Protection from Palmitate-Induced Mitochondrial DNA Damage Prevents from Mitochondrial Oxidative Stress, Mitochondrial Dysfunction, Apoptosis, and Impaired Insulin Signaling in Rat L6 Skeletal Muscle Cells

Abstract

Saturated free fatty acids have been implicated in the increase of oxidative stress, mitochondrial dysfunction, apoptosis, and insulin resistance seen in type 2 diabetes. The purpose of this study was to determine whether palmitate-induced mitochondrial DNA (mtDNA) damage contributed to increased oxidative stress, mitochondrial dysfunction, apoptosis, impaired insulin signaling, and reduced glucose uptake in skeletal muscle cells. Adenoviral vectors were used to deliver the DNA repair enzyme human 8-oxoguanine DNA glycosylase/(apurinic/apyrimidinic) lyase (hOGG1) to mitochondria in L6 myotubes. After palmitate exposure, we evaluated mtDNA damage, mitochondrial function, production of mitochondrial reactive oxygen species, apoptosis, insulin signaling pathways, and glucose uptake. Protection of mtDNA from palmitate-induced damage by overexpression of hOGG1 targeted to mitochondria significantly diminished palmitate-induced mitochondrial superoxide production, restored the decline in ATP levels, reduced activation of c-Jun N-terminal kinase (JNK) kinase, prevented cells from entering apoptosis, increased insulin-stimulated phosphorylation of serine-threonine kinase (Akt) (Ser473) and tyrosine phosphorylation of insulin receptor substrate-1, and thereby enhanced glucose transporter 4 translocation to plasma membrane, and restored insulin signaling. Addition of a specific inhibitor of JNK mimicked the effect of mitochondrial overexpression of hOGG1 and partially restored insulin sensitivity, thus confirming the involvement of mtDNA damage and subsequent increase of oxidative stress and JNK activation in insulin signaling in L6 myotubes. Our results are the first to report that mtDNA damage is the proximal cause in palmitate-induced mitochondrial dysfunction and impaired insulin signaling and provide strong evidence that targeting DNA repair enzymes into mitochondria in skeletal muscles could be a potential therapeutic treatment for insulin resistance.

Palmitate-Induced Cell Death and Mitochondrial Respiratory Dysfunction in Myoblasts are Not Prevented by Mitochondria-Targeted Antioxidants

“…We found mtDNA damage after the treatment with palmitate in both myoblasts and myotubes using the QPCR method, which is able to detect a wide range of types of DNA lesions. Our observation is in accordance with another study [18] where mtDNA damage was detected in L6 myotubes exposed to palmitate using quantitative alkaline Southern blot analysis. Authors of this study postulated that mtDNA damage is a critical event leading to mitochondrial dysfunction with consequent increase in oxidative stress and finally to apoptosis [18].…”

“…Increased oxidative stress associated with some features of mitochondrial dysfunction was reported in skeletal muscle cells after the exposure to palmitate [17,18]. However, the effects of saturated fatty acids on mitochondrial respiration have not been well studied, particularly in undifferentiated skeletal muscle cells.…”

“…There are several studies in cultured skeletal muscle cells showing an increase of mitochondrial ROS production induced by palmitate [14,15,16] accompanied by mtDNA damage and some features of mitochondrial dysfunction [17,18]. Mitochondrial DNA has an important role in the regulation of mitochondrial respiration and oxidative damage of mtDNA was postulated as an initial event leading to mitochondrial dysfunction [18]. …”

Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE

“…In this time frame, only PKR is phosphorylated in response to ceramide without any change in JNK phosphorylation state, suggesting that ceramide-induced PKR-mediated JNK phosphorylation requires more time (up to 16 h). This is not unprecedented because several studies show that incubation of myotubes from 16 -24 h is necessary to see palmitate or ceramide induce JNK phosphorylation (45)(46)(47)(48). This result confirms previous data demonstrating that, in the short term, ceramide inhibits insulin signaling through the acti-FIGURE 8.…”

Defining the role of DAG, mitochondrial function, and lipid deposition in palmitate-induced proinflammatory signaling and its counter-modulation by palmitoleate

“…We found that muscle cells incubated with palmitate exhibited a lower OCR than those exposed to palmitoleate alone but that cellular respiration and TAG accumulation was augmented in the presence of both fatty acids together. The reduced oxidative capacity associated with oversupply of SFA is likely to be a consequence of mitochondrial dysfunction given that i ) we observed a reduction in Cox4.1 and PGC1 ␣ (two proteins with important but distinct roles in mitochondrial biology) and ii ) recent studies show that palmitate causes mitochondrial damage/fragmentation in muscle cells by inducing a greater infl ammatory drive, insulin resistance, and apoptosis ( 60,61 ). In contrast, promoting oxidation and/or storage of SFAs by coprovision of MUFAs reduces the proapoptotic drive of palmitate ( 58 ), and as we and others ( 6 ) found, it also suppresses NF B-mediated proinfl ammatory signaling.…”