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

Abstract

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 glucosestimulated 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, glucoseinduced ATP production, and biphasic insulin secretion in L-cysteinetreated 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 Ca 2+ channels (VDCCs) induces an influx of calcium ions and elevated...

Inhibition of Methylglyoxal-Induced AGEs/RAGE Expression Contributes to Dermal Protection by N-Acetyl-L-Cysteine

“…In contrast, we notice in some other papers that the levels of plasma total cysteine correlates positively with the obesity or fat mass of patients [15-17]. A recent study suggested that the increased concentration of L-cysteine can inhibit insulin secretion from pancreatic β-cells through binding and inactivation of pyruvate kinase muscle isoform 2 [18]. With these conflicting reports, we intend to explore the effects of NAC on AGEs generation and diabetic wound healing.…”

Protective Effect of 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione, Isolated from Averrhoa Carambola L., Against Palmitic Acid-Induced Inflammation and Apoptosis in Min6 Cells by Inhibiting the TLR4-MyD88-NF-κB Signaling Pathway

“…Briefly, Min6 cells were seeded into a 96-well plate overnight and treated with concentrations (0 μmol/l, 10 μmol/l, 20 μmol/l, 30 μmol/l, 40 μmol/l, and 50 μmol/l) of DMDD. After incubation for 24 h, cell survival was measured by using a CCK8 as described previously [18].…”

One‐Dimensional Electrophoresis Using Nondenaturing Conditions

“…This includes the anionic dye Coomassie G-250 (referred to as Blue Native or BN; Schägger and von Jagow, 1991;Krause and Seelert, 2008;Luo et al, 2017). Recently, significant improvements in resolution have been made through use of low (<0.1%) concentrations of the anionic detergent SDS (Nowakowski, 2014;Nakatsu, 2015) or LDS (Arnold et al, 2014) as well as milder negatively charged detergents such as deoxycholate (DOC) with a variation on the Clear Native (CN) approach (Wittig et al, 2007). It should also be noted that the anionic detergent is typically left out of the sample buffer and samples are separated at temperatures from 4°to 7°C to maintain the native forms of the protein and complexes.…”