br Results br Discussion br Conclusion The flux

Results
Discussion
Conclusion The flux of glucose into cytosolic short chain acyl-CoAs was maintained in pure beta Magnolol in the presence of inhibition of ATP citrate lyase with hydroxycitrate and also in a cell line with >90% ATP citrate lyase knockdown suggesting that a pathway other than the citrate pathway is responsible for the flow of carbons into short chain acyl-CoAs during insulin secretion. The only known pathway other than through the ATP citrate lyase reaction leading to the formation of short chain acyl-CoAs in the cytosol is the acetoacetate pathway (Figure 4, Figure 5A). In the ATP citrate lyase knockdown beta cell line prior to stimulation with glucose the level of malonyl-CoA was lower compared to the control cell line, but with glucose stimulation the increase in the level of malonyl-CoA and glucose incorporated into malonyl-CoA in the ATP citrate lyase knockdown cell line was similar to that in the control cell line (Figure 4E). The results suggest that in resting beta cells the citrate pathway, with or without help from the acetoacetate pathway, maintains short chain acyl-CoA levels in the cytosol, but during insulin stimulatory conditions, such as in the presence of increased concentrations of glucose, the acetoacetate pathway is primarily responsible for supplying cytosolic short chain acyl-CoAs to the cytosol. This idea is consistent with the fact that during glucose stimulation the mitochondrial concentration of succinate increases to high levels and SCOT, which uses succinate as a substrate to form acetoacetate, has a relatively high Km for succinate [10], [15] and, therefore, the acetoacetate pathway can act as a sensor during glucose-stimulated insulin secretion. Glucose incorporation into palmitate in the ATP citrate lyase knockdown cells was even slightly higher in the ATP citrate lyase knockdown cell line than in the control cell line (Figure 5C). This also supports the idea of an active acetoacetate pathway supplying cytosolic acetyl-CoA and malonyl-CoA during glucose-stimulated insulin secretion.
Author contribution
Funding This work was supported by the National Institutes of Health [grant numbers NIH DK28348 to MJM and NIH DK046960 to RTK]; the Nowlin Family Trust of the InFaith Community Foundation (to MJM); The Michigan Regional Comprehensive Metabolomics Resource Core Grant U24 [grant number DK097153]; and the A. Alfred Taubman Institute (to C.F.B).
Acknowledgements
Introduction ATP citrate lyase (ACLY) is a cytosolic enzyme responsible for the synthesis of acetyl-CoA [1]. It catalyzes the formation of acetyl-CoA and oxaloacetate from citrate and CoA with a simultaneous hydrolysis of ATP to ADP and phosphate. The product, acetyl-CoA, is involved in several important biosynthetic pathways, including lipogenesis and cholesterogenesis. Acetyl-CoA is also required for acetylation reactions that modify proteins such as histone acetylation.
Tissue distribution and subcellular localization ACLY is most abundantly expressed in the liver and white adipose tissue [2], [3] while it exhibits low expression levels in brain, heart, small intestine and muscles [2], [4]. ACLY is also expressed and active in pancreatic beta cells [3], [5]. Additionally, over-expression of ACLY is associated with certain pathological conditions that will be discussed later in this article. ACLY is mainly a cytosolic enzyme. It is reported that ACLY is bound to endoplasmic reticulum in mammalian cells [6]. However, recently it was showed that, in addition to cytoplasm, ACLY is also detected in nuclei of different mammalian cells [7]. ACLY was identified in both nucleus and cytoplasm of human glioblastoma cells, mouse embryonic fibroblasts, murine pro-B-cell lymphoid cells and human colon carcinoma cells [7]. Citrate is a small molecule that can diffuse freely through the nuclear pore complex [8] hence, ACLY-mediated acetyl-CoA production may occur in both cytoplasmic and nuclear compartments of mammalian cells [7].