We report here studies that integrate data of respiration rate from mouse skeletal muscle in response to leptin and pharmacological interference with intermediary metabolism, together with assays for phosphatidylinositol 3-kinase (PI3K) and AMP-activated protein kinase (AMPK). Our results suggest that the direct effect of leptin in stimulating thermogenesis in skeletal muscle is mediated by substrate cycling between de novo lipogenesis and lipid oxidation, and that this cycle requires both PI3K and AMPK signaling. This substrate cycling linking glucose and lipid metabolism to thermogenesis provides a novel thermogenic mechanism by which leptin protects skeletal muscle from excessive fat storage and lipotoxicity. Keywords: Obesity; Diabetes; Lipotoxicity; Gluco-lipotoxicity; Insulin resistance; Phosphatidylinositol 3-kinase; AMP-activated protein kinase; Sterol regulatory element binding protein-1c IntroductionSkeletal muscle, which accounts for 30-40% of body mass in mammals, is an important site for glucose disposal, lipid oxidation and thermogenesis whose impairments contribute to the pathogenesis of obesity and type 2 diabetes. It has long been suspected that these metabolic events are often interdependent in normal and disease states [1,2], but a mechanistic link between glucose and lipid metabolism to skeletal muscle thermogenesis is still ill-defined. Leptin, an adipocyte-derived hormone which is well known for its role in weight regulation, has also been shown to protect insulin-sensitive tissues like skeletal muscle against excessive fat storage that can lead to functional impairments known as lipotoxicity [3]. The demonstrations that leptin can act directly on skeletal muscle, specifically via the long form of the leptin receptor (ObRb), to stimulate glucose utilization [4], lipid oxidation through AMPactivated protein kinase (AMPK) [5,6] or thermogenesis in a phosphatidylinositol 3-kinase (PI3K)-dependent manner [7], have provided the impetus to investigate the mechanisms by which muscle substrate metabolism and thermogenesis are interdependent. Although the mechanisms leading to increased fatty acid oxidation in skeletal muscle in response to leptin have been described in molecular details [6], those underlying its effects on thermogenesis are still unknown, amid continuing controversies concerning the role of novel uncoupling proteins, UCP2 and UCP3, as effectors of skeletal muscle thermogenesis [3,8,9]. Furthermore, the mechanism by which glucose and lipid metabolism are linked to thermogenesis in response to leptin's direct effect on skeletal muscle is unknown. With the objective of elucidating the mechanisms by which leptin exerts its direct effect on skeletal muscle thermogenesis, we report here a study that integrates data of respiration rate from intact mouse skeletal muscle ex vivo in response to leptin and pharmacological interference with key control points of intermediary metabolism, together with biochemical measurements for PI3K and AMPK signaling. Materials and methods Mice and ...

BackgroundVascular smooth muscle cell (VSMC) senescence and apoptosis are involved in atherosclerotic plaque vulnerability. Arginase‐II (Arg‐II) has been shown to promote vascular dysfunction and plaque vulnerability phenotypes in mice through uncoupling of endothelial nitric oxide synthase and activation of macrophage inflammation. The function of Arg‐II in VSMCs with respect to plaque vulnerability is unknown. This study investigated the functions of Arg‐II in VSMCs linking to plaque vulnerability.Methods and ResultsIn vitro studies were performed on VSMCs isolated from human umbilical veins, whereas in vivo studies were performed on atherosclerosis‐prone apolipoprotein E‐deficient (ApoE−/−) mice. In nonsenescent VSMCs, overexpressing wild‐type Arg‐II or an l‐arginine ureahydrolase inactive Arg‐II mutant (H160F) caused similar effects on mitochondrial dysfunction, cell apoptosis, and senescence, which were abrogated by silencing p66Shc or p53. The activation of p66Shc but not p53 by Arg‐II was dependent on extracellular signal‐regulated kinases (ERKs) and sequential activation of 40S ribosomal protein S6 kinase 1 (S6K1)—c‐Jun N‐terminal kinases (JNKs). In senescent VSMCs, Arg‐II and S6K1, ERK‐p66Shc, and p53 signaling levels were increased. Silencing Arg‐II reduced all these signalings and cell senescence/apoptosis. Conversely, silencing p66Shc reduced ERK and S6K1 signaling and Arg‐II levels and cell senescence/apoptosis. Furthermore, genetic ablation of Arg‐II in ApoE−/− mice reduced the aforementioned signaling and apoptotic VSMCs in the plaque of aortic roots.ConclusionsArg‐II, independently of its l‐arginine ureahydrolase activity, promotes mitochondrial dysfunction leading to VSMC senescence/apoptosis through complex positive crosstalk among S6K1‐JNK, ERK, p66Shc, and p53, contributing to atherosclerotic vulnerability phenotypes in mice.

Using a method involving repeated oxygen uptake (MO 2 ) determinations in skeletal muscle ex vivo, the addition of leptin was found to increase MO 2 in soleus muscles from lean mice. These effects were found to be inhibited by phosphatidylinositol 3-kinase inhibitors, absent in muscles from obese Lepr db mice which have the dysfunctional long form of leptin receptor, and blunted in muscles from diet-induced obese mice in the fed state but not during fasting. These findings indicate that leptin has direct thermogenic effects in skeletal muscle, and that these effects require both the long form of leptin receptors and phosphatidylinositol 3-kinase signalling. ß

Obesity is associated with development and progression of chronic kidney disease (CKD). Recent evidence demonstrates that enhanced levels of the L-arginine:ureahydrolase, including the two isoenzymes arginase-I (Arg-I) and arginase-II (Arg-II) in vascular endothelial cells promote uncoupling of endothelial nitric oxide synthase (eNOS), leading to increased superoxide radical anion and decreased NO production thereby endothelial dysfunction. Arg-II but not Arg-I is abundantly expressed in kidney and the role of Arg-II in CKD is uncertain and controversial. We aimed to investigate the role of Arg-II in renal damage associated with diet-induced obesity mouse model. Wild type (WT) C57BL/6 mice and mice deficient in Arg-II gene (Arg-II−/−) were fed with either a normal chow (NC) or a high-fat-diet (HFD) for 14 weeks (starting at the age of 7 weeks) to induce obesity. In WT mice, HFD feeding caused frequent renal lipid accumulation, enhancement of renal reactive oxygen species (ROS) levels which could be attenuated by a NOS inhibitor, suggesting uncoupling of NOS in kidney. HFD feeding also significantly augmented renal Arg-II expression and activity. All the alterations in the kidney under HFD feeding were reduced in Arg-II−/− mice. Moreover, mesangial expansion as analyzed by Periodic Acid Schiff (PAS) staining and renal expression of vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in HFD-fed WT mouse assessed by immunoblotting were reduced in the HFD-fed Arg-II−/− mice, although there was no significant difference in body weight and renal weight/body weight ratio between the WT and Arg-II−/− mice. Thus, Arg-II expression/activity is enhanced in kidney of diet-induced obesity mice. Genetic targeting of Arg-II prevents renal damage associated with obesity, suggesting an important role of Arg-II in obesity-associated renal disease development.

A highly significant linear relationship can be established across the alterations in food intake (from -50% to +100%) and the respective changes in blood pressure (BP) or HR. These data suggest that prompt changes in hemodynamics induced by alterations in food intake might be implicated in the early events during weight gain or during weight loss.