Retinoic Acid, Insulin, and Cycloheximide Alter Glycogen Homeostasis in Differentiating L6 Myoblasts

Glycogen synthesis in skeletal muscle (SM) is responsible for the majority of post-prandial glucose disposal in healthy individuals. In type 2 diabetes mellitus (T2DM), compromised skeletal muscle glycogen synthesis contributes to hyperglycemia. L6 rat SM cells can develop insulin resistance with prolonged insulin stimulation and provide an in vitro model for mechanistic dysfunction in glycogen homeostasis. SM glycogen is synthesized by glycogen synthase (GS) in response to insulin stimulation. Canonically, insulin signaling increases GS activity through inhibition of its Ser641 regulator glycogen synthase kinase-3β (GSK3β) by protein kinase B (AKT). Proximal insulin signaling activates AKT via Ser473 phosphorylation which proceeds to inhibit GSK3β via Ser9 phosphorylation. All-trans-retinoic acid (RA) has been reported to enhance insulin stimulated glycogen accumulation after 6 days of treatment in terminally differentiated L6 myotubules. We hypothesized that RA would also enhance insulin stimulated glycogen accumulation during myoblast differentiation through altered AKT-GSK3β-GS regulation. Differentiating L6 myoblasts treated with RA (0.5 µM) and insulin (10 nM) were harvested after 2 and 6 days of treatment. We reported that RA significantly enhanced insulin stimulated glycogen accumulation after 2 days of treatment without affecting pAKT (Ser473), pGS (Ser641), or pGSK3β (Ser9) expression. After 6 days of treatment, insulin induced glycogen accumulation as well as the expression of both pGS (Ser641) and pGSK3β (Ser9) without additional effects from RA. We proceeded to investigate the alterations of these key glycogen regulators further using the protein synthesis inhibitor cycloheximide (CHX) during a dose response time course study. CHX dramatically induced the expression of pGSK3β vi (Ser9) and pGS (Ser641) between 24 and 48 hours, indicating a shift in GS regulation away from GSK3β. CHX (10 µM) also inhibited GSK3β expression, dramatically induced pGSK3β (Ser9) expression, and attenuated RA (0.5 µM) and insulin (10 nM) stimulated glycogen accumulation after 48 hours. In contrast, CHX (10 nM) induced glycogen accumulation after 3 hours of treatment in rat SM ex vivo but did not alter expression of key regulatory proteins. The main findings of this dissertation indicate that the currently understood mechanisms of glycogen homeostasis in SM are far from complete.