Abstract

Background: Insulin stimulates glucose uptake in skeletal muscle and adipose tissue, as well as suppresses glucose production by the liver. Type 2 diabetes mellitus (T2DM) results from insulin insensitivity in several tissues, skeletal muscle being the most important. In obese patients with hyperlipidemia, free fatty acid (FFA) accumulation in non-adipose tissues induces insulin insensitivity in skeletal muscle and contributes thereby to T2DM. This effect is mimicked by the palmitic acid, the most abundant saturated dietary FFA. Treating cultured skeletal muscle cells with palmitic acid decreases insulin- dependent activation of glycogen synthesis and glucose transport. Insulin binding to its receptor activates the Akt pathway, which inhibits activation of the transcription factor FoxO1 in skeletal muscle. FoxO1 regulates muscle energy homeostasis by controlling glycolysis and lipolysis, and mitochondrial metabolism. Recently, we found that: (a) FoxO1 is an important mediator of diabetic cardiomyopathy (Battiprulu et al JCI 2012); (b) Insulin stimulates mitochondrial fusion and function in cardiomyocytes and skeletal muscle cells (Parra et al. Diabetes 2014); (c) In this latter cells, mitofusin-2 (Mfn2) and the complex VDAC1- Grp75-IP3R coordinate mitochondria and ER coupling, as well as regulates insulin signaling and glucose homeostasis (Del Campo et al Am J Physiol 2014). We recently proposed that dysfunctional mitochondria-endoplasmic reticulum (ER) communication plays a key role in metabolic diseases (López-Crisosto el BBA 2015). In this proposal, our research will focus on two novel players in this context: MUL1 and HERPUD1. The outer mitochondrial membrane E3 ligase MUL1 has been linked to the regulation to mitophagy, Akt regulation and regulation of mitochondrial morphology. In skeletal muscle, MUL1 promotes proteosomal degradation of Mfn2. On the other hand, HERPUD1 (or Herp) is an ER protein whose levels acutely increase in response to ER stress. HERPUD1 forms complexes with the IP3R and the RYR leading to proteasome-dependent degradation of both, and in doing so modulates Ca2+ release from the ER. These observations suggest that MUL1 and HERPUD1 may inhibit Ca2+ transfer between the ER and mitochondria. Hypothesis: "Lipotoxicity due to elevated palmitic acid levels induces insulin insensitivity in skeletal muscle cells through a mechanism involving dysfunctional ER-mitochondria communication triggered by FoxO1-dependent increased expression of HERPUD1 and MUL1". Specific aims /experimental design: Aim 1: To determine whether palmitic acid and FoxO1 control physical and functional mitochondrial-ER communication in skeletal muscle cell lines. Aim 2: To evaluate whether palmitic acid increases HERPUD1 and MUL1 expression by a FoxO1-dependent mechanism in skeletal muscle cell lines. Aim 3: To determine whether HERPUD1 and MUL1 disrupt mitochondria-ER coupling in skeletal muscle cell lines. Aim 4: To investigate whether the dysregulation of mitochondria-ER coupling is responsible for palmitic acid-induced insulin insensitivity in skeletal muscle cell lines. Aim 5: To assess the expression of HERPUD1 and MUL1 and correlation with insulin insensitivity in skeletal muscle samples obtained from patients with and without T2DM, as well as wild type and FoxO1 ko mice fed on a high fat diet (HFD, experimental model of insulin resistance and T2DM). Cultured skeletal muscle cells will be treated with palmitic acid. Mitochondria will be stained with mitotracker green and ER with anti-KDEL antibody. Colocalization of ER with mitochondria will be analyzed by confocal microscopy and electron microscopy. Functional ER-mitochondria coupling will be assessed by measuring mitochondrial Ca2+. Mitochondrial bioenergetics will be assessed by measuring mitochondrial membrane potential, ROS production, O2 consumption and ATP content. Activation of FoxO1 will be evaluated by assessing phospho- FoxO1 levels by Western blotting, nuclear translocation of FoxO1 and target gene expression. FoxO1 will be manipulated using adenoviral overexpression of wild type and dominant negative FoxO1, or FoxO1 siRNA. HERPUD1 and MUL1 mRNAs and protein levels will be assessed by RT-qPCR and Western blotting. HERPUD1 and MUL1 will be either overexpressed using available constructs or silenced using siRNA. ER- mitochondria coupling will be manipulated using a plasmid encoding an artificial ER-mitochondria linker protein. Activation of insulin signaling pathways will be evaluated by phosphorylation of insulin receptor, IRS-1, Akt, mTOR, GLUT-4 externalization and/or glucose uptake. Skeletal muscle samples from patients with and without T2DM and from FoxO1 knock out mice fed on a high fat diet will be obtained. HERPUD1 and MUL1 mRNA and/or protein levels and insulin signaling will be evaluated. Expected results: Palmitic acid or HFD should decrease in vitro and in vivo in skeletal muscle ER-mitochondrial coupling and bioenergetics by modulating signaling via the FoxO1/HERPUD1/MUL1 pathway. Relevance: The alarming expansion of T2DM associated with obesity and sedentarism is a worldwide health problem. Our research will provide new insights to obesity-induced insulin resistance and mechanisms leading to T2DM. Manipulation of ER-mitochondria coupling through the FoxO1/HERPUD1/MUL1 pathway may emerge as a novel therapeutic approach to prevent T2DM in obese patients.