Insulin Secretion In Insulinoma Cells Research Articles

Synthetic protease-activated class B GPCRs

G-protein coupled receptors (GPCRs) are the ligand detection machinery of a majority of extracellular signaling systems in metazoans. Novel chemical and biological tools to probe the structure-function relationships of GPCRs have impacted both basic and applied GPCR research. To better understand the structure-function of class B GPCRs, we generated receptor-ligand fusion chimeric proteins that can be activated by exogenous enzyme application. As a prototype, fusion proteins of the glucagon-like peptide-1 receptor (GLP-1R) with GLP-1(7–36) and exendin-4(1–39) peptides incorporating enterokinase-cleavable N-termini were generated. These receptors are predicted to generate fusion protein neo-epitopes upon proteolysis with enterokinase that are identical to the N-termini of GLP-1 agonists. This system was validated by measuring enterokinase-dependent GLP-1R mediated cAMP accumulation, and a structure-activity relationship for both linker length and peptide sequence was observed. Moreover, our results show this approach can be used in physiologically relevant cell systems, as GLP-1R-ligand chimeras were shown to induce glucose-dependent insulin secretion in insulinoma cells upon exposure to enterokinase. This approach suggests new strategies for understanding the structure-function of peptide-binding GPCRs.

Thioredoxin-mimetic peptides (TXM) reverse auranofin induced apoptosis and restore insulin secretion in insulinoma cells

The thioredoxin reductase/thioredoxin system (TrxR/Trx1) plays a major role in protecting cells from oxidative stress. Disruption of the TrxR-Trx1 system keeps Trx1 in the oxidized state leading to cell death through activation of the ASK1-Trx1 apoptotic pathway. The potential mechanism and ability of tri- and tetra-oligopeptides derived from the canonical -CxxC- motif of the Trx1-active site to mimic and enhance Trx1 cellular activity was examined. The Trx mimetics peptides (TXM) protected insulinoma INS 832/13 cells from oxidative stress induced by selectively inhibiting TrxR with auranofin (AuF). TXM reversed the AuF-effects preventing apoptosis, and increasing cell-viability. The TXM peptides were effective in inhibiting AuF-induced MAPK, JNK and p38MAPK phosphorylation, in correlation with preventing caspase-3 cleavage and thereby PARP-1 dissociation. The ability to form a disulfide-bridge-like conformation was estimated from molecular dynamics simulations. The TXM peptides restored insulin secretion and displayed Trx1 denitrosylase activity. Their potency was 10–100-fold higher than redox reagents like NAC, AD4, or ascorbic acid. Unable to reverse ERK1/2 phosphorylation, TXM-CB3 (NAc-Cys-Pro-Cys amide) appeared to function in part, through inhibiting ASK1-Trx dissociation. These highly effective anti-apoptotic effects of Trx1 mimetic peptides exhibited in INS 832/13 cells could become valuable in treating adverse oxidative-stress related disorders such as diabetes.

Platyconic acid, a saponin from Platycodi radix, improves glucose homeostasis by enhancing insulin sensitivity in vitro and in vivo

Previous research demonstrated that the crude saponins of Platycodi radix improve glucose metabolism by enhancing insulin sensitivity in type 2 diabetic animals; however, which individual saponins are the most potent insulin sensitizers is unknown. This study investigated which saponin(s) have anti-diabetic action in vitro and in vivo. The insulin-stimulated glucose uptake and PPAR-γ agonistic actions of six saponins from Platycodi radix were investigated in 3T3-L1 adipocytes, and glucose-stimulated insulin secretion was determined in Min6 cells. Four individual saponins (20 mg/kg body weight) were orally administered to low-dose streptozotocin-injected diabetic mice fed a high-fat diet for 8 weeks to evaluate glucose tolerance by oral glucose tolerance testing (OGTT), insulin sensitivity by insulin tolerance testing, and insulin signaling in the liver and adipose tissues. Platyconic acid (PA) most effectively increased insulin-stimulated glucose uptake in 3T3-L1 adipocytes, possibly in part by working as a peroxisome proliferator-activated receptors (PPAR)-γ activator; however, none of the saponins improved glucose-stimulated insulin secretion in insulinoma cells. PA-treated diabetic mice exhibited the lowest peak serum glucose levels and highest serum insulin levels during the first part of OGTT. PA also improved insulin sensitivity: PA increased glycogen accumulation and decreased triacylglycerol storage in liver, which was associated with enhanced hepatic insulin signaling, while PA potentiated the expression of adiponectin and PPAR-γ in adipose tissue, and improved insulin signaling and increased GLUT4 translocation into the membranes. PA improves glucose homeostasis in type 2 diabetic mice, partly by enhancing hepatic and adipocyte insulin sensitivity, possibly by activating PPAR-γ.

Impaired Anaplerosis and Insulin Secretion in Insulinoma Cells Caused by Small Interfering RNA-mediated Suppression of Pyruvate Carboxylase

Anaplerosis, the synthesis of citric acid cycle intermediates, by pancreatic beta cell mitochondria has been proposed to be as important for insulin secretion as mitochondrial energy production. However, studies designed to lower the rate of anaplerosis in the beta cell have been inconclusive. To test the hypothesis that anaplerosis is important for insulin secretion, we lowered the activity of pyruvate carboxylase (PC), the major enzyme of anaplerosis in the beta cell. Stable transfection of short hairpin RNA was used to generate a number of INS-1 832/13-derived cell lines with various levels of PC enzyme activity that retained normal levels of control enzymes, insulin content, and glucose oxidation. Glucose-induced insulin release was decreased in proportion to the decrease in PC activity. Insulin release in response to pyruvate alone, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine, or methyl succinate plus beta-hydroxybutyrate was also decreased in the PC knockdown cells. Consistent with a block at PC, the most PC-deficient cells showed a metabolic crossover point at PC with increased basal and/or glucose-stimulated pyruvate plus lactate and decreased malate and citrate. In addition, in BCH plus glutamine-stimulated PC knockdown cells, pyruvate plus lactate was increased, whereas citrate was severely decreased, and malate and aspartate were slightly decreased. The incorporation of 14C into lipid from [U-14C]glucose was decreased in the PC knockdown cells. The results confirm the central importance of PC and anaplerosis to generate metabolites from glucose that support insulin secretion and even suggest PC is important for insulin secretion stimulated by noncarbohydrate insulin secretagogues.

The isoflavonoid aglycone‐rich fractions of Chungkookjang, fermented unsalted soybeans, enhance insulin signaling and peroxisome proliferator‐activated receptor‐γ activity in vitro

We investigated anti-diabetic candidates and their mechanisms from the fractions of Chungkookjang (CKJ), a traditional fermented unsalted soybean, by investigating insulin signaling, peroxisome proliferator-activated receptor (PPAR)-gamma activity and glucose-stimulated insulin secretion, in vitro. Cooked soybeans (CSB) and CKJ, fermented predominantly with Bacillus subtilis, were extracted by 70% EtOH followed by an XAD-4 column chromatography with a serial mixture of solvents comprised of MeOH and water. During fermentation, the contents of isoflavonoid aglycones were elevated, and the fractions enriched with aglycones enhanced insulin-stimulated glucose uptake in 3T3-L1 adipocytes. This increase in glucose uptake resulted from stimulating a translocation of the glucose transporter (GLUT)-4 into the plasma membrane through the phosphorylation of insulin receptor substrate (IRS)-1 and Akt. Especially, daidzein enriched fractions elevated insulin-stimulated glucose uptake by acting as PPAR-gamma agonist up to levels exhibited when 10 nM insulin is administered. Fractions containing small peptides with low polarity in CKJ slightly increased glucose-stimulated insulin secretion. The data suggest that an increase in isoflavonoid aglycones in CKJ, in comparison to CSB, enhances glucose utilization via activating insulin signaling and stimulates PPAR-gamma activity in adipocytes. In addition, CKJ contains small peptides improving glucose-stimulated insulin secretion in insulinoma cells. Overall, CKJ is superior to CSB in anti-diabetic action.