Role of c-Cbl in skeletal muscle energy metabolism in relation to obesity

The obesity epidemic has already reached alarming rates in both developed and developing countries. Positive energy balance due to high calorie intake with decreased energy expenditure resulted in the current pandemic of obesity. Obesity is associated with other metabolic diseases, including type 2 diabetes (T2D). Skeletal muscle plays a major role in the control of whole-body glucose and lipid metabolism. Obese subjects have an excessive accumulation of lipid species, which interferes with the activation of insulin signaling pathways in key metabolic tissues such as the liver and skeletal muscle. Therefore, understanding the molecular mechanisms regulating energy expenditure in skeletal muscle, in particular, mitochondrial function and mitochondrial oxidative capacity has been a major research focus in obesity. It has been reported that mice lacking the c-Casitas B-lineage Lymphoma protein (c-Cbl) exhibit enhanced energy expenditure and insulin sensitivity, indicating that c-Cbl is a major factor contributing to the maintenance of whole-body energy homeostasis. c-Cbl was initially identified as an E3 ubiquitin ligase involved in the regulation of tyrosine kinase signaling. Previous studies showed that global knockout (KO) of c-Cbl in mice protects against high fat (HFat)-induced obesity and insulin resistance by increasing energy expenditure. However, this was a whole-body metabolic result and not specific to any tissue. Based on the literature review and analysis of the gaps in our current knowledge (Chapter 1), the overall aim of this thesis was established to investigate the role of c-Cbl in muscle energy metabolism. The first aim (Chapter 3) was to characterize the pathologic features and the changes of the Cbl family in different metabolic tissues during diet-induced obesity (DIO) in mice by a HFat diet. The working hypothesis was that a HFat diet would induce obesity and glucose intolerance in mice and alter Cbl expression in skeletal muscle, which may contribute to body weight gain. The first aim was addressed in C57BL/6J male mice fed a HFat diet, which induces obesity and insulin resistance. The results showed that the HFat diet induced adiposity (as indicated by more than 80% of body weight gain and a threefold increase in epididymal fat mass), glucose intolerance, lipid accumulation (increased triglycerides deposition in liver and muscle), and impaired muscle formation (suppressed myogenin expression levels). Associated with the development of metabolic syndrome, c-Cbl content was upregulated in skeletal muscle but not in the liver and white adipose tissue, and Casitas B-lineage Lymphoma protein-b (Cbl-b) was decreased in skeletal muscle by HFat-fed mice. These findings suggest that c-Cbl expression is specifically increased in skeletal muscle during HFat-induced obesity. Based on these findings the second aim (Chapter 4) was to investigate the mechanism of c-Cbl regulation in muscle mitochondrial function, mitochondrial oxidative capacity, mitochondrial biogenesis, and mitochondrial degradation. The working hypothesis was that c-Cbl knockdown (KD) may enhance muscle mitochondrial function, mitochondrial oxidative capacity and mitochondrial biogenesis and degradation in muscle cells. The second aim was addressed in the C2C12 muscle cell line by knocking down c-Cbl. Using a specific shorthairpin RNA (shRNA), expression of c-Cbl protein was reduced during muscle proliferation (myoblasts), and this reduction was maintained until muscle differentiation (myotubes). c-Cbl KD resulted in a significant increase in oxygen consumption basally and during maximal respiration in myotubes by about 20%. These data suggest that skeletal muscle is likely to be a major site of c-Cbl-mediated energy metabolism for the whole-body. We observed a significant increase in the phosphorylation of adenosine onophosphate protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in c-Cbl KD myotubes. The protein levels of mitochondrial complexes and the mitochondrial biogenesis in c-Cbl KD myotubes were increased compared with control myotubes. These findings suggest that c-Cbl KD increases the ability of energy metabolism in muscle cells. The third aim (Chapter 5) was to investigate the role of c-Cbl in muscle formation /myogenesis. The working hypothesis was that c-Cbl KD may enhance myogenesis and reduce lipid accumulation in mature muscle. This was addressed in the C2C12 muscle cell line by knocking down the expression of c-Cbl to investigate the specific effect of c-Cbl on muscle formation/myogenesis during muscle differentiation. The results determined that c-Cbl KD in myotubes enhanced myotube formation. While c-Cbl upregulation in skeletal muscle is associated with obesity, downregulation of c-Cbl promotes energy expenditure in muscle cells independent of AMPK activation. In addition, c-Cbl KD in myogenic differentiation reduced lipid accumulation under insulin-resistant conditions. These findings suggest that c-Cbl KD promotes myogenesis and may enhance insulin sensitivity. Chapter 6 summarizes the major findings and conclusions, and it discusses the limitations of studies in this thesis and how future studies could address the remaining issues and potential implications for humans. Overall, our data demonstrate that obesity affects c-Cbl expression in skeletal muscle. c-Cbl KD in mature muscle cells results in enhanced mitochondrial function. These findings suggest that c-Cbl in skeletal muscle is an important regulator of energy metabolism, and it may be targeted for the treatment of obesity and associated metabolic disorders