Endocrine Abstracts

NADPH generated by hexose-6-phosphate dehydrogenase (H6PDH) within the lumen of the endoplasmic reticulum (ER) drives the reductase activity of 11β-hydroxysteroid dehydrogenase type 1 (11-βHSD1) allowing the production of active glucocorticoids. H6PDH knockout (H6PDHKO) mice develop a vacuolating myopathy, reduced muscle mass and display activation of the ER stress response. However, the role of glucocorticoids and 11β-HSD1 in the myopathy phenotype is not clear. Previously, we have shown in murine C2C12 muscle cells that up-regulation of 11β-HSD1 may be important in differentiation. We hypothesised that down-regulation of H6PDH activity in C2C12 cells would reduce 11β-HSD1 activity leading to inhibition of myogenic differentiation and advance our understanding of signalling pathways that may be important in H6PDHKO myopathy.

Using shRNAi technology, H6PDH and scrambled control plasmids were transfected into C2C12 cells were a series of G418 resistant stable clones selected for analysis. Real-time PCR identified C2C12 clones with greater than 85% knockdown of H6PDH mRNA. Microsome analysis indicated a reduction in H6PDH protein and enzyme activity. Scrambled controls showed no change in H6PDH expression compared to un-transfected cells. H6PDH knockdown and control myoblasts were switched to 5% horse serum media at 80% confluence and differentiated for an 8-day period during which 11β-HSD1 activity and cell morphology were assessed. H6PDH knockdown clones showed the anticipated induction of 11β-HSD1 expression during differentiation but there was a >50% reduction in 11β-HSD1 activity when compared to time-matched controls. Associated with the changes in 11β-HSD1 activity was a lack of myogenic differentiation in H6PDH knockdown cells, failing to produce organised myotubes and retaining a confluent and immature myoblastic appearance.

These initial studies indicate that H6PDH is critical to 11β-HSD1 activity in C2C12 cells facilitating muscle cell differentiation. Further studies will define the mechanisms of 11β-HSD1 dependant and independent roles of H6PDH in muscle cell metabolism and differentiation.