Mitochondrial nitric oxide is increased in the mice Ob-/- modelo of metabolic syndrome

BARREYRO FJ, FINOCCHIETTO PV, FRANCO MC, HOLOD S, CARRERAS MC, PODEROSO JJ.

EEUU, Boston

Congreso; 57th Annual Meeting of the American Association for the Study of Liver Disease; 2006

Background and Aim: Insulin resistance (IR), the hallmark of non aJcoholic fatíy liver disease (NAFLD), is associated with the increase of visceral white adipose tissue (WAT). Although the mechanísm is unknown, there are severa! reports about mitochondrial abnormalities in metabolic tissues in IR, Nitric Oxide (NO) is a pleiotropic signaling moiecule, with many of its effects on cell function being elicited at the mitochondrial level. In different tissues, Nitric Oxide Synthase traslocates to mitochondria ímt- NOS)anc! synthesizes NO vectorially directed to the matrix. It is noteworíhy that NO steady-state concentration modulates electrón transfer, O2 uptake, and the reactive oxygen species yield. VVe previously reported that Insulin increases the mtNOS activity. Our aim is to determine the activity and expression of mtNOS in WAT, Muscle and Liver of lepíin deficient Ob-/- mice. Meíhods: VVe used Ob-/- and C57BL/6 VVT mice (6-9 mo oíd); were divided in fwo subgroups with no intervention or receiving (6 ¡xg bid IP 4 days) leptin replacement. Epidydimal WAT, Muscle, and Liver were exdsed and isolafion and purification of mitochondria was done by differential centrifugation. Mitochondrial NO was determined by flow citometry wiíh DAF, the expression of mtNOS by Western Blot and KT-PCR and Mitochondrial Complex I-IV activities were follovved spectrophotometrically. Complexes were separated by BN-PAGE, and tyrosine nitration was detected by Western Blot. Results: l)Ob-/- have a significant increase of mt- NOS activity in accord to 3-fold increase of expression. 2) Complex I Activity resulted markedly reduced in WAT (-81 %; p<0,05), Muscle (-72%; p<0,05), and Liver (-61%; p<0,05) from Ob-/-; there were no significant changos of the activity of Complexes II-IJI and IV. 3) According to low activity, Complex I exhibited 3-4 fold increased tyrosine nitration in WAT. Muscle and Liver mitochondria from Ób-/-. 4) Leptin replacement completely normaliyed mtNOS activity and Complex I activity, with significantly decreased nitration at same level of control». Conclusions: a)Milochondria of Ob-/- mice tissues are exposed to high NO matrix concentration that conducís lo Complex 1 nitration and subsequent reduced activity; b)Likewise, more than 60% Complex I inhibition contributes to low Ob-/- O2 uptake and obesity; c)Enhanced NO depends on high mtNOS expression and probably as well on preliminary dala of our group indicating that leptin reduces mtNOS activity and insulin increased mtNOS activity; díthus, in mis rodent mode! of metabolic syndrome, mitochondrial hypometabolísm should depend on the balance between leptin deficiency and reduced sensitivity to insulin.