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NHLH2 EXPRESSION IN MUSCLE MAY CONTRIBUTE TO ENERGY EXPENDITURE PATHWAYS

Abstract

Jessica Melvin, Dane Fausnacht, Deborah Good. Virginia Tech, Blacksburg, VA.

BACKGROUND: Obesity is a worldwide epidemic affecting billions of people every year, with one-third of the United States population considered either overweight or obese. The transcription factor nescient-helix-loop-helix-2 (NHLH2) has been implicated in neuronal pathways that regulate energy metabolism, including exercise motivation and energy uptake - both of which are associated with obesity. An RNA sequence analysis and porcine study have recently found NHLH2 expression in muscle cells. This study sought to determine if, and when, Nhlh2 was expressed in a C2C12 myoblast cell line, which can be differentiated into myotubes. METHODS: C2C12 mouse myoblast cells were cultured in vitro to determine Nhlh2 expression as the cells differentiated from myoblasts to myotubes. A real-time qPCR analysis was performed to compare RNA from undifferentiated cells to that of the differentiated cells collected 2 hours post-treatment +/- leptin stimulation (as neuronal cell expression of NHLH2responds to leptin stimulation. NHLH2 and melanocortin-4-receptor (Mc4R) target genes were both measured, as Mc4R is a neuronal target of NHL2. A two-way ANOVA test was used to determine significance (p<0.10). RESULTS: The data confirmed that NHLH2 RNA was expressed in C2C12 mouse muscle cells, but that Mc4R expression was very low. NHLH2 expression was almost doubled in differentiated cells as compared to proliferating cells (P=0.09), with again low overall expression of Mc4R. Differences in expression between +/- leptin treatment groups were not statistically significant. CONCLUSIONS: NHLH2, previously thought to be expressed only in neuronal cells, was found to be more highly expressed in differentiated C2C12 myotubes than proliferating myoblasts, confirming its expression in muscle. As NHLH2 is a basic helix-loop-helix transcription factor similar to other muscle differentiation factors, such as MyoD, these data reveal that NHLH2-regulated pathways may be implicated in muscle differentiation and metabolism. Muscle is one of the largest energy consumers of the body, and errors in metabolism can lead to the development of muscle wasting and obesity. Further research will determine when NHLH2 expression increases during the myoblast differentiation process to determine this transcription factor’s involvement, if any, in muscle differentiation. This work was supported by the NIH R-25 grant (#R5DK2735) as part of the TOUR Scholars program, directed by TOUR Program Directors Dr. Deborah Good and Dr. Samantha Harden.

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