206-LB: Increase of Hexokinase-2 Protein without Increase of Other Glycolytic Enzymes in Myotubes with High Glucose Concentration Supports the Hypothesis of Hexokinase-2-Linked Unscheduled Glycolysis as a Driver of Insulin Resistance

Abstract

Insulin resistance in skeletal muscle is the major metabolic defect leading to the development of type 2 diabetes. The initiating dysfunction driving development of insulin resistance has not been disclosed. Recently, stabilization of hexokinase-2 (HK2) to proteolysis by increased glucose concentration, increasing flux through early-stage glycolysis without increase in expression and activity of other glycolytic enzymes, was identified as the initiator of metabolic dysfunction in hyperglycemia. This creates a wave of increased glycolytic intermediates activating hexosamine and protein kinase C pathways and increasing formation of methylglyoxal. Increased glucose-6-phosphate displaces HK2 from mitochondria, producing mitochondrial hyperpolarization and dysfunction. This is termed HK2-linked unscheduled glycolysis. Skeletal muscle exposed to increased fasting plasma glucose may stimulate this. To test this, we studied glycolytic enzyme abundance in murine C2C12 myotubes incubated with 5.5 and 25 mM glucose for 5 days, determining HK2 protein changes by Western blotting, normalized to ß-actin. Protein abundances in C2C12 myotube cultures with 5.5, 11.1 and 25.0 mM glucose determined by label-free quantitative mass spectrometry proteomics were mined from published data. In C2C12 myotubes, HK2 protein increased 46 ± 5 % in 25 mM glucose (P<0.001, n = 3; Western blotting) . Proteomics corroborated this with 1.5 and 1.8 fold increase of HK2 in 11.1 and 25.0 mM glucose cultures, compared to 5.5. mM glucose control (P<0.01, n = 3) . Abundances of other glycolytic enzymes, hexokinase-1, glucose-6-phosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, were unchanged. This establishes the conditions for HK2-linked glycolytic overload. We conclude that high glucose concentration in C2C12 myotubes in vitro exhibits conditions producing hexokinase-2-linked unscheduled glycolysis Disclosure M. Xue: None. N. Rabbani: None. P. Thornalley: None. Funding Qatar Foundation, QB14Qatar University, QUHI-CMED-21_22-1