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Aims: Acetyl-coA Carboxylase 1 (ACC1) is an enzyme that couples glucose metabolism with de novo lipid synthesis in lipogenic tissues, by converting acetyl-CoA to malonyl-CoA. In beta cells, ACC1 plays a critical role in beta cell growth and insulin secretion . However, very little is known about how ACC1 activity is regulated in beta cells. The aims of the current project were to 1) assess the role of the AMP-activated protein kinase phospho-site serine 79 (ACC1S79) in beta cell function and whole body glucose homeostasis; and 2) screen for novel phospho-sites that may regulate ACC1 activity in beta cells. Methods: (i) To investigate the functional role of the ACC1S79 phospho-site in beta cell function, we studied a mouse model with ACC1S79 mutated to alanine (ACC1S79A), which were obtained by crossing ACC1 double knock-in mice 2 with wild type mice to isolate the ACC1S79A point mutation. We assessed glucose homeostasis in vivo and insulin secretion from primary islets. (ii) We purified the ACC1 protein from transformed INS1 beta cells following culture with different glucose concentrations, and performed quantitative unbiased phosphoproteomics to characterise ACC1 phospho-sites. Mass spectrometry (LC-MS/MS) was performed using a Q- Exactive MS, mascot searches run against the Uniprot data base, and data analysed using MaxQuant and Perseus. We generated phospho-specific antibodies against key phospho-sites identified that exhibited a dynamic response to glucose stimulation, and used western blotting to investigate these sites further. Results: (i) Female ACC1S79A mice exhibited normal glucose homeostasis, whilst males showed lower fasting blood glucose, and a modest reduction in blood glucose during glucose and insulin tolerance tests compared to wild type littermate controls. No significant differences in fasted or glucose-stimulated serum insulin levels were found between the genotypes in vivo. Likewise, there was no significant change in insulin secretion from isolated ACC1S79A islets at baseline (2 mmol/l glucose) or in response to glucose-stimulation (7.5 and 20 mmol/l glucose). (ii) 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 highly significant reduction in phosphorylation in response to 15 mmol/l glucose, in contrast to the more modest changes in phosphorylation of ACC1S79. In contrast, phosphorylation of ACC1S25 and ACC1S49 was lower at basal glucose and increased upon glucose stimulation. Validation by western blotting confirmed that ACC1S1215 phosphorylation was highly-regulated by glucose, with ACC1S79 phosphorylation more subtle. Conclusions: Our data demonstrate that phosphorylation of ACC1S79 does not play a major role in insulin secretion in vivo or ex vivo. However, other phospho-sites identified by our phosphoproteomic screen may play a role in regulating ACC1 activity in beta cell and other ACC1-expressing tissues. |
