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.

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.

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.

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.

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.

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 stimulates translocation of human GLUT4 to the membrane in fat bodies of transgenic Drosophila melanogaster.

The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in 'diabetic' flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin-signaling pathways in Drosophila, demonstrating the utility of TIRFM of tagged sugar transporters to monitor signaling pathways in insects.

Evidence for the regulatory role of lipocalin 2 in high-fat diet-induced adipose tissue remodeling in male mice.

Lipocalin 2 (Lcn2) has previously been characterized as an adipokine/cytokine playing a role in glucose and lipid homeostasis. In this study, we investigate the role of Lcn2 in adipose tissue remodeling during high-fat diet (HFD)-induced obesity. We find that Lcn2 protein is highly abundant selectively in inguinal adipose tissue. During 16 weeks of HFD feeding, the inguinal fat depot expanded continuously, whereas the expansion of the epididymal fat depot was reduced in both wild-type (WT) and Lcn2(-/-) mice. Interestingly, the depot-specific effect of HFD on fat mass was exacerbated and appeared more pronounced and faster in Lcn2(-/-) mice than in WT mice. In Lcn2(-/-) mice, adipocyte hypertrophy in both inguinal and epididymal adipose tissue was more profoundly induced by age and HFD when compared with WT mice. The expression of peroxisome proliferator-activated receptor-γ protein was significantly down-regulated, whereas the gene expression of extracellular matrix proteins was up-regulated selectively in epididymal adipocytes of Lcn2(-/-) mice. Consistent with these observations, collagen deposition was selectively higher in the epididymal, but not in the inguinal adipose depot of Lcn2(-/-) mice. Administration of the peroxisome proliferator-activated receptor-γ agonist rosiglitazone (Rosi) restored adipogenic gene expression. However, Lcn2 deficiency did not alter the responsiveness of adipose tissue to Rosi effects on the extracellular matrix expression. Rosi treatment led to the further enlargement of adipocytes with improved metabolic activity in Lcn2(-/-) mice, which may be associated with a more pronounced effect of Rosi treatment in reducing TGF-ß in Lcn2(-/-) adipose tissue. Consistent with these in vivo observations, Lcn2 deficiency reduces the adipocyte differentiation capacity of stromal-vascular cells isolated from HFD-fed mice in these cells. Herein Rosi treatment was again able to stimulate adipocyte differentiation to a similar extent in WT and Lcn2(-/-) inguinal and epididymal stromal-vascular cells. Thus, combined, our data indicate that Lcn2 has a depot-specific role in HFD-induced adipose tissue remodeling.

Impaired tethering and fusion of GLUT4 vesicles in insulin-resistant human adipose cells.

Systemic glucose homeostasis is profoundly influenced by adipose cell function. Here we investigated GLUT4 dynamics in living adipose cells from human subjects with varying BMI and insulin sensitivity index (Si) values. Cells were transfected with hemagglutinin (HA)-GLUT4-green fluorescent protein (GFP)/mCherry (red fluorescence), and were imaged live using total internal reflection fluorescence and confocal microscopy. HA-GLUT4-GFP redistribution to the plasma membrane (PM) was quantified by surface-exposed HA epitope. In the basal state, GLUT4 storage vesicle (GSV) trafficking to and fusion with the PM were invariant with donor subject Si, as was total cell-surface GLUT4. In cells from insulin-sensitive subjects, insulin augmented GSV tethering and fusion approximately threefold, resulting in a corresponding increase in total PM GLUT4. However, with decreasing Si, these effects diminished progressively. All insulin-induced effects on GLUT4 redistribution and trafficking correlated strongly with Si and only weakly with BMI. Thus, while basal GLUT4 dynamics and total cell-surface GLUT4 are intact in human adipose cells, independent of donor Si, cells from insulin-resistant donors show markedly impaired GSV tethering and fusion responses to insulin, even after overnight culture. This altered insulin responsiveness is consistent with the hypothesis that adipose cellular dysfunction is a primary contributor to systemic metabolic dysfunction.

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.

Decreased transcription of ChREBP-α/ß isoforms in abdominal subcutaneous adipose tissue of obese adolescents with prediabetes or early type 2 diabetes: associations with insulin resistance and hyperglycemia.

Insulin resistance associated with altered fat partitioning in liver and adipose tissues is a prediabetic condition in obese adolescents. We investigated interactions between glucose tolerance, insulin sensitivity, and the expression of lipogenic genes in abdominal subcutaneous adipose and liver tissue in 53 obese adolescents. Based on their 2-h glucose tests they were stratified in the following groups: group 1, 2-h glucose level <120 mg/dL; group 2, 2-h glucose level between 120 and 140 mg/dL; and group 3, 2-h glucose level >140 mg/dL. Liver and adipose tissue insulin sensitivity were greater in group 1 than in group 2 and group 3, and muscle insulin sensitivity progressively decreased from group 1 to group 3. The expression of the carbohydrate-responsive element-binding protein (ChREBP) was decreased in adipose tissue but increased in the liver (eight subjects) in adolescents with impaired glucose tolerance or type 2 diabetes. The expression of adipose ChREBPα and ChREBPß was inversely related to 2-h glucose level and positively correlated to insulin sensitivity. Improvement of glucose tolerance in four subjects was associated with an increase of ChREBP/GLUT4 expression in the adipose tissue. In conclusion, early in the development of prediabetes/type 2 diabetes in youth, ChREBPß expression in adipose tissue predicts insulin resistance and, therefore, might play a role in the regulation of glucose tolerance.

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.

The size of large adipose cells is a predictor of insulin resistance in first-degree relatives of type 2 diabetic patients.

Early studies reported that the size of adipose cells correlates with insulin resistance. However, a recent study comparing moderately obese, sensitive and resistant subjects, with comparable BMI (~30), did not detect any significant difference in the size of the large cells, but rather a smaller proportion of large cells in the resistant subjects, suggesting impaired adipogenesis. We hypothesize that a decreased proportion, rather than the size, of large adipose cells is also associated with insulin resistance in first-degree relatives of type 2 diabetic patients. Thirty-five leaner (BMI 18-34) subjects who were relatively healthy were recruited. Insulin sensitivity was measured by the euglycemic, hyperinsulinemic clamp. Needle biopsies of abdominal subcutaneous fat were assayed for adipose cell size by fitting the cell size distribution with two exponentials and a Gaussian function. The fraction of large cells was defined as the area of the Gaussian peak and the size of the large cells was defined as its center (c(p)). Glucose infusion rate (GIR) and c(p) were negatively correlated, but insulin sensitivity and the proportion of large cells were not correlated. BMI and c(p) were also strongly correlated, but a relationship of modest correlation between the cell size and insulin resistance was still significant after correcting for BMI. In contrast to moderately obese subjects, in the first-degree relatives of type 2 diabetic patients both BMI and the size of the large adipose cells predict the degree of insulin resistance; no correlation is found between the proportion of large adipose cells and insulin resistance.

Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.

Although erythropoietin (Epo) is the cytokine known to regulate erythropoiesis, erythropoietin receptor (EpoR) expression and associated activity beyond haematopoietic tissue remain uncertain. Here we show that mice with EpoR expression restricted to haematopoietic tissues (Tg) develop obesity and insulin resistance. Tg-mice exhibit a decrease in energy expenditure and an increase in white fat mass and adipocyte number. Conversely, Epo treatment of wild-type (WT)-mice increases energy expenditure and reduces food intake and fat mass accumulation but shows no effect in body weight of Tg-mice. EpoR is expressed at a high level in white adipose tissue and in the proopiomelanocortin (POMC) neurons of the hypothalamus. Although Epo treatment in WT-mice induces the expression of the polypeptide hormone precursor, POMC, mice lacking EpoR show reduced levels of POMC in the hypothalamus. This study provides the first evidence that mice lacking EpoR in non-haematopoietic tissue become obese and insulin resistant with loss of Epo regulation of energy homeostasis.

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.