l-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2

Increase in the concentration of plasma l-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged l-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged l-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued l-cysteine–induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, l-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. l-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N′-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in l-cysteine–treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to l-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.A metabolite, l-cysteine, is found in blood plasma, and its concentration is closely associated with an increase in fat mass and the body-mass index. These values are used as an index of obesity (1, 2), which is a major risk factor for type 2 diabetes (T2D) (3). The relationship between l-cysteine and diabetes has attracted attention because there is increasing evidence for a positive correlation between increases in plasma l-cysteine concentrations and the development and progression of diabetes. For example, increased plasma l-cysteine concentrations were associated with T2D in African American women (4), renal insufficiency [reduced glomerular filtration rate (GFR)] in T2D patients (5), obstructive sleep apnea [a risk factor for diabetes (6, 7)], and insulin resistance among Europeans (8).Reduced insulin secretion from pancreatic β-cells is the major cause of T2D (9, 10). Many investigators have studied the molecular mechanisms of glucose-stimulated insulin secretion (GSIS), which have been elucidated in detail. Elevated extracellular glucose concentration results in the enhancement of ATP production, an increased ATP/ADP ratio, the closure of ATP-sensitive K channels (K_ATP channels), and depolarization (11). The resulting activation of voltage-dependent Ca²⁺ channels (VDCCs) induces an influx of calcium ions and elevated intracellular Ca²⁺ concentrations, which triggers insulin secretion (11). Perifusion experiments have shown that insulin secretion could be categorized into two phases. The first phase involves a sharp increase in insulin secretion within ∼5 min, followed by a second phase, during which moderate insulin secretion lasts for hours (9, 12). A loss of the GSIS first phase is closely associated with the future development of T2D (9, 13, 14).Many recent studies have reported that l-cysteine is involved in GSIS. In mouse pancreatic islets and mouse insulinoma 6 (MIN6) cells, l-cysteine treatment decreased both intracellular ATP levels and insulin secretion (15). Ammon et al. also reported that the total amount and GSIS second phase were specifically inhibited by l-cysteine for rat pancreatic islets (16). Several groups showed that an increase in the H₂S moiety, which is generated from l-cysteine in cells (17), was one possible cause for l-cysteine–induced impairment of GSIS by inhibiting K_ATP channels and VDCCs (18–20). However, opposite results were reported in that l-cysteine increased the amount of total and first-phase insulin secretion by rat pancreatic islets (16, 21). Thus, the effects of l-cysteine on GSIS remain controversial. It should be noted that most of these studies on the effects of l-cysteine on GSIS were performed using experimental conditions in which insulin-secreting cells were only transiently exposed (∼1 h) to a high glucose solution that contained l-cysteine. However, in obese or T2D patients, insulin-secreting cells can be exposed to plasma that contains l-cysteine for prolonged periods of time; thus, continuous exposure to an l-cysteine–containing solution is necessary to investigate the precise effects of l-cysteine on GSIS in insulin-secreting cells.In this study, we found that l-cysteine treatment of statically incubated or perifused MIN6 cells and mouse pancreatic islets resulted in reversibly inhibiting GSIS. A comprehensive analysis of charged metabolites in l-cysteine–treated MIN6 cells by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) showed significant accumulations of l-cysteine and, concomitantly, decreased levels of pyruvate and its downstream metabolites in the tricarboxylic acid (TCA) cycle in these cells. Biochemical experiments for pyruvate kinase activity in vitro and in MIN6 cells showed that l-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of major pyruvate kinases in pancreatic islets (22) that catalyzes the glycolytic conversion of phosphoenolpyruvate (PEP) to pyruvate. PKM2 inactivation resulted from l-cysteine–induced subunit dissociation (tetramers to dimers/monomers) and inhibited glucose-induced ATP production for GSIS in MIN6 cells. Impaired GSIS due to l-cysteine was restored by treatment with methyl pyruvate, a membrane-permeable form of pyruvate, or DASA-10 (NCGC00181061, a substituted N,N′-diarylsulfonamide), a specific activator of PKM2 (23). Thus, we concluded that reduced PKM2 activity due to l-cysteine inhibited ATP production and subsequently inhibited GSIS by insulin-secreting cells.