Mitochondrial Nitric Oxide is Increased in the Mice Ob-/- Model of Metabolic Syndrome

FERNANDO J BARREYRO; PAOLA FINOCCHIETTO; FRANCO MC; SILVIA HOLOD; CARRERAS MC; JUAN J. PODEROSO

Boston, USA

Congreso; 57th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD). 2006; 2006

American Association for the Study of Liver Diseases

  Background and Aim: Insulin resistance (IR), the hallmark of non alcoholic fatty liver disease (NAFLD), is associated with the increase of visceral white adipose tissue (WAT). Although the mechanism is unknown, there are several reports about mitochondrial abnormalities in metabolic tissues in IR. Nitric Oxide (NO) is a pleiotropic signaling molecule, with many of its effects on cell function being elicited at the mitochondrial level. In different tissues, Nitric Oxide Synthase traslocates to mitochondria (mt- NOS)and synthesizes NO vectorially directed to the matrix. It is noteworthy that NO steady-state concentration modulates electron transfer, O2 uptake, and the reactive oxygen species yield. We 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 leptin deficient Ob-/- mice. Methods: We used Ob-/- and C57BL/6 WT mice (6-9 mo old); were divided in two subgroups with no intervention or receiving (6 g bid IP 4 days) leptin replacement. Epidydimal WAT, Muscle, and Liver were excised and isolation and purification of mitochondria was done by differential centrifugation. Mitochondrial NO was determined by flow citometry with DAF, the expression of mtNOS by Western Blot and RT-PCR and Mitochondrial Complex I-IV activities were followed spectrophotometrically. Complexes were separated by BN-PAGE, and tyrosine nitration was detected by Western Blot. Results: 1)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%; p0,05), Muscle (-72%; p0,05), and Liver (-61%; p0,05) from Ob-/-; there were no significant changes of the activity of Complexes II-III and IV. 3) According to low activity, Complex I exhibited 3-4 fold increased tyrosine nitration in WAT, Muscle and Liver mitochondria from Ob-/-. 4) Leptin replacement completely normalized mtNOS activity and Complex I activity, with significantly decreased nitration at same level of controls. Conclusions: a)Mitochondria of Ob-/- mice tissues are exposed to high NO matrix concentration that conducts to Complex I 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 data of our group indicating that leptin reduces mtNOS activity and insulin increased mtNOS activity; d)thus, in this rodent model of metabolic syndrome, mitochondrial hypometabolism should depend on the balance between leptin deficiency and reduced sensitivity to insulin. were no significant changes of the activity of Complexes II-III and IV. 3) According to low activity, Complex I exhibited 3-4 fold increased tyrosine nitration in WAT, Muscle and Liver mitochondria from Ob-/-. 4) Leptin replacement completely normalized mtNOS activity and Complex I activity, with significantly decreased nitration at same level of controls. Conclusions: a)Mitochondria of Ob-/- mice tissues are exposed to high NO matrix concentration that conducts to Complex I 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 data of our group indicating that leptin reduces mtNOS activity and insulin increased mtNOS activity; d)thus, in this rodent model of metabolic syndrome, mitochondrial hypometabolism should depend on the balance between leptin deficiency and reduced sensitivity to insulin. Muscle (-72%; p0,05), and Liver (-61%; p0,05) from Ob-/-; there were no significant changes of the activity of Complexes II-III and IV. 3) According to low activity, Complex I exhibited 3-4 fold increased tyrosine nitration in WAT, Muscle and Liver mitochondria from Ob-/-. 4) Leptin replacement completely normalized mtNOS activity and Complex I activity, with significantly decreased nitration at same level of controls. Conclusions: a)Mitochondria of Ob-/- mice tissues are exposed to high NO matrix concentration that conducts to Complex I 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 data of our group indicating that leptin reduces mtNOS activity and insulin increased mtNOS activity; d)thus, in this rodent model of metabolic syndrome, mitochondrial hypometabolism should depend on the balance between leptin deficiency and reduced sensitivity to insulin. were no significant changes of the activity of Complexes II-III and IV. 3) According to low activity, Complex I exhibited 3-4 fold increased tyrosine nitration in WAT, Muscle and Liver mitochondria from Ob-/-. 4) Leptin replacement completely normalized mtNOS activity and Complex I activity, with significantly decreased nitration at same level of controls. Conclusions: a)Mitochondria of Ob-/- mice tissues are exposed to high NO matrix concentration that conducts to Complex I 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 data of our group indicating that leptin reduces mtNOS activity and insulin increased mtNOS activity; d)thus, in this rodent model of metabolic syndrome, mitochondrial hypometabolism should depend on the balance between leptin deficiency and reduced sensitivity to insulin. tissues, Nitric Oxide Synthase traslocates to mitochondria (mt- NOS)and synthesizes NO vectorially directed to the matrix. It is noteworthy that NO steady-state concentration modulates electron transfer, O2 uptake, and the reactive oxygen species yield. We 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 leptin deficient Ob-/- mice. Methods: We used Ob-/- and C57BL/6 WT mice (6-9 mo old); were divided in two subgroups with no intervention or receiving (6 g bid IP 4 days) leptin replacement. Epidydimal WAT, Muscle, and Liver were excised and isolation and purification of mitochondria was done by differential centrifugation. Mitochondrial NO was determined by flow citometry with DAF, the expression of mtNOS by Western Blot and RT-PCR and Mitochondrial Complex I-IV activities were followed spectrophotometrically. Complexes were separated by BN-PAGE, and tyrosine nitration was detected by Western Blot. Results: 1)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%; p0,05), Muscle (-72%; p0,05), and Liver (-61%; p0,05) from Ob-/-; there were no significant changes of the activity of Complexes II-III and IV. 3) According to low activity, Complex I exhibited 3-4 fold increased tyrosine nitration in WAT, Muscle and Liver mitochondria from Ob-/-. 4) Leptin replacement completely normalized mtNOS activity and Complex I activity, with significantly decreased nitration at same level of controls. Conclusions: a)Mitochondria of Ob-/- mice tissues are exposed to high NO matrix concentration that conducts to Complex I 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 data of our group indicating that leptin reduces mtNOS activity and insulin increased mtNOS activity; d)thus, in this rodent model of metabolic syndrome, mitochondrial hypometabolism should depend on the balance between leptin deficiency and reduced sensitivity to insulin. were no significant changes of the activity of Complexes II-III and IV. 3) According to low activity, Complex I exhibited 3-4 fold increased tyrosine nitration in WAT, Muscle and Liver mitochondria from Ob-/-. 4) Leptin replacement completely normalized mtNOS activity and Complex I activity, with significantly decreased nitration at same level of controls. Conclusions: a)Mitochondria of Ob-/- mice tissues are exposed to high NO matrix concentration that conducts to Complex I 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 data of our group indicating that leptin reduces mtNOS activity and insulin increased mtNOS activity; d)thus, in this rodent model of metabolic syndrome, mitochondrial hypometabolism should depend on the balance between leptin deficiency and reduced sensitivity to insulin. Muscle (-72%; p0,05), and Liver (-61%; p0,05) from Ob-/-; there were no significant changes of the activity of Complexes II-III and IV. 3) According to low activity, Complex I exhibited 3-4 fold increased tyrosine nitration in WAT, Muscle and Liver mitochondria from Ob-/-. 4) Leptin replacement completely normalized mtNOS activity and Complex I activity, with significantly decreased nitration at same level of controls. Conclusions: a)Mitochondria of Ob-/- mice tissues are exposed to high NO matrix concentration that conducts to Complex I 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 data of our group indicating that leptin reduces mtNOS activity and insulin increased mtNOS activity; d)thus, in this rodent model of metabolic syndrome, mitochondrial hypometabolism should depend on the balance between leptin deficiency and reduced sensitivity to insulin. were no significant changes of the activity of Complexes II-III and IV. 3) According to low activity, Complex I exhibited 3-4 fold increased tyrosine nitration in WAT, Muscle and Liver mitochondria from Ob-/-. 4) Leptin replacement completely normalized mtNOS activity and Complex I activity, with significantly decreased nitration at same level of controls. Conclusions: a)Mitochondria of Ob-/- mice tissues are exposed to high NO matrix concentration that conducts to Complex I 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 data of our group indicating that leptin reduces mtNOS activity and insulin increased mtNOS activity; d)thus, in this rodent model of metabolic syndrome, mitochondrial hypometabolism should depend on the balance between leptin deficiency and reduced sensitivity to insulin.