The regulation of insulin secretion by the pancreatic beta-cell is unique in that it requires the metabolic breakdown of the main physiological stimulus, glucose. The events which link the metabolism to the electrical activity of the beta-cell and to depolarization-induced Ca2+ influx are named “triggering pathway” (Henquin, 2000). However, it has been shown that additional signals emanating from the metabolism are needed for the full extent of glucose-induced insulin secretion. This hitherto incompletely understood pathway was named “amplifying pathway”, since it remains ineffective without depolarization-induced Ca2+ influx (Henquin, 2000).
Both, triggering and amplifying pathways initiate at the glucose phosphorylation by the beta-cell glucokinase, which increases the glycolytic pyruvate production (Jitrapakdee et al., 2010, Prentki et al., 2013). Thereby the supply of acetyl-CoA by pyruvate dehydrogenase and of oxaloacetate by pyruvate carboxylase is enhanced (see Fig. 1). The provision of reducing equivalents by the citrate cycle activates the respiratory chain and ultimately elevates the cytosolic ATP/ADP ratio (Jitrapakdee et al., 2010, Prentki et al., 2013). In response to elevation of this ratio, the ATP-sensitive K+ channels in the beta-cell plasma membrane are closed and, in conjunction with inward currents, depolarize the beta-cell. The ensuing opening of Ca2+ channels raises the cytosolic free Ca2+ concentration and triggers insulin release (Rorsman and Ashcroft, 2018).
Beta-cell mitochondria have a high activity of pyruvate carboxylase, which is unusual for non-gluconeogenic cells (MacDonald et al., 2005). The generation of oxaloacetate by pyruvate carboxylase ensures adequate supply of citrate cycle intermediates for the rapid increase in citrate cycle activity and export of intermediates into the cytosol (cataplerosis) (Jitrapakdee et al., 2010, Prentki et al., 2013). The export generates signals which amplify the insulin-releasing efficiency of the elevated cytosolic Ca2+ concentration (Henquin, 2000). This metabolic amplification starts within a few min of fuel application and persists during the first- and second-phase secretion (Henquin, 2009). Multiple metabolic intermediates in the beta-cell cytosol have been considered as putative mediators of glucose-induced amplification and objections have been raised to most of them (Jitrapakdee et al., 2010, Jensen et al., 2008, Rustenbeck et al., 2021).
In addition to glucose only a few metabolic fuels (calorigenic nutrients) by themselves trigger insulin release (MacDonald et al., 2005). Alpha-ketoisocaproate (KIC), the transamination product of leucine, has gained interest because its metabolism does not involve glycolysis and its insulin-releasing potency is about as high as the potency of glucose (Lenzen and Panten, 1980, Hutton et al., 1980, Panten et al., 1981). In the absence of other exogenous fuels KIC triggers insulin release at extracellular concentrations > 2–3 mmol/L and is maximally effective at 10–20 mmol/L (Lenzen and Panten, 1980, Hutton et al., 1980, Panten et al., 1981). KIC triggers insulin release by serving as substrate of mitochondrial branched-chain aminotransferase in the beta-cells and thus generating alpha-ketoglutarate as long as mitochondrial glutamate is available (Zhou et al., 2010). As KIC and its catabolites were found to inhibit the alpha-ketoglutarate dehydrogenase (Pizarro-Delgado et al., 2009) and the pyruvate dehydrogenase in hepatocytes (Walajtys-Rode and Williamson, 1980), the GABA shunt and the oxidation of KIC (Lenzen and Panten, 1980, Hutton et al., 1980, Pizarro-Delgado et al., 2009) supply most of the oxaloacetate and acetyl-CoA required for activating the citrate cycle (see Fig. 1). This leads to the same events as outlined above, elevation of the cytosolic ATP/ADP ratio and closure of the ATP-sensitive K+ channels. In addition, KIC (> 5 mmol/L) intensifies the closure of ATP-sensitive K+ channels via its binding to the sulfonylurea receptor site (Heissig et al., 2005).
In a series of preceding investigations, we have examined the mechanisms of the metabolic amplification during stimulation by glucose and KIC (Urban and Panten, 2005, Panten and Rustenbeck, 2008, Panten et al., 2013, Panten et al., 2016, Schulze et al., 2017). To selectively influence the amplification, all ATP-sensitive K+ channels were closed by a maximally effective sulfonylurea concentration. Amplification is often examined during potassium depolarization when all ATP-sensitive K+ channels are opened by diazoxide (Henquin, 2000), but to avoid complications resulting from interaction of diazoxide and KIC at the ATP-sensitive K+ channels, we prefer the depolarization by sulfonylureas. We chose mouse islets since they lack the cytosolic malic enzyme activity (MacDonald, 2002), thus narrowing down the metabolites which might mediate the amplification. In islets exposed to the sulfonylurea glipizide at a maximally effective concentration throughout the experiment and pretreated by the prolonged absence of exogenous fuel, 10 mmol/L KIC within a few min strongly amplified the secretion of insulin, whereas the metabolic amplification by glucose stimulation was prevented (Urban and Panten, 2005, Panten et al., 2013, see also Fig. 2).
Activation of the glycerolipid/non-esterified fatty acid (NEFA) cycle by increase in glycerol 3-phosphate production and generation of NADPH by increase in supply of cytosolic isocitrate have been suggested to act as key amplifying mechanisms (Prentki et al., 2020, Campbell and Newgard, 2021). But the strong amplification by 10 mmol/L KIC in mouse islets, all ATP-sensitive K+ channels of which were closed by sulfonylureas (Heissig et al., 2005, Urban and Panten, 2005, Panten et al., 2013, Schulze et al., 2017), argues against these views, since KIC does not generate glycolytic glycerol 3-phosphate and since KIC amplified the secretion without increasing the islet NADPH/NADP+ ratio (Panten and Rustenbeck, 2008).
Assuming that the metabolic amplification is brought about by a final mechanism common to glucose and KIC, none of the previously considered mediators fits into this role. Since the metabolism of glucose and KIC supports the mitochondrial export of citrate and acetoacetate and since both are sources of cytosolic acetyl-CoA in insulin-secreting cells (see Fig. 1) we have expected that increases in fuel-induced amplification of insulin secretion coincide with increases in acetyl-CoA content. However, dissociations between fuel-induced amplification and increase in islet content of acetyl-CoA were observed after 20 min incubations (Panten et al., 2016). Using a modified methodology, the current study tested the hypothesis that the mitochondrial metabolism of fuel secretagogues in normal pancreatic islets enhances the supply of cytosolic acetyl-CoA both at an early and later stage of stimulated secretion. The observations support the view that supply of cytosolic acetyl-CoA mediates metabolic amplification.