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

Abstract

ObjectiveGlucose-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.MethodsWe 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.ResultsIn both instances glucose-stimulated insulin release was not inhibited. Mass spectrometry analysis showed that the flux of carbon from [U-13C]glucose and/or [U-13C]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 13C-glucose and 13C-KIC introduced substantial 13C 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.ConclusionThe results establish the involvement of the acetoacetate pathway in insulin secretion in pancreatic beta cells.

Lipid-associated metabolic signalling networks in pancreatic beta cell function

“…Similarly, the ACL inhibitor hydroxycitrate lacked effect on GSIS in INS1(832/13) cells and rat islets [68]. In 13 C isotopomer flux measurements, inhibiting the flux of glucose carbons into malonyl-CoA by hydroxycitrate had no effect on GSIS in INS1 cells [40]. Finally, the use of the FA-CoA synthase inhibitor, triascin C, did not reveal a modification of GSIS in INS-1 cells [69] or INS 832/13 cells [70].…”

“…Suppression of ACL ACL knockdown was reported to have no effect on GSIS in INS-1(832/13) cells when measured only at maximal 16.7 mmol/l glucose, possibly due to the absence of NEFA in the incubation medium for insulin secretion as well as GSIS not being tested at submaximal glucose [68]. The lack of effect could also be due to the presence of alternate routes that provide cytosolic acetyl-CoA, as mentioned above [40]. We reported that ACL knockdown in the same cell line under similar experimental conditions does result in reduced GSIS but that the inhibitory effect is most prominent in the presence of exogenous NEFA or at intermediate glucose [34].…”

“…The possibility of cytosolic acetyl-CoA being generated from glucose-derived pyruvate by alternative routes was suggested by the observation that inhibition of ACL still allows glucose carbon incorporation into lipids without compromising GSIS, probably via the acetoacetate pathway [40]. Thus, Fig.…”

Multi-dimensional Roles of Ketone Bodies in Fuel Metabolism, Signaling, and Therapeutics

“…Anaerobic bacterial fermentation of complex polysaccharides yields butyrate, which is absorbed by colonocytes in mammalians for terminal oxidation or ketogenesis (Cherbuy et al, 1995), which may play a role in colonocyte differentiation (Wang et al, 2016). Excluding gut epithelial cells and hepatocytes, HMGCS2 is nearly absent in almost all other mammalian cells, but the prospect of extrahepatic ketogenesis has been raised in tumor cells, astrocytes of the central nervous system, the kidney, pancreatic β cells, retinal pigment epithelium (RPE), and even in skeletal muscle (Adijanto et al, 2014; Avogaro et al, 1992; El Azzouny et al, 2016; Grabacka et al, 2016; Kang et al, 2015; Le Foll et al, 2014; Nonaka et al, 2016; Takagi et al, 2016a; Thevenet et al, 2016; Zhang et al, 2011). Ectopic HMGCS2 has been observed in tissues that lack net ketogenic capacity (Cook et al, 2016; Wentz et al, 2010), and HMGCS2 exhibits prospective ketogenesis-independent ‘moonlighting’ activities, including within the cell nucleus (Chen et al, 2016; Kostiuk et al, 2010; Meertens et al, 1998).…”

Disruption of beta cell acetyl-CoA carboxylase-1 in mice impairs insulin secretion and beta cell mass

“…For example, altered acetylation of proteins in the glycolytic pathway and tricarboxylic acid cycle, as in the ACC-null liver [20], may impair metabolic coupling in ACC1-null beta cells. Likewise, acetoacetate breakdown provides acetyl-CoA to ACC1 in beta cells via a pathway distinct from ATP citrate lyase [45]. It is therefore plausible that loss of ACC1 activity could result in persistent elevations of acetoacetate, contributing to defective beta cell function in βACC1KO and tmx-βACC1KO mice.…”