Abnormal Leptin-AMPK Pathway provokes NO Release and Mitochondrial Dysfunction and Obesity in ob/ob Mice

FINOCCHIETTO PV, HOLOD S, BARREYRO F, PERALTA JG, ALIPPE Y, GIOVAMBATTISTA A, CARRERAS MC, PODEROSO JJ

Sao Pedro

Congreso; VII Meeting of the SFRBM South American Group. I Sao Paulo Advanced School on Redox Processes in Biomedicine.; 2011

SFRBM South American Group

Obesity results from an imbalance between energy intake and expenditure. Insulin resistance is a feature of obesity and the metabolic syndrome. Leptin regulates the balance of energy by AMPK pathway. A model of Metabolic Syndrome is the leptin deficient ob/ob mouse. The mitochondrial Nitric Oxide (NO) represents a biochemical pathway regulating the supply of O2 and energy to tissues under dynamic conditions and metabolism. We hypothesized that leptin deficiency and insulin resistance induces mitochondrial dysfunction through mitochondrial NO excess, insufficient activation of the AMPK signalling pathways and obesity. Male ob/ob and C57BL/6 wt mice (6-9 months old) were divided in groups: no intervention or mouse recombinant leptin (1 mg/kg ip. for 4 d). Mitochondria were isolated and purificated from homogenized adipose tissue by differential centrifugation and gradients. nNOS expression and activity was detected by Real Time PCR, Western-blot, citrulline assay, electronic and confocal microscopy, flow-citometry and NO-electrode sensor. A 3-4 fold increase in nNOS expression and activity was detected in epididymal white adipose tissue (WAT) mitochondria (+270%) and cytosol (+160%) from ob/ob mice (n: 5) p< 0.05. Increase of cytosol (+2-3 fold) and mitochondrial NO (+3-4 fold) yield in ob/ob respect to control samples was measured in adipose and liver tissues. Ob/ob mitochondrial complex I activity was reduced by 50 to 75% in white adipose tissue, muscle and liver. Western blotting with an anti-3-nitrotyrosine antibody revealed nitration at complex I in ob/ob mitochondria from these tissues. High level of NO restricted β-oxidation in obese mitochondria and displaced acetyl-CoA to fat synthesis and decreased AMPK-P expression ; siRNAnNOS caused fat reduction in ob/ob adipocytes. Leptin administration to ob/ob mice prevented nNOS overexpression and mitochondrial dysfunction in vivo, while leptin-Ob-Rb disruption increased the formation of mitochondrial NO in control adipocytes. We demonstrated that in ob/ob, hypoleptinemia, is associated to low mitochondrial p-AMPK and that, oppositely to p-Akt2, p-AMPK is a negative modulator of nNOS. Thereby, defective leptin-AMPK pathway links mitochondrial NO to obesity with complex I syndrome, and dysfunctional mitochondria.