Glycogen synthesis from lactate in skeletal muscle of the painted turtle in vitro.

Abstract

Painted turtles (Chrysemys picta) can accumulate lactate loads approaching 200 mM while over wintering in ice‐covered ponds, a feat achieved, in part, because of large glycogen stores in the liver and skeletal muscle. Mammals cannot directly synthesize glycogen from lactate in muscle and rely on liver Cori cycling for this purpose. To investigate the potential for gluconeogenesis from lactate in skeletal muscle in this species, turtles were made anoxic for 24 hours at 20°C to deplete skeletal muscle glycogen. Bundles of iliofibularis muscle were isolated and incubated in HEPES or CO2/HCO3− buffered solutions for 8 hours to determine the dependence of glycogen repletion on the buffering system at 20°C. Bundles were also incubated for 4 hours in solutions containing L‐[14C(U)]‐lactate with varying concentrations of glucose and lactate, and then analyzed for the amount of 14C that was oxidized or incorporated into muscle glycogen. Muscle glycogen synthesis required the presence of CO2 and HCO3−. The rates of 14C‐lactate oxidation and 14C‐incorporation into glycogen were independent of extracellular glucose, but dependent on extracellular lactate concentrations. The involvement of PEPCK, a key enzyme in gluconeogenesis, was also assessed using the specific inhibitor, 3‐mercaptopicolinic acid (MPA). 100 μM MPA inhibited 40% of the 14C‐incorporation into muscle glycogen when added to lactate incubations. To confirm the specificity of MPA on turtle PEPCK, enzyme assays were carried out with liver tissue homogenate. 100 μM MPA inhibited 86% of cytosolic PEPCK activity and 61% of mitochondrial PEPCK activity. This work confirms that the skeletal muscle mass of the turtle is important in the direct metabolism of lactate and provides additional support for the hypothesis that in situ skeletal muscle gluconeogenesis is the norm among vertebrates, rather than the exception.Support or Funding InformationThis study was funded by a National Institutes of Health R15 awarded to DEW.