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.

The present study evaluated the antidiabetic activity of the Combretum lanceolatum Pohl ex Eichler, Combretaceae, flowers extract (ClEtOH) in diabetic rats. Streptozotocin-diabetic rats were divided into four groups: diabetic control, diabetic treated with 500 mg/kg of metformin and diabetic treated with 250 or 500 mg/kg of ClEtOH for 21 days. The treatment of diabetic rats with 500 mg/kg of ClEtOH promoted an increase in the weight of liver, white adipose tissues and skeletal muscles, improving body weight gain. Diabetic rats treated with 500 mg/kg of ClEtOH also presented reduction in glycemia, glycosuria and urinary urea levels, and increase in liver glycogen content. HPLC chromatogram showed that quercetin is the major compound in the extract. The phosphorylation levels of adenosine monophosphate-activated protein kinase were increased in liver slices incubated in vitro with 50 µg/mL of ClEtOH, similarly to the incubation with metformin (50 µg/mL) or quercetin (10 µg/mL). The antihyperglycemic effect of ClEtOH was similar to that of metformin and appears to be through inhibition of gluconeogenesis, since urinary urea was reduced and skeletal muscle mass was increased. These data indicate that the antidiabetic activity of the Combretum lanceolatum extract could be mediated, at least in part, through activation of adenosine monophosphateactivated protein kinase by quercetin

The recurrent gain-of-function JAK2V617F mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). The lack of complete response in most patients treated with the JAK1/2 inhibitor ruxolitinib indicates the need for identifying novel therapeutic strategies. Metformin is a biguanide that exerts selective antineoplastic activity in hematological malignancies. In the present study, we investigate and compare effects of metformin and ruxolitinib alone and in combination on cell signaling and cellular functions in JAK2V617F-positive cells. In JAK2V617F-expressing cell lines, metformin treatment significantly reduced cell viability, cell proliferation, clonogenicity, and cellular oxygen consumption and delayed cell cycle progression. Metformin reduced cyclin D1 expression and RB, STAT3, STAT5, ERK1/2 and p70S6K phosphorylation. Metformin plus ruxolitinib demonstrated more intense reduction of cell viability and induction of apoptosis compared to monotherapy. Notably, metformin reduced Ba/F3 JAK2V617F tumor burden and splenomegaly in Jak2V617F knock-in-induced MPN mice and spontaneous erythroid colony formation in primary cells from polycythemia vera patients. In conclusion, metformin exerts multitarget antileukemia activity in MPN: downregulation of JAK2/STAT signaling and mitochondrial activity. Our exploratory study establishes novel molecular mechanisms of metformin and ruxolitinib action and provides insights for development of alternative/complementary therapeutic strategies for MPN.

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Intracellular long-chain acyl-CoA synthetases (ACSL) activate fatty acids to produce acyl-CoA, which undergoes β-oxidation and participates in the synthesis of esterified lipids such as triacylglycerol (TAG). Imbalances in these metabolic routes are closely associated with the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Triacsin C is one of the few compounds that inhibit TAG accumulation into lipid droplets (LD) by suppressing ACSL activity. Here we report that treatment of primary rat hepatocytes with triacsin C at concentrations lower than the IC (4.1 μM) for LD formation: (i) diminished LD number in a concentration-dependent manner; (ii) increased mitochondrial amount; (iii) markedly improved mitochondrial metabolism by enhancing the β-oxidation efficiency, electron transport chain capacity, and degree of coupling - treatment of isolated rat liver mitochondria with the same triacsin C concentrations did not affect the last two parameters; (iv) decreased the GSH/GSSG ratio and elevated the protein carbonyl level, which suggested an increased reactive oxygen species production, as observed in isolated mitochondria. The hepatocyte mitochondrial improvements were not related to either the transcriptional levels of PGC-1α or the content of mTOR and phosphorylated AMPK. Triacsin C at 10 μM induced hepatocyte death by necrosis and/or apoptosis through mechanisms associated with mitochondrial permeability transition pore opening, as demonstrated by experiments using isolated mitochondria. Therefore, triacsin C at sub-IC concentrations modulates the lipid imbalance by shifting hepatocytes to a more oxidative state and enhancing the fatty acid consumption, which can in turn accelerate lipid oxidation and reverse NAFLD in long-term therapies.

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