INVESTIGADORES
PODEROSO Juan Jose
congresos y reuniones científicas
Título:
Modulation of mitochondrial nitric oxide synthase by insulin in rat skeletal muscle.
Autor/es:
FINOCCHIETTO PV, GALLI S, BARREYRO F, FRANCO MC, CARRERAS MC, PODEROSO JJ
Lugar:
Atenas, Grecia
Reunión:
Congreso; 41st EASD Meeting; 2005
Institución organizadora:
EASD
Resumen:
Post translational modifications as N- terminal acylation and Serin 1412 phosphorylation are associated to Nitric Oxide Synthase traslocation to mitochondria in different tissues (mtNOS).The mtNOS synthesize nitric oxide (NO) vectorially directed to the mitochondrial matrix. It is noteworthy that NO steady-state concentration modulates the electrons transfer, O2 uptake, redox enzymes and reactive oxygen species. Serin 1412 is an  AKT-dependent domain and considery that insulin activates this Kinase, we analyzed insulin effects on rats skeletal muscles in mitochondrial Nitric Oxide Synthase (mtNOS ) Materials and Methods Wistar rats  (200-250g) were subcutaneously inoculated with 0.1U/Kg insulin glargine or ClNa 0.9%  and afterwards surgically excised extensoris digitorum and soleus muscles at 3 and 48 hours. The isolation and purification of mitochondria was made by differential centrifugation at 3 and 48hours and in isolated muscles incubated with insulin and LY294002 ( PI3K specific inhibitor ). The mtNOS activity was determined by conversion of L-[ H3] arginine to L-[H3] citrulline and the expression of mtNOS, p-AKT, AKT 1 and AKT 2 were determined by Western Blot. We measured the p-AKT activity and phosphorylation of mtNOS by inmunoprecipitation. The  mitochondrial p-AKT and Nitric oxide were determined by flow cytometry. Mitochondrial oxygen uptake was determined polarographically with a Clark-type electrode  placed in chamber at 30ºC in reaction medium, saturated with room air with 0,3mg mitochondria protein/ml and it was determined with malate-glutamate in presence or absence of phosphate acceptor (ADP) and expressed in nanogram atom oxygen minute mg/protein. Data were expressed as means ± SE and analyzed by ANOVA and DUNNETT’s test. Statistical significance was accepted at  p< 0.05 Results:1) Insulin increased the mtNOS activity at 48hs ( in pmoles /min/mg prot: C: 30 ± 0.7  3hs: 35 ± 2.6, 48hs: 145.8 ± 3.05 ) without expression changes analyzed by Western Blot.  Accordantly this activity was inhibited by LY294002 in muscle homogenates ( C: 27.5 ± 2.9 , I: 100.4 ± 7.5, LY: 49.5 ± 5.26, LY + I : 20.2 ± 3.09 ). 2) The mitochondrial NO production  rate  was increased at 48hs and the mitochondrial oxygen uptake was lower at 48hs respect to controls and 3 hs ( C: 53± 10.9, 3 : 87.5 ± 5, 48 : 33 ± 5.7). 3) p-AKT traslocated to mitochondria and its expression was increased at 3 hours in mitochondria (38,8 %) and citosol ( 59,7%) respect to controls and 48 hours. 4) p-AKT activity was higher at 3hours in mitochondria and citosol respect to controls and 48 hours. 5)The AKT2 protein levels were increased by 59% in citosol and 20,8% in mitochondria at 3 hs but the AKT 1 expression was unaltered. 6) mtNOS was phosphorylated by p-AKT. Conclusions: P-AKT activated by PI3K-Insulin in  mitochondria  from skeletal muscle increases the mtNOS activity by phosphorylation. Likely, this effect reduces the O2 uptake and favors the biosynthetic metabolism pathways as  glucogenosynthesis.