Melatonin has a number of beneficial metabolic actions and reduced levels of melatonin may contribute to type 2 diabetes. The present study investigated the metabolic pathways involved in the effects of melatonin on mitochondrial function and insulin resistance in rat skeletal muscle. The effect of melatonin was tested both in vitro in isolated rats skeletal muscle cells and in vivo using pinealectomized rats (PNX). Insulin resistance was induced in vitro by treating primary rat skeletal muscle cells with palmitic acid for 24 hr. Insulin-stimulated glucose uptake was reduced by palmitic acid followed by decreased phosphorylation of AKT which was prevented my melatonin. Palmitic acid reduced mitochondrial respiration, genes involved in mitochondrial biogenesis and the levels of tricarboxylic acid cycle intermediates whereas melatonin counteracted all these parameters in insulin-resistant cells. Melatonin treatment increases CAMKII and p-CREB but had no effect on p-AMPK. Silencing of CREB protein by siRNA reduced mitochondrial respiration mimicking the effect of palmitic acid and prevented melatonin-induced increase in p-AKT in palmitic acid-treated cells. PNX rats exhibited mild glucose intolerance, decreased energy expenditure and decreased p-AKT, mitochondrial respiration, and p-CREB and PGC-1 alpha levels in skeletal muscle which were restored by melatonin treatment in PNX rats. In summary, we showed that melatonin could prevent mitochondrial dysfunction and insulin resistance via activation of CREB-PGC-1 alpha pathway. Thus, the present work shows that melatonin play an important role in skeletal muscle mitochondrial function which could explain some of the beneficial effects of melatonin in insulin resistance states.

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid, palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers, including cutaneous melanoma, in which its levels of expression are associated with tumor invasion and poor prognosis. We have previously shown that FASN inhibition with orlistat significantly reduces the number of spontaneous mediastinal lymph node metastases following the implantation of B16-F10 mouse melanoma cells in the peritoneal cavity of C57BL/6 mice. In this study, we investigate the biological mechanisms responsible for the FASN inhibition-induced apoptosis in B16-F10 cells. Both FASN inhibitors, cerulenin and orlistat, significantly reduced melanoma cell proliferation and activated the intrinsic pathway of apoptosis, as demonstrated by the cytochrome c release and caspase-9 and -3 activation. Further, apoptosis was preceded by an increase in both reactive oxygen species production and cytosolic calcium concentrations and independent of p53 activation and mitochondrial permeability transition. Taken together, these findings demonstrate the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells.

Atherosclerotic disease remains a leading cause of death in westernized societies, and reactive oxygen species (ROS) play a pivotal role in atherogenesis. Mitochondria are the main intracellular sites of ROS generation and are also targets for oxidative damage. Here, we show that mitochondria from atherosclerosis-prone, hypercholesterolemic low-density lipoprotein (LDL) receptor knockout mice have oxidative phosphorylation efficiency similar to that from control mice but have a higher net production of ROS and susceptibility to develop membrane permeability transition. Increased ROS production was observed in mitochondria isolated from several tissues, including liver, heart, and brain, and in intact mononuclear cells from spleen. In contrast to control mitochondria, knockout mouse mitochondria did not sustain a reduced state of matrix NADPH, the main source of antioxidant defense against ROS. Experiments in vivo showed faster liver secretion rates and de novo synthesis of triglycerides and cholesterol in knockout than in control mice, suggesting that increased lipogenesis depleted the reducing equivalents from NADPH and generated a state of oxidative stress in hypercholesterolemic knockout mice. These data provide the first evidence of how oxidative stress is generated in LDL receptor defective cells and could explain the increased LDL oxidation, cell death, and atherogenesis seen in familiar hypercholesterolemia.

BACKGROUND AND PURPOSEObesity is associated with structural and functional changes in perivascular adipose tissue (PVAT), favouring release of reactive oxygen species (ROS), vasoconstrictor and proinflammatory factors. The cytokine TNF-α induces vascular dysfunction and is produced by PVAT. We tested the hypothesis that obesity-associated PVAT dysfunction was mediated by augmented mitochondrial ROS (mROS) generation due to increased TNF-α production in this tissue. EXPERIMENTAL APPROACHC57Bl/6J and TNF-α receptor-deficient mice received control or high fat diet (HFD) for 18 weeks. We used pharmacological tools to determine the participation of mROS in PVAT dysfunction. Superoxide anion (O 2 .-) and H 2 O 2 were assayed in PVAT and aortic rings were used to assess vascular function. KEY RESULTSAortae from HFD-fed obese mice displayed increased contractions to phenylephrine and loss of PVAT anti-contractile effect. Inactivation of O 2 .-, dismutation of mitochondria-derived H 2 O 2 , uncoupling of oxidative phosphorylation and Rho kinase inhibition, decreased phenylephrine-induced contractions in aortae with PVAT from HFD-fed mice. O 2 .-and H 2 O 2 were increased in PVAT from HFD-fed mice. Mitochondrial respiration analysis revealed decreased O 2 consumption rates in PVAT from HFD-fed mice. TNF-α inhibition reduced H 2 O 2 levels in PVAT from HFD-fed mice. PVAT dysfunction, i.e. increased contraction to phenylephrine in PVAT-intact aortae, was not observed in HFD-obese mice lacking TNF-α receptors. Generation of H 2 O 2 was prevented in PVAT from TNF-α receptor deficient obese mice. CONCLUSION AND IMPLICATIONSTNF-α-induced mitochondrial oxidative stress is a key and novel mechanism involved in obesity-associated PVAT dysfunction. These findings elucidate molecular mechanisms whereby oxidative stress in PVAT could affect vascular function.

TNFalpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNFalpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNFalpha in the hypothalamus leads to increased O(2) consumption/CO(2) production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNFalpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNFalpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNFalpha action to be important mediator of the wastage syndrome in cachexia.

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O(2) consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.

Mitochondrial redox imbalance has been implicated in mechanisms of aging, various degenerative diseases and drug-induced toxicity. Statins are safe and well-tolerated therapeutic drugs that occasionally induce myotoxicity such as myopathy and rhabdomyolysis. Previous studies indicate that myotoxicity caused by statins may be linked to impairment of mitochondrial functions. Here, we report that 1-h incubation of permeabilized rat soleus muscle fiber biopsies with increasing concentrations of simvastatin (1–40 μM) slowed the rates of ADP-or FCCP-stimulated respiration supported by glutamate/malate in a dose-dependent manner, but caused no changes in resting respiration rates. Simvastatin (1 μM) also inhibited the ADP-stimulated mitochondrial respiration supported by succinate by 24% but not by TMPD/ascorbate. Compatible with inhibition of respiration, 1 μM simvastatin stimulated lactate release from soleus muscle samples by 26%. Co-incubation of muscle samples with 1 mM L-carnitine, 100 μM mevalonate or 10 μM coenzyme Q10 (Co-Q10) abolished simvastatin effects on both mitochondrial glutamate/malate-supported respiration and lactate release. Simvastatin (1 μM) also caused a 2-fold increase in the rate of hydrogen peroxide generation and a decrease in Co-Q10 content by 44%. Mevalonate, Co-Q10 or L-carnitine protected against stimulation of hydrogen peroxide generation but only mevalonate prevented the decrease in Co-Q10 content. Thus, independently of Co-Q10 levels, L-carnitine prevented the toxic effects of simvastatin. This suggests that mitochondrial respiratory dysfunction induced by simvastatin, is associated with increased generation of superoxide, at the levels of complexes-I and II of the respiratory chain. In all cases the damage to these complexes, presumably at the level of 4Fe-4S clusters, is prevented by L-carnitine.

. Cold-induced PGC-1␣ expression modulates muscle glucose uptake through an insulin receptor/Akt-independent, AMPK-dependent pathway. Am J Physiol Endocrinol Metab