Knockdown of ATP citrate lyase in pancreatic beta cells does not inhibit insulin secretion or glucose flux and implicates the acetoacetate pathway in insulin secretion

Mahmoud El Azzouny; Melissa J Longacre; Israr-Ul H Ansari; Robert T Kennedy; Charles F Burant; Michael J MacDonald

doi:10.1016/j.molmet.2016.07.011

Knockdown of ATP citrate lyase in pancreatic beta cells does not inhibit insulin secretion or glucose flux and implicates the acetoacetate pathway in insulin secretion

Mol Metab. 2016 Aug 8;5(10):980-987. doi: 10.1016/j.molmet.2016.07.011. eCollection 2016 Oct.

Authors

Mahmoud El Azzouny¹, Melissa J Longacre², Israr-Ul H Ansari², Robert T Kennedy³, Charles F Burant¹, Michael J MacDonald⁴

Affiliations

¹ Metabolism, Endocrinology and Diabetes, University of Michigan Medical School, Ann Arbor, MI 48109, United States.
² Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, United States.
³ Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, United States.
⁴ Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, United States. Electronic address: mjmacdon@wisc.edu.

Abstract

Objective: Glucose-stimulated insulin secretion in pancreatic beta cells requires metabolic signals including the generation of glucose-derived short chain acyl-CoAs in the cytosol from mitochondrially-derived metabolites. One concept of insulin secretion is that ATP citrate lyase generates short chain acyl-CoAs in the cytosol from mitochondrially-derived citrate. Of these, malonyl-CoA, is believed to be an important signal in insulin secretion. Malonyl-CoA is also a precursor for lipids. Our recent evidence suggested that, in the mitochondria of beta cells, glucose-derived pyruvate can be metabolized to acetoacetate that is exported to the cytosol and metabolized to the same short chain acyl-CoAs and fatty acids that can be derived from citrate. We tested for redundancy of the citrate pathway.

Methods: We inhibited ATP citrate lyase activity using hydroxycitrate as well as studying a stable cell line generated with shRNA knockdown of ATP citrate lyase in the pancreatic beta cell line INS-1 832/13.

Results: In both instances glucose-stimulated insulin release was not inhibited. Mass spectrometry analysis showed that the flux of carbon from [U-(13)C]glucose and/or [U-(13)C]α-ketoisocaproic acid (KIC) into short chain acyl-CoAs in cells with hydroxycitrate-inhibited ATP citrate lyase or in the cell line with stable severe (>90%) shRNA knockdown of ATP citrate lyase was similar to the controls. Both (13)C-glucose and (13)C-KIC introduced substantial (13)C labeling into acetyl-CoA, malonyl-CoA, and HMG-CoA under both conditions. Glucose flux into fatty acids was not affected by ATP citrate lyase knockdown.

Conclusion: The results establish the involvement of the acetoacetate pathway in insulin secretion in pancreatic beta cells.

Keywords: ACC, acetyl-CoA carboxylase; ACLY, ATP citrate lyase; Acetoacetate pathway; Acetyl-CoA; Citrate; KIC, α-ketoisocaproic acid; Malonyl-CoA; Mass spectrometry; Mitochondrial biosynthesis; Palmitate; SCOT, succinyl-CoA:3-ketoacid-CoA transferase; ZMP, 5-aminoimidazole-4-carboxamide ribonucleotide.

Abstract

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