Roberto Fabris

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.

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.

Neuroendocrine regulation of eating behavior

The dual center hypothesis in the central control of energy balance originates from the first observations performed more than 5 decades ago with brain lesioning and stimulation experiments. On the basis of these studies the “satiety center” was located in the ventromedial hypothalamic nucleus, since lesions of this region caused overfeeding and excessive weight gain, while its electrical stimulation suppressed eating. On the contrary, lesioning or stimulation of the lateral hypothalamus elicited the opposite set of responses, thus leading to the conclusion that this area represented the “feeding center”. The subsequent expansion of our knowledge of specific neuronal subpopulations involved in energy homeostasis has replaced the notion of specific “centers” controlling energy balance with that of discrete neuronal pathways fully integrated in a more complex neuronal network. The advancement of our knowledge on the anatomical structure and the function of the hypothalamic regions reveals the great complexity of this system. Given the aim of this review, we will focus on the major structures involved in the control of energy balance.

Lactate infusion in anesthetized rats produces insulin resistance in heart and skeletal muscles

Plasma lactate is elevated in many physiological and pathological conditions, such as physical exercise, obesity, and diabetes, in which a reduction of insulin sensitivity is also present. Furthermore, an increased production of lactate from muscle and adipose tissue together with increased gluconeogenic substrate flux to the liver plays a primary role in enhancing hepatic glucose production (HGP) in diabetes. It has been shown that lactate may interfere with the utilization and oxidation of other substrates such as free fatty acids (FFAs). The aim of this study was to investigate if lactate infusion affects peripheral glucose utilization in rats. Animals were acutely infused with lactate to achieve a final lactate concentration of 4 mmol/L. They were then submitted to a euglycemic-hyperinsulinemic clamp to study HGP and overall glucose metabolism (rate of disappearance [Rd]). At the end of the clamp, a bolus of 2-deoxy-[1-3H]-glucose was injected to study insulin-dependent glucose uptake in different tissues. The results show that lactate infusion did not affect HGP either in the basal state or at the end of clamp, whereas glucose utilization significantly decreased in lactate-infused rats (26.6 +/- 1.1 v 19.5 +/- 1.4 mg.kg-1.min-1, P < .01). A reduction in the tissue glucose utilization index was noted in heart (18.01 +/- 4.44 v 46.21 +/- 6.51 ng.mg-1.min-1, P < .01), diaphragm (5.56 +/- 0.74 v 9.01 +/- 0.93 ng.mg-1.min-1, P < .01), soleus (13.62 +/- 2.29 v 34.05 +/- 6.08 ng.mg-1.min-1, P < .01), and red quadricep (4.43 +/- 0.73 v 5.88 +/- 0.32 ng.mg-1.min-1, P < .05) muscle in lactate-infused animals, whereas no alterations were observed in other muscles or in adipose tissue. Therefore, we suggest that acute lactate infusion induces insulin resistance in the heart and some muscles, thus supporting a role for lactate in the regulation of peripheral glucose metabolism.

Hyperlactatemia reduces muscle glucose uptake and GLUT-4 mRNA while increasing (E1α)PDH gene expression in rat

An increased basal plasma lactate concentration is present in many physiological and pathological conditions, including obesity and diabetes. We previously demonstrated that acute lactate infusion in rats produced a decrease in overall glucose uptake. The present study was carried out to further investigate the effect of lactate on glucose transport and utilization in skeletal muscle. In chronically catheterized rats, a 24-h sodium lactate or bicarbonate infusion was performed. To study glucose uptake in muscle, a bolus of 2-deoxy-[3H]glucose was injected in basal condition and during euglycemic-hyperinsulinemic clamp. Our results show that hyperlactatemia decreased glucose uptake in muscles (i.e., red quadriceps; P< 0.05). Moreover in red muscles, both GLUT-4 mRNA (-30% in red quadriceps and -60% in soleus; P < 0.025) and protein (-40% in red quadriceps; P < 0.05) were decreased, whereas the (E1α)pyruvate dehydrogenase (PDH) mRNA was increased (+40% in red quadriceps; P< 0.001) in lactate-infused animals. PDH protein was also increased (4-fold in red gastrocnemius and 2-fold in red quadriceps). These results indicate that chronic hyperlactatemia reduces glucose uptake by affecting the expression of genes involved in glucose metabolism in muscle, suggesting a role for lactate in the development of insulin resistance.

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.

Lipolytic effect of beta-endorphin in human fat cells.

Recently, a role of beta-Endorphin on peripheral tissue metabolism has been suggested. A lipolytic effect of beta-Endorphin has been observed both in vivo and in vitro in animals but, at present, there is no evidence for a similar effect in humans. In this study, we investigated the lipolytic effect of beta-Endorphin in isolated human adipocytes. beta-Endorphin induced a significant increase in glycerol release in isolated human fat cells. Naloxone was able to inhibit the beta-Endorphin-induced lipolysis. The opioid antagonist alone had no effect on basal lipolysis and on Epinephrine-stimulated lipolysis when administered together with this hormone. Our results suggest that beta-Endorphin may play a role on lipolysis also in human fat cells and that this effect may be mediated by a specific opiate receptor.

Substrate competition and insulin action in animal models.

Increased basal plasma FFA and lactate concentrations are often present in obesity and may deeply affect insulin action. The inhibition of glucose transport or phosphorylation is thought to be involved in this phenomenon, but the molecular mechanisms on the basis are still unknown. In our laboratory we observed that a chronic infusion of Intralipid plus heparin in rats significantly decreased the insulin dependent-glucose uptake, as well as GLUT4 gene expression in muscular tissue. On the other hand it has been shown that an enhanced plasma lactate concentration may increase insulin secretion and hepatic insulin clearance. Moreover we observed that chronic hyperlactatemia in rats is able to decrease glucose uptake in muscles, while reducing GLUT4 mRNA and protein in the same tissues. In obesity, lactate and FFA overproduction from visceral fat may therefore play a synergic role in reducing insulin sensitivity.

3-Hydroxy-3-methylglutaric, adipic, and 2-oxoglutaric acids measured by HPLC in the plasma from diabetic patients.

A method for the measurement of organic acids in human plasma is presented. The analytical procedure consists of plasma protein precipitation with acetonitrile, acid extraction by chromatography through a DEAE-cellulose column eluted with 100 mM perchloric acid, HPLC by cation-exchange column Aminex HPX-87 eluted with 6.5 mM sulfuric acid. Adipic, 3-hydroxy-3-methylglutaric, 2-oxoglutaric, and citric acids were determined in the plasma of diabetic patients. The concentrations of all the measured acids, but particularly those of adipic and 3-hydroxy-3-methylglutaric acids, were significantly higher than those of healthy controls. These results suggest that in diabetics the omega-oxidation of fatty acids is enhanced.

Presence of anti-ADAMTS13 antibodies in obesity

Eur J Clin Invest 2012; 42 (11): 1197–1204