Abstract
BackgroundIn the metabolic syndrome with hyperinsulinemia, mitochondrial inhibition facilitates muscle fat and glycogen accumulation and accelerates its progression. In the last decade, nitric oxide (NO) emerged as a typical mitochondrial modulator by reversibly inhibiting citochrome oxidase and oxygen utilization. We wondered whether insulin-operated signaling pathways modulate mitochondrial respiration via NO, to alternatively release complete glucose oxidation to CO2 and H2O or to drive glucose storage to glycogen.Methodology/Principal FindingsWe illustrate here that NO produced by translocated nNOS (mtNOS) is the insulin-signaling molecule that controls mitochondrial oxygen utilization. We evoke a hyperinsulinemic-normoglycemic non-invasive clamp by subcutaneously injecting adult male rats with long-lasting human insulin glargine that remains stable in plasma by several hours. At a precise concentration, insulin increased phospho-Akt2 that translocates to mitochondria and determines in situ phosphorylation and substantial cooperative mtNOS activation (+4–8 fold, P<.05), high NO, and a lowering of mitochondrial oxygen uptake and resting metabolic rate (−25 to −60%, P<.05). Comparing in vivo insulin metabolic effects on gastrocnemius muscles by direct electroporation of siRNA nNOS or empty vector in the two legs of the same animal, confirmed that in the silenced muscles disrupted mtNOS allows higher oxygen uptake and complete (U-14C)-glucose utilization respect to normal mtNOS in the vector-treated ones (respectively 37±3 vs 10±1 µmolO2/h.g tissue and 13±1 vs 7.2±1 µmol 3H2O/h.g tissue, P<.05), which reciprocally restricted glycogen-synthesis by a half.Conclusions/SignificanceThese evidences show that after energy replenishment, insulin depresses mitochondrial respiration in skeletal muscle via NO which permits substrates to be deposited as macromolecules; at discrete hyperinsulinemia, persistent mtNOS activation could contribute to mitochondrial dysfunction with insulin resistance and obesity and therefore, to the progression of the metabolic syndrome.
Highlights
The powerhouse of the cell, mitochondria are responsible for sustaining energy levels
Insulin increases p-Akt2 in skeletal muscle mitochondria Under fairly constant stimulation, insulin early increased the expression of Akt2 and phospho-Akt (p-Akt) in cytosol and mitochondria from skeletal muscle; thereafter, only mitochondrial Akt2 and p-Akt remained very high up to twelve hours after insulin
Flow cytometry of the isolated and purified mitochondria confirmed a net increase of p-Akt fluorescence, with a similar temporal kinetics to that detected by western blotting. (Figure1B)
Summary
The powerhouse of the cell, mitochondria are responsible for sustaining energy levels. Critical reduction of respiration takes part as well in the mechanisms of prevalent illnesses; a reduction of mitochondrial activity and the decrease in energy expenditure contribute substantially to metabolic dysfunction in aging, insulin resistance and diabetes and conducts to lipid accumulation [3]. Of the many studies focused on insulin resistance and mitochondrial dysfunction in the last decade [4], few have critically examined mitochondrial activity in terms of alternation between complete substrate oxidation and storage as macromolecular deposits [glycogen or fat stores]. Lipid and glycogen accumulation further increases insulin resistance [6], a clear mechanism for reduction of mitochondrial oxidations with displacement of substrates to storage was not defined in this context yet. We wondered whether insulin-operated signaling pathways modulate mitochondrial respiration via NO, to alternatively release complete glucose oxidation to CO2 and H2O or to drive glucose storage to glycogen
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