Endocrine Abstracts

Background: Acetyl-CoA-Carboxylase (ACC1), the rate-limiting enzyme of de novo lipogenesis, plays a critical role in beta cell growth and insulin secretion. In spite of plethora evidence for the role of ACC1 in insulin secretion and beta cells function both in vivo and in vitro, very little is known about how ACC1 activity is regulated in pancreatic beta cells. The aim of the current study was to screen for novel phospho-sites that may regulate ACC1 activity in beta cell.

Methods: ACC1 protein was purified from INS1 beta cells cultured with different glucose concentrations. Unbiased quantitative phosphoproteomics was performed to characterise ACC1 phospho-sites: LC-MS/MS was undertaken using a Q-Executive mass spectrometer and data analysed using MaxQuant and Perseus. We generated phospho-specific antibodies against key phospho-sites identified by our screen that exhibited a dynamic response to glucose stimulation, which were validated by western blotting.

Results: Using quantitative phosphoproteomics, we identified twenty phospho-sites on the ACC1 protein in beta cells. ACC1S1215 was highly phosphorylated at 2 mmol/l glucose and showed a marked and significant reduction in phosphorylation in response to 15 mmol/l glucose, in contrast to the more modest changes in phosphorylation of ACC1S79. Phosphorylation of ACC1S25 was lower at basal glucose and increased upon glucose stimulation. Validation by western blotting confirmed that ACC1S1215 phosphorylation was highly regulated by glucose.

Conclusions: This study was undertaken to expand our understanding of ACC1 regulation in beta cells. To this aim, we have generated a high-resolution phosphoproteomics profile for ACC1 protein in beta cells. We identified novel phospho-sites that show significant and dynamic regulation by glucose and may play a role in regulating beta cell ACC1 activity and insulin secretion.