Marnie Granzotto

Exercise Training Induces Mitochondrial Biogenesis and Glucose Uptake in Subcutaneous Adipose Tissue Through eNOS-Dependent Mechanisms

Insulin resistance and obesity are associated with a reduction of mitochondrial content in various tissues of mammals. Moreover, a reduced nitric oxide (NO) bioavailability impairs several cellular functions, including mitochondrial biogenesis and insulin-stimulated glucose uptake, two important mechanisms of body adaptation in response to physical exercise. Although these mechanisms have been thoroughly investigated in skeletal muscle and heart, few studies have focused on the effects of exercise on mitochondria and glucose metabolism in adipose tissue. In this study, we compared the in vivo effects of chronic exercise in subcutaneous adipose tissue of wild-type (WT) and endothelial NO synthase (eNOS) knockout (eNOS−/−) mice after a swim training period. We then investigated the in vitro effects of NO on mouse 3T3-L1 and human subcutaneous adipose tissue–derived adipocytes after a chronic treatment with an NO donor: diethylenetriamine-NO (DETA-NO). We observed that swim training increases mitochondrial biogenesis, mitochondrial DNA content, and glucose uptake in subcutaneous adipose tissue of WT but not eNOS−/− mice. Furthermore, we observed that DETA-NO promotes mitochondrial biogenesis and elongation, glucose uptake, and GLUT4 translocation in cultured murine and human adipocytes. These results point to the crucial role of the eNOS-derived NO in the metabolic adaptation of subcutaneous adipose tissue to exercise training.

Loss-of-Function Mutation of the GPR40 Gene Associates with Abnormal Stimulated Insulin Secretion by Acting on Intracellular Calcium Mobilization

BACKGROUND Free fatty acids (FFAs) acutely stimulate but chronically impair glucose-stimulated insulin secretion from beta-cells. The G protein-coupled transmembrane receptor 40 (GPR40) mediates both acute and chronic effects of FFAs on insulin secretion and plays a role in glucose homeostasis. Limited information is available on the effect of GPR40 genetic abnormalities on insulin secretion and metabolic regulation in human subjects. STUDY DESIGN AND RESULTS For in vivo studies, we screened 734 subjects for the coding region of GPR40 and identified a new single-nucleotide mutation (Gly180Ser). The mean allele frequency was 0.75%, which progressively increased (P < 0.05) from nonobese subjects (0.42%) to moderately obese (body mass index = 30-39.9 kg/m2, 1.07%) and severely obese patients (body mass index > or = 40 kg/m2, 2.60%). The relationship between the GPR40 mutation, insulin secretion, and metabolic alterations was studied in 11 Gly/Ser mutation carriers. In these subjects, insulin secretion (insulinogenic index derived from oral glucose tolerance test) was significantly lower than in 692 Gly/Gly carriers (86.0 +/- 48.2 vs. 183.7 +/- 134.4, P < 0.005). Moreover, a case-control study indicated that plasma insulin and C-peptide responses to a lipid load were significantly (P < 0.05) lower in six Gly/Ser than in 12 Gly/Gly carriers. In vitro experiments in HeLa cells cotransfected with aequorin and the mutated Gly/Ser GPR40 indicated that intracellular Ca2+ concentration increase after oleic acid was significantly lower than in Gly/Gly GPR40-transfected cells. This fact was confirmed using fura-2 acetoxymethyl ester. CONCLUSIONS This newly identified GPR40 variant results in a loss of function that prevents the beta-cell ability to adequately sense lipids as an insulin secretory stimulus because of impaired intracellular Ca2+ concentration increase.

Changes in FAT=CD36, UCP2, UCP3 and GLUT4 gene expression during lipid infusion in rat skeletal and heart muscle

Objective: It has been reported that an increased availability of free fatty acids (NEFA) not only interferes with glucose utilization in insulin-dependent tissues, but may also result in an uncoupling effect of heart metabolism. We aimed therefore to investigate the effect of an increased availability of NEFA on gene expression of proteins involved in transmembrane fatty acid (FAT/CD36) and glucose (GLUT4) transport and of the uncoupling proteins UCP2 and 3 at the heart and skeletal muscle level.Study Design: Euglycemic hyperinsulinemic clamp was performed after 24 h Intralipid® plus heparin or saline infusion in lean Zucker rats. Skeletal and heart muscle glucose utilization was calculated by 2-deoxy-[1-3H]-D-glucose technique. Quantification of FAT/CD36, GLUT4, UCP2 and UCP3 mRNAs was obtained by Northern blot analysis or RT-PCR.Results: In Intralipid® plus heparin infused animals a significant decrease in insulin-mediated glucose uptake was observed both in the heart (22.62±2.04 vs 10.37±2.33 ng/mg/min; P<0.01) and in soleus muscle (13.46±1.53 vs 6.84±2.58 ng/mg/min; P<0.05). FAT/CD36 mRNA was significantly increased in skeletal muscle tissue (+117.4±16.3%, P<0.05), while no differences were found at the heart level in respect to saline infused rats. A clear decrease of GLUT4 mRNA was observed in both tissues. The 24 h infusion of fat emulsion resulted in a clear enhancement of UCP2 and UCP3 mRNA levels in the heart (99.5±15.3 and 80±4%) and in the skeletal muscle (291.5±24.7 and 146.9±12.7%).Conclusions: As a result of the increased availability of NEFA, FAT/CD36 gene expression increases in skeletal muscle, but not at the heart level. The augmented lipid fuel supply is responsible for the depression of insulin-mediated glucose transport and for the increase of UCP2 and 3 gene expression in both skeletal and heart muscle.

Increased adipogenic conversion of muscle satellite cells in obese Zucker rats

Aims/hypothesis:Visceral and intermuscular adipose tissue (IMAT) depots account for most obesity-related metabolic and cardiovascular complications. Muscle satellite cells (SCs) are mesenchymal stem cells giving rise to myotubes and also to adipocytes, suggesting their possible contribution to IMAT origin and expansion. We investigated the myogenic differentiation of SCs and the adipogenic potential of both preadipocytes and SCs from genetically obese Zucker rats (fa/fa), focusing on the role of Wnt signaling in these differentiation processes.Methods:SCs were isolated by single-fiber technique from flexor digitorum brevis muscle and preadipocytes were extracted from subcutaneous adipose tissue (AT). Morphological features and gene expression profile were evaluated during in vitro myogenesis and adipogenesis. Wingless-type MMTV integration site family member 10b (Wnt10b) expression was quantified by quantitative PCR in skeletal muscle and AT.Results:We did not observe any difference in the proliferation rate and in the myogenic differentiation of SCs from obese and lean rats. However, a decreased insulin-induced glucose uptake was present in myotubes originating from fa/fa rats. Under adipogenic conditions, preadipocytes and SCs of obese animals displayed an enhanced adipogenesis. Wnt10b expression was reduced in obese rats in both muscle and AT.Conclusions/interpretation:Our data suggest that the increase in different fat depots including IMAT and the reduced muscle insulin sensitivity, the major phenotypical alteration of obese Zucker rats, could be ascribed to an intrinsic defect, either genetically determined or acquired, still present in both muscle and fat precursors. The involvement of Wnt10b as a regulator of both adipogenesis and muscle-to-fat conversion is suggested.

Exercise training boosts eNOS-dependent mitochondrial biogenesis in mouse heart: role in adaptation of glucose metabolism

Endurance exercise training increases cardiac energy metabolism through poorly understood mechanisms. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) in cardiomyocytes contributes to cardiac adaptation. Here we demonstrate that the NO donor diethylenetriamine-NO (DETA-NO) activated mitochondrial biogenesis and function, as assessed by upregulated peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial transcription factor A (Tfam) expression, and by increased mitochondrial DNA content and citrate synthase activity in primary mouse cardiomyocytes. DETA-NO also induced mitochondrial biogenesis and function and enhanced both basal and insulin-stimulated glucose uptake in HL-1 cardiomyocytes. The DETA-NO-mediated effects were suppressed by either PGC-1α or Tfam small-interference RNA in HL-1 cardiomyocytes. Wild-type and eNOS(-/-) mice were subjected to 6 wk graduated swim training. We found that eNOS expression, mitochondrial biogenesis, mitochondrial volume density and number, and both basal and insulin-stimulated glucose uptake were increased in left ventricles of swim-trained wild-type mice. On the contrary, the genetic deletion of eNOS prevented all these adaptive phenomena. Our findings demonstrate that exercise training promotes eNOS-dependent mitochondrial biogenesis in heart, which behaves as an essential step in cardiac glucose transport.

Obesity Reduces the Expression of GLUT4 in the Endometrium of Normoinsulinemic Women Affected by the Polycystic Ovary Syndrome

Abstract: GLUT4 is the most important glucose transporter in insulin‐dependent tissues. A decrease of its expression by the adipocytes was reported in polycystic ovary syndrome (PCOS), regardless of obesity and glucose tolerance. In PCOS, abnormal menstrual cycles, abnormal insulin secretory patterns, and obesity, which are risk factors for endometrial diseases, frequently coexist. The endometrial effects of insulin are direct through specific insulin receptors. However, it is unknown whether the endometrium expresses GLUT4 and can be considered an insulin‐regulated tissue. In this study, we investigated this question, and we investigated whether obesity modulates this expression in PCOS normoinsulinemic patients. We assayed GLUT4 in the endometrial samples from 18 normoinsulinemic PCOS patients and 9 controls in the advanced follicular phase of the cycle; 10 patients were lean and 8 obese, and all were aged between 23 and 32 years. Most tissue was immediately frozen for RT‐PCR; some tissue was saved for histology and immunohistochemistry. GLUT4 mRNA expression was measured in three samples for every patient and expressed as mean ± SE of an arbitrary unit. In obese PCOS subjects, endometrial GLUT4 expression was significantly lower than in the lean ones (24.0 ± 6.8 vs. 65.2 ± 24.4; P < 0.005) and the controls (53.2 ± 10.7). Lean PCOS and control subjects showed similar values. GLUT4 immunostaining was strong in the epithelial and absent in the stromal cells. We demonstrated endometrial GLUT4 expression. The similar results in lean PCOS and control subjects suggest that endometrial GLUT4 expression is not affected by PCOS itself, whereas it is reduced by obesity in PCOS patients.

Insulin induces rapid changes of plasma leptin in lean but not in genetically obese (fa/fa) rats

OBJECTIVES: To evaluate the plasma leptin concentration in lean and genetically obese fa/fa rats and to assess the response to 2 h hyperinsulinaemia. BACKGROUND: The recently discovered peptide leptin is a putative link between the size of the adipose mass and the hypothalamic centres controlling feeding behaviour. Several genetic models of animal obesity have been characterized as carriers of mutations of either the ob gene or leptin receptor. EXPERIMENTAL DESIGN: Lean (+/?) and obese (fa/fa) Zucker rats were studied under pentobarbital anaesthesia and underwent a 2 h euglycaemic hyperinsulinaemic clamp. Plasma leptin was measured in basal condition and at the end of the clamp study. Glucose rate of disappearance was evaluated by means of the isotope dilution technique using 3-3H-glucose as tracer. RESULTS: fa/fa rats showed a 40 fold higher leptin concentration compared to lean littermates (0.47±0.10 vs 19.55±1.50 ng/ml, P<0.0001). Euglycaemic hyperinsulinaemia increased plasma leptin in lean but not in genetically obese rats. CONCLUSIONS: Our results suggest that insulin may be a regulator of in vivo leptin secretion by adipose tissue of lean rats whereas it is ineffective in increasing plasma leptin in obese Zucker rats.

Effects of intravenous neuropeptide Y on insulin secretion and insulin sensitivity in skeletal muscle in normal rats

Summary Intracerebroventricular administration of neuropeptide Y to normal rats induces a syndrome characterised by obesity, hyperinsulinaemia, insulin resistance and over expression of the adipose tissue ob gene. Little is known about the effect of circulating neuropeptide Y on glucose metabolism, insulin secretion and leptin. We therefore aimed to evaluate the effect of an intravenous infusion of neuropeptide Y on glucose disposal, endogenous glucose production, whole body glycolytic flux, and glucose storage as assessed during euglycaemic hyperinsulinaemic clamp. In addition, the insulin-stimulated glucose utilisation index in individual tissues was measured by the 2-deoxy-[1-3H]-glucose technique. The effect of neuropeptide Y on insulin secretion was evaluated by hyperglycaemic clamp. Infusion did not induce any change in endogenous glucose production during basal conditions or at the end of the clamp. Glucose disposal was significantly increased in the rats given neuropeptide Y compared with controls (27.8 ± 1.3 vs 24.3 ± 1.6 mg · min–1· kg–1; p < 0.05) as was the glycolytic flux (18.9 ± 1.6 vs 14.4 ± 0.8 mg · min–1· kg–1; p < 0.05), while glucose storage was comparable in the two groups. In skeletal muscle, the glucose utilisation index was increased significantly in rats given neuropeptide Y. The glucose utilisation index in subcutaneous and epididimal adipose tissue was not significantly different between the two groups. Plasma leptin was significantly increased by hyperinsulinaemia, but was not affected by neuropeptide Y infusion. Both the early and late phase of the insulin response to hyperglycaemia were significantly reduced by neuropeptide Y. In conclusion neuropeptide Y infusion may increase insulin-induced glucose disposal in normal rats, accelerating its utilisation through the glycolytic pathway. Neuropeptide Y reduces both phases of the insulin response to hyperglycaemia. [Diabetologia (1998) 41: 1361–1367]

Insulin receptor and glucose transporters mRNA expression throughout the menstrual cycle in human endometrium: aphysiological and cyclical condition of tissue insulin resistance

The expression of insulin receptor (IR), together with that of glucose transporters 1 and 4 (GLUT1-4) and of Insulin Growth Factor-I and -II (IGF-I,-II) in the endometrium of healthy and young women in both phases of menstrual cycle was assessed. Sixteen out of 20 healthy and normal menstruating volunteers were studied. Endometrial samplings were performed in every subject, twice in the same cycle, during the follicular and luteal phase respectively. The mRNA expression of IR, GLUT1-4, IGF-I and -II were evaluated by real-time quantitative RT-PCR and immunostaining reactions. Our results indicate that IR, GLUT1-4, IGF-I and -II mRNAs were expressed in both phases of the endometrial cycle: GLUT4 and IGF-I mRNA expression were significantly higher in the follicular phase and localized at the epithelial and stromal cell level, respectively, whereas IR, GLUT1 and IGF-II mRNA expression were mostly present in the secretory phase and mainly localized at the stromal level. An inverse tendency of IR and GLUT4 mRNA expression was respectively observed from follicular to luteal phase. In conclusion our data suggest that IR, glucose transporters and IGFs are significantly and differently expressed at the endometrial level throughout the menstrual cycle and that human endometrium cyclically undergoes through a transitory condition from normal to an insulin-resistance state.

Systemic and Local Adipose Tissue in Knee Osteoarthritis: OBESITY AND FAT PAD IN KNEE OSTEOARTHRITIS

Osteoarthritis is a common chronic joint disorder affecting older people. The knee is the major joint affected. The symptoms of osteoarthritis include limited range of motion, joint swelling, and pain causing disability. There are no disease modifying drugs available, and treatments are mainly focused on pain management. Total knee replacement performed at the end stage of the disease is considered the only cure available. It has been found that obese people have an increased risk to develop not only knee but also hand osteoarthritis. This supports the concept that adipose tissue might be related to osteoarthritis not only through overloading. As matter of fact, obesity induces a low grade systemic inflammatory state characterized by the production and secretion of several adipocytokines that may have a role in osteoarthritis development. Furthermore, hypertension, impaired glucose, and lipid metabolism, which are comorbidities associated with obesity, have been shown to alter the joint tissue homeostasis. Moreover, infrapatellar fat pad in the knee has been demonstrated to be a local source of adipocytokines and potentially contribute to osteoarthritis pathogenesis. Here, we discuss the role of systemic and local adipose tissue in knee osteoarthritis. J. Cell. Physiol. 232: 1971–1978, 2017.