Abstract

The main objective of energy metabolism is the generation of adenosine triphosphate (ATP) through the reduction of both exogenous and endogenous nutrients. Skeletal muscle uses a wide variety of nutrients to generate ATP depending on the state it is in, also several reactions regulate the transformation of these nutrients to metabolic fuel for the cell, herein insulin signaling plays a key role. Insulin is the primary regulator of the postprandial metabolic response. This hormone promotes a metabolic shift towards the uptake and utilization of glucose into skeletal muscle, which is essential for maintaining glucose homeostasis. Moreover, mitochondria are organelles responsible for the conversion of glucose to ATP via the Krebs cycle. Both insulin and mitochondria are then firmly connected in the control of cell metabolism. Recent studies have shown that mitochondria form a highly dynamic interconnected network which is governed by fusion and fission processes that control their morphology depending on cell type. For this reason, in recent years the role of mitochondrial morphology and proteins that regulate, as Mfn2, Drp1 and Opa-1, in muscle energy metabolism has begun to be study. Particularly low levels of Mfn2 have been found in skeletal muscle from obese patients. Meanwhile obesity, insulin resistance and type 2 diabetes are also associated with a decrease in mitochondrial mass, which is accompanied by a lower rate of oxidative phosphorylation and abnormalities in morphology of the mitochondrial network. In summary, various studies have shown that the structure of the mitochondrial membrane and mitochondrial function depend on the balance between fusion and mitochondrial fission processes. To find the relationship between the events of mitochondrial morphology, insulin signaling and insulin resistance, a cell line from rat skeletal muscle named L6-GLUT- 4myc was used as a model. The L6-GLUT-4myc cells were transduced with an antisense adenovirus for Mitofusin 2 (AsMfn2), Micro RNA for Opa-1 or a dominant 21 negative adenovirus of Drp1 (Drp1K38A). Energy metabolism was studied through the determination of mitochondrial membrane potential, intracellular ATP content and the oxygen consumption rate. Insulin signaling was determined by Akt phosphorylation and glucose uptake, while the mitochondrial morphology was assessed by confocal microscopy. Our results show that insulin increased in mitochondrial metabolism given by an increase in oxygen consumption after 3 h of stimulation. Moreover, this metabolic response was accompanied by increases in mitochondrial fusion and mitochondrial protein Opa-1 levels, a protein critical for fusion and mitochondrial cristae remodeling. The results also showed that changes in mitochondrial morphology towards a phenotype fissioned by the use of adenovirus and microRNA against AsMfn2 and Opa- 1, promoted a reduction in the response to insulin as reflected in decreased phosphorylation of Akt , the principal protein of insulin signaling cascade, and the consequent decrease in glucose uptake. On the other hand, one of the most important findings of this research shows that insulin promotes an increase in mitochondrial Ca2+ uptake, which has been described as a mechanism for regulating the activity of the Krebs cycle, an increase that is completely inhibited by the use of mitochondrial uniporter channel inhibitor, Ruthenium Red. At the same time the pre-incubation of the myoblasts with Ruthenium Red produces a significant decrease in Akt phosphorylation together with a increased phosphorylation of AMPK. Based on these results, we concluded that there is a bidirectional control between mitochondrial morphology and insulin signaling and action, where mitochondrial Ca2+ has a key role. Thus this research promotes new targets investigation and potential therapeutic targets in the treatment of diseases of the metabolic syndrome.