In our studies of leucine and BCH in

In our studies of leucine and BCH in pancreatic islets and clonal insulin cell lines, we noticed that leucine was a slightly stronger stimulant of insulin release. Previous studies showed that leucine can be metabolized to CO2[11], [12], [13], [14] and can increase acetoacetate in pancreatic islets [15] and multiple short chain acyl-CoAs in INS-1 fak pathway [16], but were not meant to directly answer the question of whether leucine's metabolism contributes to its insulinotropism. No one has previously reported that leucine is a stronger insulin stimulant than BCH. We thought that the idea of leucine's metabolism to acetoacetate might converge with a concept about the role of acetoacetate as a carrier of acyl groups from mitochondria to cytosol in the beta cell [16]. We hypothesized that discerning the differences between leucine and BCH might reveal more information about the metabolic pathways involved in insulin secretion. In the present project, we studied the response of pancreatic beta cells to incubation with leucine and BCH in more detail. We compared the effects of BCH and leucine on the activation of glutamate dehydrogenase enzyme activity, on insulin release and on the levels of acetoacetate, α-ketoglutarate and short chain acyl-CoA levels in pancreatic islets and/or the INS-1 832/13 cell line. We also compared leucine's and BCH's ability to stimulate insulin release in an INS-1 832/13 cell line we made deficient in ATP citrate lyase so that the cells were unable to convert citrate exported from the mitochondria into acetyl-CoA in the cytosol. The results support the idea that in addition to leucine enhancing flux through glutamate dehydrogenase, leucine's metabolism to acetoacetate and acetyl-CoA contributes to its insulinotropism. Besides being informative about leucine-induced insulin release, the results strengthen the theory that the normal beta cell can export acyl carbon as acetoacetate, in addition to citrate, from the mitochondria to the cytosol to form short chain acyl-CoAs which may have roles in insulin secretion.
Materials and methods
Results
Discussion Part of the mechanism by which leucine stimulates insulin secretion is by its allosterically activating glutamate dehydrogenase (Fig. 5, reaction 11) which increases metabolism of endogenous glutamate [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. This conclusion is supported by experiments in which BCH, a non-metabolizable analog of leucine, activates glutamate dehydrogenase and stimulates insulin release from pancreatic beta cells (references [1], [2], [3], [4] and Table 1 and Fig. 1, Fig. 2). However, leucine can be metabolized by islet cells (Table 2, Table 3 and references [11], [12], [13], [14], [15]) and the contribution of leucine metabolism to its insulinotropism has not been thoroughly studied. The results of the current study as discussed below suggest two points about insulin secretion. First they indicate that leucine's conversion to acetoacetate and acetyl-CoA and to other short chain acyl-CoAs (Table 4), as well as its oxidation to CO2 (Table 2), contributes to its ability to stimulate insulin secretion and explain why it is a stronger insulin secretagogue than BCH even though BCH and leucine are equally strong as activators of glutamate dehydrogenase (Fig. 3 and reference [25]). Second, experiments with agents that supplied acetoacetate to rescue decreased BCH-induced insulin release (Fig. 4) in cells made deficient in ATP citrate lyase (Fig. 5, reaction 2) strengthen the recent conclusion [16] that the pathways of formation of cytosolic short chain acyl-CoAs in the beta cell involve the transfer of acyl carbon from mitochondria to the cytosol as acetoacetate in addition to citrate.
Acknowledgements