Coordinated activation of mitochondrial respiration and exocytosis mediated by PKC signaling in pancreatic β cells

Abstract

Mitochondria play a central role in pancreatic β-cell nutrient sensing by coupling their metabolism to plasma membrane excitability and insulin granule exocytosis. Whether non-nutrient secretagogues stimulate mitochondria as part of the molecular mechanism to promote insulin secretion is not known. Here, we show that PKC signaling, which is employed by many non-nutrient secretagogues, augments mitochondrial respiration in INS-1E (rat insulinoma cell line clone 1E) and human pancreatic β cells. The phorbol ester, phorbol 12-myristate 13-acetate, accelerates mitochondrial respiration at both resting and stimulatory glucose concentrations. A range of inhibitors of novel PKC isoforms prevent phorbol ester-induced respiration. Respiratory response was blocked by oligomycin that demonstrated PKC-dependent acceleration of mitochondrial ATP synthesis. Enhanced respiration was observed even when glycolysis was bypassed or fatty acid transport was blocked, which suggested that PKC regulates mitochondrial processes rather than upstream catabolic fluxes. A phosphoproteome study of phorbol ester-stimulated INS-1E cells maintained under resting (2.5 mM) glucose revealed a large number of phosphorylation sites that were altered during short-term activation of PKC signaling. The data set was enriched for proteins that are involved in gene expression, cytoskeleton remodeling, secretory vesicle transport, and exocytosis. Interactome analysis identified PKC, C-Raf, and ERK1/2 as the central phosphointeraction cluster. Prevention of ERK1/2 signaling by using a MEK1 inhibitor caused a marked decreased in phorbol 12-myristate 13-acetate-induced mitochondrial respiration. ERK1/2 signaling module therefore links PKC activation to downstream mitochondrial activation. We conclude that non-nutrient secretagogues act, in part, PKC and downstream ERK1/2 signaling to stimulate mitochondrial energy production to compensate for energy expenditure that is linked to β-cell activation.-Santo-Domingo, J., Chareyron, I., Dayon, L., Galindo, A. N., Cominetti, O., Giménez, M. P. G., De Marchi, U., Canto, C., Kussmann, M., Wiederkehr, A. Coordinated activation of mitochondrial respiration and exocytosis mediated by PKC signaling in pancreatic β cells.

Basal hypersecretion of glucagon and insulin from palmitate-exposed human islets depends on FFAR1 but not decreased somatostatin secretion

“…The potential mechanism for this complementary effect is that FFAR1 signaling via PLC, DAG and PKC/PKD1 leads to enhanced Ca 2+ mobilization from intracellular Ca 2+ -stores, activation of L-type calcium-channels and enhanced islet respiration and ATP-production, which can facilitate higher secretion of both insulin and glucagon 28, 39, 46–49 . In favor of FFAR1 signaling and our previous findings with regards to the role of PKC on respiration in beta-cells 16 , these coordinated cellular events were verified concerning their effect on insulin release and mitochondrial respiration 50 . A likely scenario is that activation of FFAR1 alone at low or fasting glucose concentrations is not sufficient for maintaining increased secretion or respiration without additional substrates for the enhanced mitochondrial activity.…”

Glucose-dependent phosphorylation signaling pathways and crosstalk to mitochondrial respiration in insulin secreting cells

“…6e). Consistent with our earlier published results [28], PKC inhibition (Go6983) reduced glucose-induced respiration (Fig. 6f).…”

“…The data suggests kinases of the PKC family are upstream regulators of mitochondrial function. Consistent with these findings, we recently demonstrated that short-term regulation of PKC rapidly modulates glucose-stimulated mitochondrial respiration [28]. For a number of other signal transduction pathways, their role in glucose-induced respiration has not been investigated to date.…”

Feedback regulation of insulin secretion by extended synaptotagmin‐1

“…PKC is also a well‐characterized amplifier of insulin secretion, and the observed augmentation of glucose‐stimulated insulin secretion in cells with reduced E‐Syt1 expression is consistent with earlier work involving pharmacological activation of PKC (42–44). Numerous PKC targets of importance for insulin secretion have been identified, including proteins involved in glucose metabolism and exocytosis (45). One recently characterized PKC effector is the ER‐localized lipid transport protein TMEM24 (35).…”