue of lean and genetically obese (fa/fa) Zucker rats was used to study the expression, subcellular distribution and insulin-induced recruitment of the glucose transporter GLUT4 and to elucidate possible molecular alterations of the translocation process. Hearts were removed from basal and insulin-treated (20 min) lean and obese Zucker rats, and processed for subcellular fractionation and Western blotting of proteins. In obese rats, the total GLUT4 content in a crude membrane fraction was reduced to 75 +/- 8% (P = 0.019) of lean controls. In contrast, GLUT4 abundance in plasma membranes was not significantly different between lean and obese rats with a concomitant decrease (47 +/- 3%) in the microsomal fraction of obese animals. In plasma membranes of lean animals insulin was found to increase the GLUT4 abundance to 294 +/- 43% of control with a significantly (P = 0.009) reduced effect in the obese group (139 +/- 10% of control). In these animals insulin failed to recruit GLUT4 from the microsomal fraction, whereas the hormone induced a significant decrease (41 +/- 4%) of microsomal GLUT4 in lean controls. In GLUT4-enriched membrane vesicles, obtained from cardiac microsomes of lean rats, a 24 kDa GTP-binding protein could be detected, whereas no significant labelling of this species was observed in GLUT4 vesicles prepared from obese animals. In addition to the translocation of GLUT4, insulin was found to promote the movement of the small GTP-binding protein rab4A from the cytosol (decrease to 61 +/- 13% of control) to the plasma membrane (increase to 177 +/- 19% of control) in lean rats with no effect of the hormone on rab4A redistribution in the obese group. In conclusion, cardiac glucose uptake of insulin-resistant obese Zucker rats is subject to multiple cellular abnormalities involving a reduced expression, altered redistribution and defective recruitment of GLUT4. We show here an association of the latter defect with alterations at the level of small GTP-binding proteins possibly leading to an impaired trafficking of GLUT4 in the insulin-resistant state.