Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle

Inducible nitric oxide synthase (iNOS) is induced by inflammatory cytokines in skeletal muscle and fat. It has been proposed that chronic iNOS induction may cause muscle insulin resistance. Here we show that iNOS expression is increased in muscle and fat of genetic and dietary models of obesity. Moreover, mice in which the gene encoding iNOS was disrupted (Nos2-/- mice) are protected from high-fat-induced insulin resistance. Whereas both wild-type and Nos2-/- mice developed obesity on the high-fat diet, obese Nos2-/- mice exhibited improved glucose tolerance, normal insulin sensitivity in vivo and normal insulin-stimulated glucose uptake in muscles. iNOS induction in obese wild-type mice was associated with impairments in phosphatidylinositol 3-kinase and Akt activation by insulin in muscle. These defects were fully prevented in obese Nos2-/- mice. These findings provide genetic evidence that iNOS is involved in the development of muscle insulin resistance in diet-induced obesity.     

The adipogenic function and other biological effects of insulin

Studies on experimental animals with knockout of the insulin receptor gene (Insr) in the whole body or in certain tissues and/or related genes encoding proteins involved in realization of insulin signal transduction in target cells, have made an important contribution to the elucidation of insulin regulation of metabolism, particularly fat metabolism. Since the whole insulin secreted by β-cells, together with the products of gastrointestinal tract digestion of proteins, fats, and carbohydrates reaches in the liver, the latter is the first organ on which this hormone acts. The liver employs released amino acids for synthesis of proteins, including apo-proteins for various lipoproteins. Glucose is used for synthesis of glycogen, fatty acids, and triglycerides, which enter all the organs in very low density lipoproteins (VLDL). The LIRKO mice with knockout of the insr gene in the liver demonstrated inhibition of synthesis of macromolecular compounds from amino acids, glucose, and fatty acids. Low molecular weight substances demonstrated increased entry to circulation, and together with other disorders induced hyperglycemia. In LIRKO mice blood glucose levels and glucose tolerance demonstrated time-dependent normalization and at later stages the increase in glucose levels was replaced by hypoglycemia. These changes can be well explained if we take into consideration that one of the main functions of insulin consists in stimulation of energy accumulation by means of activation of triglyceride deposition in adipose tissue. FIRKO mice with selective knockout of adipose tissue Insr were characterized by decreased uptake of glucose in adipocytes, and its transformation into lipids. However, the level of body fat in animals remained normal, possibly due to preserved insulin receptor in the liver and insulin-induced activation of triglyceride production which maintained normal levels of body fat stores, the effective functioning of adipose tissue and secretion of leptin by adipocytes during inhibition of glucose transformation into triglyceride in adipose tissue. Knockout of the Insr gene in muscles blocked glucose uptake by myocytes, but it did not induce hyperglycemia, probably due to the increase in glucose uptake by other organs, which retained the insulin receptor, and induced some increase in fat resources in adipose tissue. Similar results were obtained in mice with knockout the glucose transporter 4 GLUT4 in muscle and/or adipose tissue. Insulin microinjections in the brain, in the cerebral ventricle 4 (CVI) and mediobasal hypothalamus (MBH) did not affect the insulin levels in the general circulation, but effectively activate lipogenesis and inhibited lipolysis in adipose tissue. They induced obesity, similar to conventional obesity when the insulin levels increased. These results may serve as an additional confirmation of the importance of the adipogenic insulin function in mechanisms of regulation of general metabolism.     

Subcutaneous fat modulates insulin sensitivity in mice by regulating TNF-alpha expression in visceral fat.

The distribution of fat in obese persons is related to the risk of developing various metabolic disorders, such as glucose intolerance, dyslipidemia and hypertension, and the combination of these conditions is known as the metabolic syndrome. The aim of this study was to investigate the role of subcutaneous fat in regulating insulin resistance and its influence on TNF-alpha expression in visceral fat, by using mice that were subjected to subcutaneous lipectomy with or without subsequent fat transplantation. After partial subcutaneous lipectomy, mice showed significantly greater accumulation of visceral fat compared with sham-operated control mice. Lipectomy led to higher plasma insulin and lower plasma glucose levels after loading with glucose and insulin, respectively, compared with the levels in control mice. Insulin-induced phosphorylation of IRS-1 was decreased in the skeletal muscles of lipectomized mice. Subcutaneous transplantation of fat pads into lipectomized mice reversed the above-mentioned changes indicating insulin resistance in these animals. The fat storage area of adipocytes and TNF- alpha expression by adipocytes in visceral fat were significantly higher in the lipectomized mice than in controls, while subcutaneous transplantation of fat reduced both the fat storage area and TNF-alpha expression. The insulin resistance of lipectomized mice was also ameliorated by systemic neutralization of TNF-alpha activity using a specific antibody. These findings obtained in mice subjected to subcutaneous lipectomy with/without subsequent fat transplantation indicate that subcutaneous fat regulates systemic insulin sensitivity, possibly through altering fat storage and the expression of TNF-alpha by adipocytes in visceral fat. The balance between accumulation of subcutaneous fat and visceral fat may be important with respect to the occurrence of systemic insulin resistance in the metabolic syndrome.     

Differential gene expression between visceral and subcutaneous fat depots.

Abdominal obesity has been linked to the development of insulin resistance and Type 2 diabetes mellitus (DM2). By surgical removal of visceral fat (VF) in a variety of rodent models, we prevented insulin resistance and glucose intolerance, establishing a cause-effect relationship between VF and the metabolic syndrome. To characterize the biological differences between visceral and peripheral fat depots, we obtained perirenal visceral (VF) and subcutaneous (SC) fat from 5 young rats. We extracted mRNA from the fat tissue and performed gene array hybridization using Affymetrix technology with a platform containing 9 000 genes. Out of the 1 660 genes that were expressed in fat tissue, 297 (17.9 %) genes show a two-fold or higher difference in their expression between the two tissues. We present the 20 genes whose expression is higher in VF fat (by 3 - 7 fold) and the 20 genes whose expression is higher in SC fat (by 3 - 150 fold), many of which are predominantly involved in glucose homeostasis, insulin action, and lipid metabolism. We confirmed the findings of gene array expression and quantified the changes in expression in VF of genes involved in insulin resistance (PPARgamma leptin) and its syndrome (angiotensinogen and plasminogen activating inhibitor-1, PAI-1) by real-time PCR (qRT-PCR) technology. Finally, we demonstrated increased expression of resistin in VF by around 12-fold and adiponectin by around 4-fold, peptides that were not part of the gene expression platform. These results indicate that visceral fat and subcutaneous fat are biologically distinct.     

Increased insulin mediated glucose metabolism in fat cells from I versus C57BL mice

The purpose of this study was to determine whether adipocytes from I strain mice, which are characterized by a greater in vivo glucose tolerance than most other strains, had a higher capacity to utilize glucose in response to physiological concentrations of insulin. Using C57BL mice as a control strain, we examined the effect of insulin on glucose metabolism in epididymal and inguinal adipocytes from 2-month-old male mice. Body weight was only slightly less (7%) for the I mice than for the C57BL mice, but fat pad sizes were 60 and 20% less for epididymal and inguinal depots, respectively, in the I mice. Fat cell size was also smaller in epididymal adipocytes from the I mice than from the C57BL mice. Fat cell size of inguinal adipocytes was similar in the two strains. Without insulin the rates of [U-14C]glucose incorporation into CO2 or lipids were twofold higher in cells from the I mice than in those from the C57BL mice. Maximal insulin concentration (2.5 nM) increased glucose metabolism by 140 and 500% in epididymal and inguinal adipose cells, respectively, in the I mice versus 30 and 50% in the C57BL mice. The maximal effect of insulin was reached at a much higher insulin concentration in the I mice than in the C57BL mice. The activity of fatty acid synthetase was four- to sixfold higher in fat cells from I than in those from C57BL mice. These results demonstrate an increased insulin responsiveness of glucose metabolism in fat cells from the I mice related to an increased lipogenic capacity. Furthermore, they show that adipose tissue in mice exhibits significant regional differences in terms of insulin responsiveness of glucose metabolism.     

Effects of the presence, absence, and overexpression of uncoupling protein-3 on adiposity and fuel metabolism in congenic mice

Uncoupling protein-3 (UCP3) is a poorly understood mitochondrial inner membrane protein expressed predominantly in skeletal muscle. The aim of this study was to examine the effects of the absence or constitutive physiological overexpression of UCP3 on whole body energy metabolism, glucose tolerance, and muscle triglyceride content. Congenic male UCP3 knockout mice (Ucp3-/-), wild-type, and transgenic UCP3 overexpressing (UCP3Tg) mice were fed a 10% fat diet for 4 or 8 mo after they were weaned. UCP3Tg mice had lower body weights and were less metabolically efficient than wild-type or Ucp3-/- mice, but they were not hyperphagic. UCP3Tg mice had smaller epididymal white adipose tissue and brown adipose tissue (BAT) depots; however, there were no differences in muscle weights. Glucose and insulin tolerance tests revealed that both UCP3Tg and Ucp3-/- mice were protected from development of impaired glucose tolerance and were more sensitive to insulin. 2-Deoxy-D-[1-3H]glucose tracer studies showed increased uptake of glucose into BAT and increased storage of liver glycogen in Ucp3-/- mice. Assessments of intramuscular triglyceride (IMTG) revealed decreases in quadriceps of UCP3Tg mice compared with wild-type and Ucp3-/- mice. When challenged with a 45% fat diet, Ucp3-/- mice showed increased accumulation of IMTG compared with wild-type mice, which in turn had greater IMTG than UCP3Tg mice. Results are consistent with a role for UCP3 in preventing accumulation of triglyceride in both adipose tissue and muscle.     

Biological properties of antibodies against rat adipocyte intrinsic membrane proteins. Dependence on multivalency for insulin-like activity.

Antisera from rabbits injected with rat adipocyte plasma membranes or intrinsic proteins from such membranes, obtained by a dimethylmaleic anhydride extraction step, mimicked the action of insulin on both glucose transport and lipolysis in intact adipocytes. Biological activity in both types of antisera was mediated by immunoglobulin binding to one or more intrinsic proteins of the adipocyte plasma membrane since fat cells were unresponsive to all antisera absorbed with dimethylmaleic anhydride-extracted membranes. Acid treatment of immunoprecipitates released antibodies which activated glucose uptake and reacted with solubilized adipocyte membranes on immunodiffusion plates. The biologically active immunoglobulin preparations failed to form immunoprecipitin lines when tested against membranes from brain, liver, lung, muscle, kidney, and spleen. Insulin-sensitive glucose uptake in rat soleus muscle did not respond to the antisera. The antibodies activated hexose uptake into fat cells and reacted with solubilized adipocyte membranes on immunodiffusion plates when rat or mouse adipocytes were studied, but not when monkey fat cells were used. The anti-membrane antibody preparations readily activated hexose uptake in trypsinized fat cells which had lost the capacity to bind or respond to insulin. These data are consistent with the concept previously proposed (Pillion, D.J., and Czech, M.P. (1978) J. Biol. Chem. 253, 3761-3764) that the anti-membrane immunoglobulins do not interact with the insulin binding site of the insulin receptor. Monovalent Fab fragments of the biologically active antisera, prepared by papain digestion of the native anti-membrane immunoglobulins, were ineffective in enhancing glucose uptake in adipocytes. However, biological activity of the anti-membrane Fab fragments was restored by the addition of goat anti-rabbit Fab antisera to cells treated with the Fab fraction. Anti-rabbit Fab antisera alone or in combination with Fab fragments prepared from control rabbit sera exhibited no biological activity. These results demonstrate that the ability of anti-membrane antisera to mimic the biological activity of insulin on isolated fat cells is critically dependent on immunoglobulin binding to one or more intrinsic plasma membrane proteins and the multivalent nature of immunoglobulin structure.     

Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance

The prevalence of type 2 diabetes mellitus is growing worldwide. By the year 2020, 250 million people will be afflicted. Most forms of type 2 diabetes are polygenic with complex inheritance patterns, and penetrance is strongly influenced by environmental factors. The specific genes involved are not yet known, but impaired glucose uptake in skeletal muscle is an early, genetically determined defect that is present in non-diabetic relatives of diabetic subjects. The rate-limiting step in muscle glucose use is the transmembrane transport of glucose mediated by glucose transporter (GLUT) 4 (ref. 4), which is expressed mainly in skeletal muscle, heart and adipose tissue. GLUT4 mediates glucose transport stimulated by insulin and contraction/exercise. The importance of GLUT4 and glucose uptake in muscle, however, was challenged by two recent observations. Whereas heterozygous GLUT4 knockout mice show moderate glucose intolerance, homozygous whole-body GLUT4 knockout (GLUT4-null) mice have only mild perturbations in glucose homeostasis and have growth retardation, depletion of fat stores, cardiac hypertrophy and failure, and a shortened life span. Moreover, muscle-specific inactivation of the insulin receptor results in minimal, if any, change in glucose tolerance. To determine the importance of glucose uptake into muscle for glucose homeostasis, we disrupted GLUT4 selectively in mouse muscles. A profound reduction in basal glucose transport and near-absence of stimulation by insulin or contraction resulted. These mice showed severe insulin resistance and glucose intolerance from an early age. Thus, GLUT4-mediated glucose transport in muscle is essential to the maintenance of normal glucose homeostasis.     

Depot-specific regulation of glucose uptake and insulin sensitivity in HIV-lipodystrophy

Altered fat distribution is associated with insulin resistance in HIV, but little is known about regional glucose metabolism in fat and muscle depots in this patient population. The aim of the present study was to quantify regional fat, muscle, and whole body glucose disposal in HIV-infected men with lipoatrophy. Whole body glucose disposal was determined by hyperinsulinemic clamp technique (80 mU x m(-2) x min(-1)) in 6 HIV-infected men and 5 age/weight-matched healthy volunteers. Regional glucose uptake in muscle and subcutaneous (SAT) and visceral adipose tissue (VAT) was quantified in fasting and insulin-stimulated states using 2-deoxy-[18F]fluoro-D-glucose positron emission tomography. HIV-infected subjects with lipoatrophy had significantly increased glucose uptake into SAT (3.8 +/- 0.4 vs. 2.3 +/- 0.5 micromol x kg tissue(-1) x min(-1), P < 0.05) in the fasted state. Glucose uptake into VAT did not differ between groups. VAT area was inversely related with whole body glucose disposal, insulin sensitivity, and muscle glucose uptake during insulin stimulation. VAT area was highly predictive of whole body glucose disposal (r2 = 0.94, P < 0.0001). This may be mediated by adiponectin, which was significantly associated with VAT area (r = -0.75, P = 0.008), and whole body glucose disposal (r = 0.80, P = 0.003). This is the first study to directly demonstrate increased glucose uptake in subcutaneous fat of lipoatrophic patients, which may partially compensate for loss of SAT. Furthermore, we demonstrate a clear relationship between VAT and glucose metabolism in multiple fat and muscle depots, suggesting the critical importance of this depot in the regulation of glucose and highlighting the significant potential role of adiponectin in this process.     

Mice deficient in the insulin-regulated membrane aminopeptidase show substantial decreases in glucose transporter GLUT4 levels but maintain normal glucose homeostasis

The insulin-regulated aminopeptidase (IRAP) is a zinc-dependent membrane aminopeptidase. It is the homologue of the human placental leucine aminopeptidase. In fat and muscle cells, IRAP colocalizes with the insulin-responsive glucose transporter GLUT4 in intracellular vesicles and redistributes to the cell surface in response to insulin, as GLUT4 does. To address the question of the physiological function of IRAP, we generated mice with a targeted disruption of the IRAP gene (IRAP-/-). Herein, we describe the characterization of these mice with regard to glucose homeostasis and regulation of GLUT4. Fed and fasted blood glucose and insulin levels in the IRAP-/- mice were normal. Whereas IRAP-/- mice responded to glucose administration like control mice, they exhibited an impaired response to insulin. Basal and insulin-stimulated glucose uptake in extensor digitorum longus muscle, and adipocytes isolated from IRAP-/- mice were decreased by 30-60% but were normal for soleus muscle from male IRAP-/- mice. Total GLUT4 levels were diminished by 40-85% in the IRAP-/- mice in the different muscles and in adipocytes. The relative distribution of GLUT4 in subcellular fractions of basal and insulin-stimulated IRAP-/- adipocytes was the same as in control cells. We conclude that IRAP-/- mice maintain normal glucose homeostasis despite decreased glucose uptake into muscle and fat cells. The absence of IRAP does not affect the subcellular distribution of GLUT4 in adipocytes. However, it leads to substantial decreases in GLUT4 expression.     

Partial Inhibition of Adipose Tissue Lipolysis Improves Glucose Metabolism and Insulin Sensitivity Without Alteration of Fat Mass

When energy is needed, white adipose tissue (WAT) provides fatty acids (FAs) for use in peripheral tissues via stimulation of fat cell lipolysis. FAs have been postulated to play a critical role in the development of obesity-induced insulin resistance, a major risk factor for diabetes and cardiovascular disease. However, whether and how chronic inhibition of fat mobilization from WAT modulates insulin sensitivity remains elusive. Hormone-sensitive lipase (HSL) participates in the breakdown of WAT triacylglycerol into FAs. HSL haploinsufficiency and treatment with a HSL inhibitor resulted in improvement of insulin tolerance without impact on body weight, fat mass, and WAT inflammation in high-fat-diet–fed mice. In vivo palmitate turnover analysis revealed that blunted lipolytic capacity is associated with diminution in FA uptake and storage in peripheral tissues of obese HSL haploinsufficient mice. The reduction in FA turnover was accompanied by an improvement of glucose metabolism with a shift in respiratory quotient, increase of glucose uptake in WAT and skeletal muscle, and enhancement of de novo lipogenesis and insulin signalling in liver. In human adipocytes, HSL gene silencing led to improved insulin-stimulated glucose uptake, resulting in increased de novo lipogenesis and activation of cognate gene expression. In clinical studies, WAT lipolytic rate was positively and negatively correlated with indexes of insulin resistance and WAT de novo lipogenesis gene expression, respectively. In obese individuals, chronic inhibition of lipolysis resulted in induction of WAT de novo lipogenesis gene expression. Thus, reduction in WAT lipolysis reshapes FA fluxes without increase of fat mass and improves glucose metabolism through cell-autonomous induction of fat cell de novo lipogenesis, which contributes to improved insulin sensitivity.     

Vaspin gene expression in human adipose tissue: association with obesity and type 2 diabetes

Recently, vaspin was identified as an adipokine with insulin-sensitizing effects, which is predominantly secreted from visceral adipose tissue in a rat model of type 2 diabetes. In this study, we examined whether vaspin mRNA expression is a marker of visceral obesity and correlates with anthropometric and metabolic parameters in paired samples of visceral and subcutaneous adipose tissue from 196 subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance. Vaspin mRNA expression was only detectable in 23% of the visceral and in 15% of the subcutaneous (SC) adipose tissue samples. Vaspin mRNA expression was not detectable in lean subjects (BMI<25) and was more frequently detected in patients with type 2 diabetes. No significant correlations were found between visceral vaspin gene expression and visceral fat area or SC vaspin expression. However, visceral vaspin expression significantly correlates with BMI, % body fat, and 2 h OGTT plasma glucose. Subcutaneous vaspin mRNA expression is significantly correlated with WHR, fasting plasma insulin concentration, and glucose infusion rate during steady state of an euglycemic-hyperinsulinemic clamp. Multivariate linear regression analysis revealed % body fat as strongest predictor of visceral vaspin and insulin sensitivity as strongest determinant of SC vaspin mRNA expression. In conclusion, our data indicate that induction of human vaspin mRNA expression in adipose tissue is regulated in a fat depot-specific manner and could be associated with parameters of obesity, insulin resistance, and glucose metabolism.     

Running Exercise Improves Metabolic Abnormalities and Fat Accumulation in Sucrose-Induced Insulin-Resistant Rats

NARA, MAKOTO, MASAKI TAKAHASHI, TSUGIYASU KANDA, YOUNOSUKE SHIMOMURA, ISAO KOBAYASHI. Running exercise improves metabolic abnormalities and fat accumulation in sucrose-induced insulin-resistant rats. Insulin resistance and hyperinsulinemia are observed in rats fed a high sucrose diet. Insulin resistance is thought to be related to abnormal fat distribution. We previously reported the metabolic characteristics and the fat distribution in rats with sucrose-induced insulin resistance. This study was designed to examine the effects of exercise in these rats. The rats were divided into three groups: those receiving a starch-based diet (control), those receiving a high-sucrose diet (sucrose fed), and those receiving a high-sucrose diet and wheel-running exercise (exercised). Animals were killed after 4 weeks or 12 weeks. After 4 weeks, the three groups did not differ with respect to gain in adipose tissues. The portal vein (PV) insulin concentration was significantly increased in the sucrose-fed and the exercised rats compared with the control rats. The inferior vena cava (IVC) glucose concentration and the PV free fatty acid (FFA) were significantly lower in the exercised rats than in the sucrose-fed rats. After 12 weeks, the exercised rats had significantly lower mesenteric fat (MS) and subcutaneous fat (SC) and a lower MS:SC ratio than the sucrose-fed rats. The glucose levels in IVC, PV, and FFA in PV were significantly reduced in the exercised rats as compared with the sucrose-fed rats. These findings suggest that long-term exercise improves insulin resistance by reducing the accumulation of MS as well as SC. It is also suggested that short-term exercise improves glucose metabolism without change of fat accumulation.     

Adipocyte insulin resistance: effects of aging, obesity, exercise, and food restriction

This study examined the effects of aging, exercise training, and food restriction on epididymal fat cell size and resistance to insulin in rats. The exercise group was given access to voluntary running wheels at age 6 mo. The rats were studied at ages 12 and 28 mo. Sedentary free-eating (SFE) rats were obese and their fat cells were extremely insulin resistant, showing minimal increases in glucose oxidation and 2-deoxy-D-glucose (2-DOG) uptake in response to high insulin concentrations. The runners' adipocytes were smaller and had a greater responsiveness to insulin (approximately 9-fold for 2-DOG uptake and approximately 30-fold for glucose oxidation) than those of the SFE rats. Sedentary rats that were food restricted to keep their body weights the same as those of the runners had fat cells that were intermediate both in size and insulin responsiveness relative to those of the SFE rats and runners. There was a close correlation between fat cell size and responsiveness to insulin of 2-DOG uptake and glucose oxidation independent of age. There were no significant differences in fat cell size, insulin sensitivity, or insulin responsiveness between the adult (12 mo) and old (28 mo) rats in the same treatment groups. We conclude that aging alone has little or no effect on the responsiveness to insulin of glucose metabolism in fat cells and that the insulin resistance of adipocytes from obese older rats is due to fat cell hypertrophy, not aging. Exercise is effective in protecting against development of fat cell hypertrophy and insulin resistance.     

Wnt signaling activation in adipose progenitors promotes insulin-independent muscle glucose uptake

Adipose tissues provide circulating nutrients and hormones. We present in?vivo mouse studies highlighting roles for Wnt signals in both aspects of metabolism. β-catenin activation in PPARγ-expressing fat progenitors (PBCA) decreased fat mass and induced fibrotic replacement of subcutaneous fat specifically. In spite of lipodystrophy, PBCA mice did not develop the expected diabetes and hepatosteatosis, but rather exhibited improved glucose metabolism and normal insulin sensitivity. Glucose uptake was increased in muscle independently of insulin, associated with cell-surface translocation of glucose transporters and AMPK activation. Ex?vivo assays showed these effects were likely secondary to blood-borne signals since PBCA sera or conditioned media from PBCA fat progenitors enhanced glucose uptake and activated AMPK in muscle cultures. Thus, adipose progenitor Wnt activation dissociates lipodystrophy from dysfunctional metabolism and highlights a fat-muscle endocrine axis, which may represent a potential therapy to lower blood glucose and improve metabolism.     

High sodium intake enhances insulin-stimulated glucose uptake in rat epididymal adipose tissue

Objective: This study investigated the effect of different sodium content diets on rat adipose tissue carbohydrate metabolism and insulin sensitivity. Methods and Procedures: Male Wistar rats were fed on normal‐ (0.5% Na⁺; NS), high‐ (3.12% Na⁺; HS), or low‐sodium (0.06% Na⁺; LS) diets for 3, 6, and 9 weeks after weaning. Blood pressure (BP) was measured using a computerized tail‐cuff system. An intravenous insulin tolerance test (ivITT) was performed in fasted animals. At the end of each period, rats were killed and blood samples were collected for glucose and insulin determinations. The white adipose tissue (WAT) from abdominal and inguinal subcutaneous (SC) and periepididymal (PE) depots were weighed and processed for adipocyte isolation and measurement of in vitro rates of insulin‐stimulated 2‐deoxy‐d ‐[³H]‐glucose uptake (2DGU) and conversion of ‐[U‐¹⁴C]‐glucose into ¹⁴CO₂. Results: After 6 weeks, HS diet significantly increased the BP, SC and PE WAT masses, PE adipocyte size, and plasma insulin concentration. The sodium dietary content did not influence the whole‐body insulin sensitivity. A higher half‐maximal effective insulin concentration (EC₅₀) from the dose‐response curve of 2DGU and an increase in the insulin‐stimulated glucose oxidation rate were observed in the isolated PE adipocytes from HS rats. Discussion: The chronic salt overload enhanced the adipocyte insulin sensitivity for glucose uptake and the insulin‐induced glucose metabolization, contributing to promote adipocyte hypertrophy and increase the mass of several adipose depots, particularly the PE fat pad.     

Role of ABCA1 on membrane cholesterol content,insulin-dependent Akt phosphorylation and glucose uptake in adult skeletal muscle fibers from mice

The ATP-binding cassette transporter A1 (ABCA1) promotes cellular cholesterol efflux, leading to cholesterol binding to the extracellular lipid-free apolipoprotein A-I. ABCA1 regulates lipid content, glucose tolerance and insulin sensitivity in adipose tissue. In skeletal muscle, most GLUT4-mediated glucose transport occurs in the transverse tubule, a system composed by specialized cholesterol-enriched invaginations of the plasma membrane. We have reported that insulin resistant mice have higher cholesterol levels in transverse tubule from adult skeletal muscle. These high levels correlate with decreased GLUT4 trafficking and glucose uptake; however, the role of ABCA1 on skeletal muscle insulin-dependent glucose metabolism remains largely unexplored. Here, we evaluated the functional role of the ABCA1 on insulin-dependent signaling pathways, glucose uptake and cellular cholesterol content in adult skeletal muscle. Male mice were fed for 8?weeks with normal chow diet (NCD) or high fat diet (HFD). Compared to NCD-fed mice, ABCA1 mRNA levels and protein content were lower in muscle homogenates from HFD-fed mice. In Flexor digitorum brevis muscle from NCD-fed mice, shABCA1-RFP in vivo electroporation resulted in 65% reduction of ABCA1 protein content, 1.6-fold increased fiber cholesterol levels, 74% reduction in insulin-dependent Akt (Ser⁴⁷³) phosphorylation, total suppression of insulin-dependent GLUT4 translocation and decreased 2-NBDG uptake compared to fibers electroporated with the scrambled plasmid. Pre-incubation with methyl-β cyclodextrin reestablished both GLUT4 translocation and 2-NBDG transport. Based on the present results, we suggest that decreased ABCA1 contributes to the anomalous cholesterol accumulation and decreased glucose transport displayed by skeletal muscle membranes in the insulin resistant condition.     

Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance

Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive beta-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial beta-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus, mcd(-/-) mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.     

Improved glucose homeostasis in mice with muscle-specific deletion of protein-tyrosine phosphatase 1B

Obesity and type 2 diabetes are characterized by insulin resistance. Mice lacking the protein-tyrosine phosphatase PTP1B in all tissues are hypersensitive to insulin but also have diminished fat stores. Because adiposity affects insulin sensitivity, the extent to which PTP1B directly regulates glucose homeostasis has been unclear. We report that mice lacking PTP1B only in muscle have body weight and adiposity comparable to those of controls on either chow or a high-fat diet (HFD). Muscle triglycerides and serum adipokines are also affected similarly by HFD in both groups. Nevertheless, muscle-specific PTP1B(-/-) mice exhibit increased muscle glucose uptake, improved systemic insulin sensitivity, and enhanced glucose tolerance. These findings correlate with and are most likely caused by increased phosphorylation of the insulin receptor and its downstream signaling components. Thus, muscle PTP1B plays a major role in regulating insulin action and glucose homeostasis, independent of adiposity. In addition, rosiglitazone treatment of HFD-fed control and muscle-specific PTP1B(-/-) mice revealed that rosiglitazone acts additively with PTP1B deletion. Therefore, combining PTP1B inhibition with thiazolidinediones should be more effective than either alone for treating insulin-resistant states.     

Diets Rich in Saturated and Polyunsaturated Fatty Acids Induce Morphological Alterations in the Rat Ventral Prostate

Aim

To evaluate the influence of dietary lipid quality on the body mass, carbohydrate metabolism and morphology of the rat ventral prostate.

Materials and Methods

Wistar rats were divided into four groups: SC (standard chow), HF-S (high-fat diet rich in saturated fatty acids), HF-P (high-fat diet rich in polyunsaturated fatty acids) and HF-SP (high-fat diet rich in saturated and polyunsaturated fatty acids). We analyzed body mass, fat mass deposits, plasma blood, insulin resistance and the ventral prostate structure.

Results

Groups that received high-fat diets were heavier and presented larger fat deposits than SC group. The HF-S and HF-SP groups had higher glucose, insulin and total cholesterol serum levels and insulin resistance compared with the SC. The acinar area, epithelium height and area density of the lumen were higher in the HF-SP than in the other groups. The epithelium area density and epithelial cell proliferation were greater in the HF-P and HF-SP than in the SC group. All of the groups that received high-fat diets had greater area density of the stroma, area density of smooth muscle cells and stromal cell proliferation compared with the SC group.

Conclusion

Diets rich in saturated and/or polyunsaturated fatty acids induced overweight. Independently of insulin resistance, polyunsaturated fatty acids increased prostate stromal and epithelial cell proliferation. Saturated fatty acids influenced only stromal cellular proliferation. These structural and morphometric alterations may be considered risk factors for the development of adverse remodeling process in the rat ventral prostate.