BMI and waist circumference cut-offs for corresponding levels of insulin sensitivity in a Middle Eastern immigrant versus a native Swedish population - the MEDIM population based study.

BACKGROUND: The aim of this study was to identify corresponding body mass index (BMI) and waist circumference cut-offs for equivalent levels of insulin sensitivity in a Middle Eastern immigrant population compared with native Swedes. METHODS: Citizens of Malmö, Sweden aged 30 to 75 years, who were born in Iraq or Sweden, were in 2010-2012 invited to participate in a health examination including anthropometrics, oral glucose tolerance test, fasting samples and interviews concerning sociodemographic factors and lifestyle behaviours. RESULTS: In total, 1176 individuals born in Iraq and 688 born in Sweden, without previously diagnosed type 2 diabetes, participated in the study. In normal weight participants (BMI < 25 kg/m2), 21.2% of Iraqis vs 9.3% of Swedes were insulin resistant. Corresponding figures in participants without abdominal obesity (waist circumference, men < 94 cm, women < 80 cm) were 28.2% of Iraqis vs 9.4% of Swedes. The age-adjusted insulin sensitivity index (ISI) for obese Swedes (BMI 30 kg/m2) corresponded in Iraqi men with BMI of 28.5 kg/m2, and in Iraqi women with BMI of 27.5 kg/m2. The ISI level in abdominally obese Swedes corresponded with waist circumference cut-offs of 84.0 cm and 71.0 cm in Iraqi men and women, respectively. In men only, larger waist circumference (P interaction = 0.026) presented a stronger association with impaired ISI in Iraqis as compared to Swedes. CONCLUSIONS: Our data shows that the impact of BMI and waist circumference on ISI is ethnic- and gender-specific, indicating a disturbed fat metabolism in Iraqi males in particular. Our data suggests that 10 cm lower cut-off values for abdominal obesity, than is currently recommended by major organisations, should be considered when estimating diabetes risk in Middle Eastern populations.

In vivo 2H2O administration reveals impaired triglyceride storage in adipose tissue of insulin-resistant humans.

Indirect evidence suggests that impaired triglyceride storage in the subcutaneous fat depot contributes to the development of insulin resistance via lipotoxicity. We directly tested this hypothesis by measuring, in vivo, TG synthesis, de novo lipogenesis (DNL), adipocyte proliferation, and insulin suppression of lipolysis in subcutaneous adipose tissue of BMI-matched individuals classified as insulin resistant (IR) or insulin sensitive (IS). Nondiabetic, moderately obese subjects with BMI 25-35 kg/m(2), classified as IR or IS by the modified insulin suppression test, consumed deuterated water ((2)H2O) for 4 weeks. Deuterium incorporation into glycerol, palmitate, and DNA indicated TG synthesis, DNL, and adipocyte proliferation, respectively. Net TG synthesis and DNL in adipose cells were significantly lower in IR as compared with IS subjects, whereas adipocyte proliferation did not differ significantly. Plasma FFAs measured during an insulin suppression test were 2.5-fold higher in IR subjects, indicating resistance to insulin suppression of lipolysis. Adipose TG synthesis correlated directly with DNL but not with proliferation. These results provide direct in vivo evidence for impaired TG storage in subcutaneous adipose tissue of IR as compared with IS. Relative inability to store TG in the subcutaneous depot may represent a mechanism contributing to the development of insulin resistance in the setting of obesity.

Single injections of apoA-I acutely improve in vivo glucose tolerance in insulin-resistant mice.

AIMS/HYPOTHESIS: Apolipoprotein A-I (apoA-I), the main protein constituent of HDL, has a central role in the reverse cholesterol-transport pathway, which together with the anti-inflammatory properties of apoA-I/HDL provide cardioprotection. Recent findings of direct stimulation of glucose uptake in muscle by apoA-I/HDL suggest that altered apoA-I and HDL functionality may be a contributing factor to the development of diabetes. We have studied the in vivo effects of short treatments with human apoA-I in a high-fat diet fed mouse model. In addition to native apoA-I, we investigated the effects of the cardioprotective Milano variant (Arg173Cys). METHODS: Male C57Bl6 mice on a high-fat diet for 2 weeks that received a single injection of human apoA-I proteins (wild-type and Milano) were analysed for blood glucose and insulin levels during a 3 h incubation followed by glucose tolerance tests. Incorporation of injected human apoA-I protein into HDLs was analysed by native gel electrophoresis. RESULTS: ApoA-I treatment significantly improved insulin secretion and blood glucose clearance in the glucose tolerance test, with an efficiency exceeding that of lean control animals, and led to decreased basal glucose during the 3 h incubation. Notably, the two apoA-I variants triggered insulin secretion and glucose clearance to the same extent. CONCLUSIONS/INTERPRETATION: ApoA-I treatment leads to insulin- and non-insulin-dependent effects on glucose homeostasis. The experimental model of short-term (2 weeks) feeding of a high-fat diet to C57Bl6 mice provides a suitable and time-efficient system to unravel the resulting tissue-specific mechanisms of acute apoA-I treatment that lead to improved glucose homeostasis.

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice.

Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and T-tubules and analyzed insulin-stimulated exocytosis at the single vesicle level by total internal reflection fluorescence and confocal microscopy. We found that only 10% of the intracellular GLUT4 pool comprised mobile vesicles, whereas most of the GLUT4 structures remained stationary or tethered at the sarcolemma or T-tubules. In fact, most of the insulin-stimulated exocytosis emanated from pretethered vesicles, whereas the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action but rather locally distributed. Most likely, pretethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis.

A common variant in the patatin-like phospholipase 3 gene (PNPLA3) is associated with fatty liver disease in obese children and adolescents.

UNLABELLED: The genetic factors associated with susceptibility to nonalcoholic fatty liver disease (NAFLD) in pediatric obesity remain largely unknown. Recently, a nonsynonymous single-nucleotide polymorphism (rs738409), in the patatin-like phospholipase 3 gene (PNPLA3) has been associated with hepatic steatosis in adults. In a multiethnic group of 85 obese youths, we genotyped the PNLPA3 single-nucleotide polymorphism, measured hepatic fat content by magnetic resonance imaging and insulin sensitivity by the insulin clamp. Because PNPLA3 might affect adipogenesis/lipogenesis, we explored the putative association with the distribution of adipose cell size and the expression of some adipogenic/lipogenic genes in a subset of subjects who underwent a subcutaneous fat biopsy. Steatosis was present in 41% of Caucasians, 23% of African Americans, and 66% of Hispanics. The frequency of PNPLA3(rs738409) G allele was 0.324 in Caucasians, 0.183 in African Americans, and 0.483 in Hispanics. The prevalence of the G allele was higher in subjects showing hepatic steatosis. Surprisingly, subjects carrying the G allele showed comparable hepatic glucose production rates, peripheral glucose disposal rate, and glycerol turnover as the CC homozygotes. Carriers of the G allele showed smaller adipocytes than those with CC genotype (P = 0.005). Although the expression of PNPLA3, PNPLA2, PPARγ2(peroxisome proliferator-activated receptor gamma 2), SREBP1c(sterol regulatory element binding protein 1c), and ACACA(acetyl coenzyme A carboxylase) was not different between genotypes, carriers of the G allele showed lower leptin (LEP)(P = 0.03) and sirtuin 1 (SIRT1) expression (P = 0.04). CONCLUSION: A common variant of the PNPLA3 gene confers susceptibility to hepatic steatosis in obese youths without increasing the level of hepatic and peripheral insulin resistance. The rs738409 PNPLA3 G allele is associated with morphological changes in adipocyte cell size.

Using pre-existing microarray datasets to increase experimental power: application to insulin resistance.

Although they have become a widely used experimental technique for identifying differentially expressed (DE) genes, DNA microarrays are notorious for generating noisy data. A common strategy for mitigating the effects of noise is to perform many experimental replicates. This approach is often costly and sometimes impossible given limited resources; thus, analytical methods are needed which increase accuracy at no additional cost. One inexpensive source of microarray replicates comes from prior work: to date, data from hundreds of thousands of microarray experiments are in the public domain. Although these data assay a wide range of conditions, they cannot be used directly to inform any particular experiment and are thus ignored by most DE gene methods. We present the SVD Augmented Gene expression Analysis Tool (SAGAT), a mathematically principled, data-driven approach for identifying DE genes. SAGAT increases the power of a microarray experiment by using observed coexpression relationships from publicly available microarray datasets to reduce uncertainty in individual genes' expression measurements. We tested the method on three well-replicated human microarray datasets and demonstrate that use of SAGAT increased effective sample sizes by as many as 2.72 arrays. We applied SAGAT to unpublished data from a microarray study investigating transcriptional responses to insulin resistance, resulting in a 50% increase in the number of significant genes detected. We evaluated 11 (58%) of these genes experimentally using qPCR, confirming the directions of expression change for all 11 and statistical significance for three. Use of SAGAT revealed coherent biological changes in three pathways: inflammation, differentiation, and fatty acid synthesis, furthering our molecular understanding of a type 2 diabetes risk factor. We envision SAGAT as a means to maximize the potential for biological discovery from subtle transcriptional responses, and we provide it as a freely available software package that is immediately applicable to any human microarray study.

Hypertrophy-driven adipocyte death overwhelms recruitment under prolonged weight gain.

Fat pads dynamically regulate energy storage capacity under energy excess and deficit. This remodeling process is not completely understood, with controversies regarding differences between fat depots and plasticity of adipose cell number. We examined changes of mouse adipose cell-size distributions in epididymal, inguinal, retroperitoneal, and mesenteric fat under both weight gain and loss. With mathematical modeling, we specifically analyzed the recruitment, growth/shrinkage, and loss of adipose cells, including the size dependence of these processes. We found a qualitatively universal adipose tissue remodeling process in all four fat depots: 1), There is continuous recruitment of new cells under weight gain; 2), the growth and shrinkage of larger cells (diameter >50 µm) is proportional to cell surface area; and 3), cell loss occurs under prolonged weight gain, with larger cells more susceptible. The mathematical model gives a predictive integrative picture of adipose tissue remodeling in obesity.

Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.

Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

Hypertrophy and/or Hyperplasia: Dynamics of Adipose Tissue Growth.

Adipose tissue grows by two mechanisms: hyperplasia (cell number increase) and hypertrophy (cell size increase). Genetics and diet affect the relative contributions of these two mechanisms to the growth of adipose tissue in obesity. In this study, the size distributions of epididymal adipose cells from two mouse strains, obesity-resistant FVB/N and obesity-prone C57BL/6, were measured after 2, 4, and 12 weeks under regular and high-fat feeding conditions. The total cell number in the epididymal fat pad was estimated from the fat pad mass and the normalized cell-size distribution. The cell number and volume-weighted mean cell size increase as a function of fat pad mass. To address adipose tissue growth precisely, we developed a mathematical model describing the evolution of the adipose cell-size distributions as a function of the increasing fat pad mass, instead of the increasing chronological time. Our model describes the recruitment of new adipose cells and their subsequent development in different strains, and with different diet regimens, with common mechanisms, but with diet- and genetics-dependent model parameters. Compared to the FVB/N strain, the C57BL/6 strain has greater recruitment of small adipose cells. Hyperplasia is enhanced by high-fat diet in a strain-dependent way, suggesting a synergistic interaction between genetics and diet. Moreover, high-fat feeding increases the rate of adipose cell size growth, independent of strain, reflecting the increase in calories requiring storage. Additionally, high-fat diet leads to a dramatic spreading of the size distribution of adipose cells in both strains; this implies an increase in size fluctuations of adipose cells through lipid turnover.

Intact glucose uptake despite deteriorating signaling in adipocytes with high-fat feeding.

To capture immediate cellular changes during diet-induced expansion of adipocyte cell volume and number, we characterized mature adipocytes during a short-term high-fat diet (HFD) intervention. Male C57BL6/J mice were fed chow diet, and then switched to HFD for 2, 4, 6 or 14 days. Systemic glucose clearance was assessed by glucose tolerance test. Adipose tissue was dissected for RNA-seq and cell size distribution analysis using coulter counting. Insulin response in isolated adipocytes was monitored by glucose uptake assay and Western blotting, and confocal microscopy was used to assess autophagic activity. Switching to HFD was accompanied by an immediate adipocyte size expansion and onset of systemic insulin resistance already after two days, followed by recruitment of new adipocytes. Despite an initially increased non-stimulated and preserved insulin-stimulated glucose uptake, we observed a decreased phosphorylation of insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB). After 14 days of HFD, both the insulin-stimulated phosphorylation of Akt substrate of 160 kDa (AS160) and glucose uptake was blunted. RNA-seq analysis of adipose tissue revealed transient changes in gene expression at day four, including highly significant upregulation of Trp53inp, previously demonstrated to be involved in autophagy. We confirmed increased autophagy, measured as an increased density of LC3-positive puncta and decreased p62 expression after 14 days of HFD. In conclusion, HFD rapidly induced systemic insulin resistance, whereas insulin-stimulated glucose uptake remained intact throughout 6 days of HFD feeding. We also identified autophagy as an early cellular process that potentially influences adipocyte function upon switching to HFD.

Exercise differentially affects metabolic functions and white adipose tissue in female letrozole- and dihydrotestosterone-induced mouse models of polycystic ovary syndrome.

Here we hypothesized that exercise in dihydrotestosterone (DHT) or letrozole (LET)-induced polycystic ovary syndrome mouse models improves impaired insulin and glucose metabolism, adipose tissue morphology, and expression of genes related to adipogenesis, lipid metabolism, Notch pathway and browning in inguinal and mesenteric fat. DHT-exposed mice had increased body weight, increased number of large mesenteric adipocytes. LET-exposed mice displayed increased body weight and fat mass, decreased insulin sensitivity, increased frequency of small adipocytes and increased expression of genes related to lipolysis in mesenteric fat. In both models, exercise decreased fat mass and inguinal and mesenteric adipose tissue expression of Notch pathway genes, and restored altered mesenteric adipocytes morphology. In conclusion, exercise restored mesenteric adipocytes morphology in DHT- and LET-exposed mice, and insulin sensitivity and mesenteric expression of lipolysis-related genes in LET-exposed mice. Benefits could be explained by downregulation of Notch, and modulation of browning and lipolysis pathways in the adipose tissue.

Adipose tissue macrophages impair preadipocyte differentiation in humans.

AIM: The physiologic mechanisms underlying the relationship between obesity and insulin resistance are not fully understood. Impaired adipocyte differentiation and localized inflammation characterize adipose tissue from obese, insulin-resistant humans. The directionality of this relationship is not known, however. The aim of the current study was to investigate whether adipose tissue inflammation is causally-related to impaired adipocyte differentiation. METHODS: Abdominal subcutaneous(SAT) and visceral(VAT) adipose tissue was obtained from 20 human participants undergoing bariatric surgery. Preadipocytes were isolated, and cultured in the presence or absence of CD14+ macrophages obtained from the same adipose tissue sample. Adipocyte differentiation was quantified after 14 days via immunofluorescence, Oil-Red O, and adipogenic gene expression. Cytokine secretion by mature adipocytes cultured with or without CD14+macrophages was quantified. RESULTS: Adipocyte differentiation was significantly lower in VAT than SAT by all measures (p<0.001). With macrophage removal, SAT preadipocyte differentiation increased significantly as measured by immunofluorescence and gene expression, whereas VAT preadipocyte differentiation was unchanged. Adipocyte-secreted proinflammatory cytokines were higher and adiponectin lower in media from VAT vs SAT: macrophage removal reduced inflammatory cytokine and increased adiponectin secretion from both SAT and VAT adipocytes. Differentiation of preadipocytes from SAT but not VAT correlated inversely with systemic insulin resistance. CONCLUSIONS: The current results reveal that proinflammatory immune cells in human SAT are causally-related to impaired preadipocyte differentiation, which in turn is associated with systemic insulin resistance. In VAT, preadipocyte differentiation is poor even in the absence of tissue macrophages, pointing to inherent differences in fat storage potential between the two depots.

Adipose cell hypertrophy precedes the appearance of small adipocytes by 3 days in C57BL/6 mouse upon changing to a high fat diet.

Adipose tissue is the energy buffer in mammals. The cellularity of adipose tissue has a major role in determining the response of adipose tissue to insulin action. A reduction in the ability of adipose tissue to store ingested caloric excess can lead to dyslipidemia and lipotoxicity, impacting insulin action systemically. The dynamic response of adipose tissue to changes in diet is therefore a crucial aspect of metabolism, and has attracted attention in the context of the ongoing worldwide increase in overweight and obesity and resulting metabolic syndrome dysfunctions. We investigated in a mouse model if there is a specific delay between an increase in caloric intake and the recruitment of new adipocytes, and if there are other changes in adipose tissue dynamics concomitant with such a diet change. By developing a dynamic mathematical model, we found that there is a delay of 3 days between the start of a high fat diet and the recruitment of new adipocytes, and that the rate of fat mass increase modulates lipid turnover and adipose cell hypertrophy.

Dual Actions of Apolipoprotein A-I on Glucose-Stimulated Insulin Secretion and Insulin-Independent Peripheral Tissue Glucose Uptake Lead to Increased Heart and Skeletal Muscle Glucose Disposal.

Apolipoprotein A-I (apoA-I) of HDL is central to the transport of cholesterol in circulation. ApoA-I also provides glucose control with described in vitro effects of apoA-I on ß-cell insulin secretion and muscle glucose uptake. In addition, apoA-I injections in insulin-resistant diet-induced obese (DIO) mice lead to increased glucose-stimulated insulin secretion (GSIS) and peripheral tissue glucose uptake. However, the relative contribution of apoA-I as an enhancer of GSIS in vivo and as a direct stimulator of insulin-independent glucose uptake is not known. Here, DIO mice with instant and transient blockade of insulin secretion were used in glucose tolerance tests and in positron emission tomography analyses. Data demonstrate that apoA-I to an equal extent enhances GSIS and acts as peripheral tissue activator of insulin-independent glucose uptake and verify skeletal muscle as an apoA-I target tissue. Intriguingly, our analyses also identify the heart as an important target tissue for the apoA-I-stimulated glucose uptake, with potential implications in diabetic cardiomyopathy. Explorations of apoA-I as a novel antidiabetic drug should extend to treatments of diabetic cardiomyopathy and other cardiovascular diseases in patients with diabetes.

Adipose Cell Size and Regional Fat Deposition as Predictors of Metabolic Response to Overfeeding in Insulin-Resistant and Insulin-Sensitive Humans.

Obesity is associated with insulin resistance, but significant variability exists between similarly obese individuals, pointing to qualitative characteristics of body fat as potential mediators. To test the hypothesis that obese, insulin-sensitive (IS) individuals possess adaptive adipose cell/tissue responses, we measured subcutaneous adipose cell size, insulin suppression of lipolysis, and regional fat responses to short-term overfeeding in BMI-matched overweight/obese individuals classified as IS or insulin resistant (IR). At baseline, IR subjects exhibited significantly greater visceral adipose tissue (VAT), intrahepatic lipid (IHL), plasma free fatty acids, adipose cell diameter, and percentage of small adipose cells. With weight gain (3.1 ± 1.4 kg), IR subjects demonstrated no significant change in adipose cell size, VAT, or insulin suppression of lipolysis and only 8% worsening of insulin-mediated glucose uptake (IMGU). Alternatively, IS subjects demonstrated significant adipose cell enlargement; decrease in the percentage of small adipose cells; increase in VAT, IHL, and lipolysis; 45% worsening of IMGU; and decreased expression of lipid metabolism genes. Smaller baseline adipose cell size and greater enlargement with weight gain predicted decline in IMGU, as did increase in IHL and VAT and decrease in insulin suppression of lipolysis. Weight gain in IS humans causes maladaptive changes in adipose cells, regional fat distribution, and insulin resistance. The correlation between development of insulin resistance and changes in adipose cell size, VAT, IHL, and insulin suppression of lipolysis highlight these factors as potential mediators between obesity and insulin resistance.

Endofacial competitive inhibition of glucose transporter-4 intrinsic activity by the mitogen-activated protein kinase inhibitor SB203580.

The translocation of glucose transporter-4 (GLUT4) to the cell surface is a complex multistep process that involves movement of GLUT4 vesicles from a reservoir compartment, and docking and fusion of the vesicles with the plasma membrane. It has recently been proposed that a p38 mitogen-activated protein kinase (MAPK)-dependent step may lead to intrinsic activation of the transporters exposed at the cell surface. In contrast to data obtained in muscle and adipocyte cell lines, we found that no insulin activation of p38 MAPK occurred in rat adipose cells. However, the p38 MAPK inhibitor SB203580 consistently inhibited transport activity after preincubation with the adipose cells. These apparently contradictory findings led us to hypothesize that the inhibitor may have a direct effect on the transport catalytic activity of GLUT4 that was independent of inhibition of the kinase. Kinetic analysis of 3-O-methyl-d-glucose transport activity revealed that SB203580 was a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but was a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport. This pattern of inhibition of GLUT4 was also observed with cytochalasin B. The pattern of inhibition is consistent with interaction at the endofacial surface, but not the exofacial surface of the transporter. Occupation of the endofacial substrate site reduces maximum velocity under zero-trans conditions, because return of the substrate site to the outside is blocked, and no substrate is present inside to displace the inhibitor. Under equilibrium-exchange conditions, internal substrate competitively displaces the inhibitor, and the transport K(m) is increased.

7-hydroxystaurosporine (UCN-01) inhibition of Akt Thr308 but not Ser473 phosphorylation: a basis for decreased insulin-stimulated glucose transport.

7-hydroxystaurosporine (UCN-01) infused for 72 hours by continuous i.v. infusion induced insulin resistance during phase I clinical trials. To understand the mechanism for this observation, we examined the effect of UCN-01 on insulin-stimulated glucose transport activity with 3-O-methylglucose in isolated rat adipose cells. UCN-01 inhibits glucose transport activity in a dose-dependent manner at all insulin concentrations. At the clinically relevant concentration of 0.25 mumol/L UCN-01, glucose transport is inhibited 66, 29, and 26% at insulin concentrations of 10, 50, and 100,000 (100K) microunits/mL respectively, thus shifting the dose-response curve to the right. Increasing concentrations of UCN-01 up to 2.5 mumol/L progressively shift the insulin dose-response curve even further. As Akt is known to mediate in part action initiated at the insulin receptor, we also studied the effect of UCN-01 on Akt activation in whole-cell homogenates of these cells. Decreased glucose transport activity directly parallels decreased Akt Thr308 phosphorylation in both an insulin and UCN-01 dose-dependent manner, whereas Akt Ser473 phosphorylation is inhibited only at the lowest insulin concentration, and then, only modestly. UCN-01 also inhibits insulin-induced Thr308 but not Ser473 phosphorylation of Akt associated with the plasma membranes and low-density microsomes and inhibits translocation of GLUT4 from low-density microsomes to plasma membranes as expected from the glucose transport activity measurements. These data suggest that UCN-01 induces clinical insulin resistance by blocking Akt activation and subsequent GLUT4 translocation in response to insulin, and this effect appears to occur by inhibiting Thr308 phosphorylation even in the face of almost completely unaffected Ser473 phosphorylation.

Human adipose cells in vitro are either refractory or responsive to insulin, reflecting host metabolic state.

While intercellular communication processes are frequently characterized by switch-like transitions, the endocrine system, including the adipose tissue response to insulin, has been characterized by graded responses. Yet here individual cells from adipose tissue biopsies are best described by a switch-like transition between the basal and insulin-stimulated states for the trafficking of the glucose transporter GLUT4. Two statistically-defined populations best describe the observed cellular heterogeneity, representing the fractions of refractive and responsive adipose cells. Furthermore, subjects exhibiting high systemic insulin sensitivity indices (SI) have high fractions of responsive adipose cells in vitro, while subjects exhibiting decreasing SI have increasing fractions of refractory cells in vitro. Thus, a two-component model best describes the relationship between cellular refractory fraction and subject SI. Since isolated cells exhibit these different response characteristics in the presence of constant culture conditions and milieu, we suggest that a physiological switching mechanism at the adipose cellular level ultimately drives systemic SI.

Amelioration of insulin resistance by rosiglitazone is associated with increased adipose cell size in obese type 2 diabetic patients.

Early studies reported that the size of adipose cells positively correlates with insulin resistance, but recent evidence suggests that the relationship between adipose cell size and insulin resistance is more complex. We previously reported that among BMI-matched moderately obese subjects who were either insulin sensitive or resistant insulin resistance correlated with the proportion of small adipose cells, rather than the size of the large adipose cells, whereas the size of large adipose cells was found to be a predictor of insulin resistance in the first-degree relatives of type 2 diabetic (T2D) patients. The relationship between adipose cellularity and insulin resistance thus appears to depend on the metabolic state of the individual. We did a longitudinal study with T2D patients treated with the insulin-sensitizer rosiglitazone to test the hypothesis that improved insulin sensitivity is associated with increased adipocyte size. Eleven T2D patients were recruited and treated with rosiglitazone for 90 days. Blood samples and needle biopsies of abdominal subcutaneous fat were taken at six time points and analyzed for cell size distributions. Rosiglitazone treatment ameliorated insulin resistance as evidenced by significantly decreased fasting plasma glucose and increased index of insulin sensitivity, QUICKI. In association with this, we found significantly increased size of the large adipose cells and, with a weaker effect, increased proportion of small adipose cells. We conclude rosiglitazone treatment both enlarges existing large adipose cells and recruits new small adipose cells in T2D patients, improving fat storage capacity in adipose tissue and thus systemic insulin sensitivity.

Insulin regulates Glut4 confinement in plasma membrane clusters in adipose cells.

Insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) is the hallmark of glucose metabolism. In this study we examined insulin's effects on GLUT4 organization in PM of adipose cells by direct microscopic observation of single monomers tagged with photoswitchable fluorescent protein. In the basal state, after exocytotic delivery only a fraction of GLUT4 is dispersed into the PM as monomers, while most of the GLUT4 stays at the site of fusion and forms elongated clusters (60-240 nm). GLUT4 monomers outside clusters diffuse freely and do not aggregate with other monomers. In contrast, GLUT4 molecule collision with an existing cluster can lead to immediate confinement and association with that cluster. Insulin has three effects: it shifts the fraction of dispersed GLUT4 upon delivery, it augments the dissociation of GLUT4 monomers from clusters ∼3-fold and it decreases the rate of endocytic uptake. All together these three effects of insulin shift most of the PM GLUT4 from clustered to dispersed states. GLUT4 confinement in clusters represents a novel kinetic mechanism for insulin regulation of glucose homeostasis.