Endocrine Abstracts

Introduction: Phosphoenolpyruvate carboxykinase (PEPCK) is the key rate determining enzymes of gluconeogenesis pathway. PEPCK exists in cytosolic (PEPCK-C) and mitochondrial (PEPCK-M) isoforms. PEPCK isoforms maintain glucose/lipid homeostasis and is being explored as a therapeutic target for treating metabolic diseases. We tested the influence of a naturally occurring compound Genistein (a soy derived isoflavone) to constitutively regulate PEPCK isoforms to address its potential use as a therapeutic molecule.

Methods: HepG2 and fibroblasts were tested for the effects of genistein by western blot; RNA stability was examined by RT-PCR using actinomycin treated cells; transcriptional regulation was performed by reporter assays and CpG DNA methylation was analysed by bisulfite sequencing.

Results: Expression and activity of PEPCK isoforms in fibroblasts was lower than in HepG2 hepatoma cells. In HepG2 cells, genistein induced degradation of PEPCK-C transcript and proteins but increased PEPCK-M expression. In fibroblasts, genistein increased PEPCK-C expression through promoter demethylation but PEPCK-M was unaffected. Reporter gene and RT-PCR assays suggested genistein mediated RNA degradation of PEPCK-C in HepG2 cells is due to the existence of AU-rich elements (AREs) in 3′-UTR of the gene.

Conclusion: We show an evidence for the regulation of PEPCK-C gene by promoter DNA methylation in human fibroblasts, which might be responsible for maintaining baseline PEPCK activity in non-gluconeogenic tissues. We show selective effect of soy isoflavone genistein on PEPCK isoform genes in hepatic and extra hepatic cell types by increasing the expression of PEPCK-M as a compensatory mechanism and there by maintaining glucose homeostasis in cells as a pro-survival effect.

Disclosure: Department of Biotechnology, Government of India.