Glibenclamide-induced Insulin Secretion Research Articles

Glibenclamide-Induced Autophagy Inhibits Its Insulin Secretion-Improving Function in β Cells

Diabetes is a metabolic disease, partly due to hypoinsulinism, which affects ∼8% of the world's adult population. Glibenclamide is known to promote insulin secretion by targeting β cells. Autophagy as a self-protective mechanism of cells has been widely studied and has particular physiological effects in different tissues or cells. However, the interaction between autophagy and glibenclamide is unclear. In this study, we investigated the role of autophagy in glibenclamide-induced insulin secretion in pancreatic β cells. Herein, we showed that glibenclamide promoted insulin release and further activated autophagy through the adenosine 5′-monophosphate (AMP) activated protein kinase (AMPK) pathway in MIN-6 cells. Inhibition of autophagy with autophagy inhibitor 3-methyladenine (3-MA) potentiated the secretory function of glibenclamide further. These results suggest that glibenclamide-induced autophagy plays an inhibitory role in promoting insulin secretion by activating the AMPK pathway instead of altering the mammalian target of rapamycin (mTOR).

The brown seaweed Sargassum hemiphyllum exhibits α-amylase and α-glucosidase inhibitory activity and enhances insulin release in vitro.

Diabetes is one of the most prevalent chronic diseases globally. In this study, major polyphenols (17.35±0.93-36.66±2.01mg/g) and minor fucoxanthin (non detected 15.12±0.09mg/g) were isolated from water, ethanol, and acetone extracts (WES, EES, and AES, respectively) of Sargassum hemiphyllum. Inhibition of α-amylase, α-glucosidase, sucrose, and maltase activities and stimulation of insulin secretion was greater with AES than with WES or EES and correlated with polyphenol and fucoxanthin concentrations in extracts. Moreover, 250μg/ml EES and AES significantly increased insulin secretion in the presence of 25mg/ml glibenclamide to higher levels than those obtained with 50mg/ml glibenclamide. None of the extracts exhibited cytotoxicity, exacerbated the side effects of glibenclamide, or inhibited glibenclamide-induced insulin secretion. These results suggested that the S. hemiphyllum extracts WES, EES, and AES could be used as pharmaceuticals and functional foods to reduce dosages of synthetic diabetes drugs.

Enhanced GLP-1- and Sulfonylurea-Induced Insulin Secretion in Islets Lacking Leptin Signaling

We have previously reported that the absence of leptin signaling in β-cells enhances glucose-stimulated insulin secretion and improves glucose tolerance in vivo. To investigate the relevance of β-cell leptin signaling in the context of postprandial or therapeutic insulin secretion, we examined the cross talk between leptin and glucagon-like peptide (GLP)-1 and sulfonylurea actions. Single and size-matched islets isolated from control or pancreas-specific leptin receptor knockout (pancreas-ObR-KO) mice were treated either with GLP-1 or with glibenclamide. Leptin suppressed GLP-1-stimulated intracellular Ca(2+) concentrations ([Ca(2+)](i)) increase that paralleled the decrease in insulin secretion in controls. In contrast, and as expected, the ObR-KO islets were nonresponsive to leptin, and instead, showed a 2.8-fold greater GLP-1-stimulated [Ca(2+)](i) increase and a 1.7-fold greater insulin secretion. Phosphorylation of cAMP-responsive element binding protein was enhanced, and phosphodiesterase enzymatic activity was suppressed in MIN6 β-cells with ObR knockdown compared with controls. The ObR-KO islets also showed significantly higher glibenclamide-induced insulin secretion compared with control islets, whereas [Ca(2+)](i) was similar to the controls. These data support enhanced insulinotropic effects of glucose, GLP-1, and sulfonylureas in the islets lacking leptin signaling with potential therapeutic implications.

Mechanisms of p-Methoxycinnamic Acid-induced Increase in Insulin Secretion

p-Methoxycinnamic acid (p-MCA) is a cinnamic acid derivative that shows various pharmacologic actions such as hepatoprotective and antihyperglycemic activities. The present study was to elucidate the mechanisms by which p-MCA increases [Ca²⁺]i and insulin secretion in INS-1 cells. p-MCA (100 μM) increased [Ca²⁺]i in INS-1 cells. The p-MCA-induced insulin secretion and rise in [Ca²⁺]i were markedly inhibited in the absence of extracellular Ca²⁺ or in the presence of an L-type Ca²⁺ channel blocker nimodipine. These results suggested that p-MCA increased Ca²⁺ influx via the L-type Ca²⁺ channels. Diazoxide, an ATP-sensitive K⁺ channel opener, did not alter p-MCA-induced insulin secretion, nor [Ca²⁺]i response. In addition, p-MCA enhanced glucose-, glibenclamide-induced insulin secretion whereas it also potentiated the increase in insulin secretion induced by arginine, and Bay K 8644, an L-type Ca²⁺ channel agonist. Taken together, our results suggest that p-MCA stimulated insulin secretion from pancreatic β-cells by increasing Ca²⁺ influx via the L-type Ca²⁺ channels, but not through the closure of ATP-sensitive K⁺ channels.

Video rate bioluminescence imaging of secretory proteins in living cells: Localization, secretory frequency, and quantification

We have developed a method of video rate bioluminescence imaging to investigate protein secretion from a single mammalian cell and analyzed the localization, secretory frequency, and quantification of secreted protein. By detecting the luminescence signals from the Gaussia luciferase (GLase) reaction using a high-speed electron-multiplying charge-coupled device (EM-CCD) camera, video rate imaging was performed with a time resolution within 500 ms/image over 30 min in living cells. As a model study, we applied the method to visualize the glucose-stimulated insulin secretion from clustered pancreatic MIN6 β cells using the fused protein of GLase with preproinsulin. High-quality video images clearly showed that the glucose-stimulated insulin secretion from the clustered MIN6 β cells oscillated within a period of a few minutes over 10 min. In addition, the glibenclamide-induced insulin secretion from the clustered MIN6 β cells was visualized, suggesting that bioluminescence video rate imaging is a useful method for validating drug action in living cells.

Quercetin potentiates insulin secretion and protects INS‐1 pancreatic β‐cells against oxidative damage via the ERK1/2 pathway

Quercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic β-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting β-cells. Using the INS-1 β-cell line, the effects of quercetin were determined on glucose- or glibenclamide-induced insulin secretion and on β-cell dysfunctions induced by hydrogen peroxide (H(2)O(2)). These effects were analysed along with the activation of the extracellular signal-regulated kinase (ERK)1/2 pathway. N-acetyl-L-cysteine (NAC) and resveratrol, two antioxidants also known to exhibit some anti-diabetic properties, were used for comparison. Insulin release was quantified by the homogeneous time resolved fluorescence method and ERK1/2 activation tested by Western blot experiments. Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay. KEY RESULTS Quercetin (20 µmol·L(-1)) potentiated both glucose (8.3 mmol·L(-1))- and glibenclamide (0.01 µmol·L(-1))-induced insulin secretion and ERK1/2 phosphorylation. The ERK1/2 (but not the protein kinase A) signalling pathway played a crucial role in the potentiation of glucose-induced insulin secretion by quercetin. In addition, quercetin (20 µmol·L(-1)), protected β-cell function and viability against oxidative damage induced by 50 µmol·L(-1) H(2)O(2) and induced a major phosphorylation of ERK1/2. In the same conditions, resveratrol or NAC were ineffective. Quercetin potentiated glucose and glibenclamide-induced insulin secretion and protected β-cells against oxidative damage. Our study suggested that ERK1/2 played a major role in those effects. The potential of quercetin in preventing β-cell dysfunction associated with diabetes deserves further investigation.

Sulfonylurea and glinide reduce insulin content, functional expression of K ATP channels, and accelerate apoptotic β-cell death in the chronic phase

We previously found that chronic exposure to glibenclamide inhibits acute glibenclamide-induced insulin secretion by reducing the number of functional ATP-sensitive K + (K ATP) channels on the plasma membrane of pancreatic β-cells. In the present study, we compared sulfonylurea-induced and glinide-induced insulin secretion in pancreatic β-cells chronically exposed to these widely used oral hypoglycemic agents. Chronic exposure of pancreatic β-cells to sulfonylureas (glibenclamide or tolbutamide) and glinide (nateglinide) similarly impaired their acute effectiveness by reducing the insulin content and the number of functional K ATP channels on the plasma membrane. Functional expression of the voltage-dependent Ca 2+ channels (VDCCs), ion channels that play a critical role in the K ATP channel dependent insulin secretory pathway, was similar to that in drug-untreated cells. Chronic exposure to each of the three agents similarly accelerated apoptotic β-cell death. Thus, reduction of the insulin content, reduction of the number of functional K ATP channels on the plasma membrane, and acceleration of apoptotic β-cell death all are involved in impaired insulinotropic agent-induced acute insulin secretion in the chronic phase of sulfonylurea and glinide treatment. These findings help to clarify the mechanism of secondary failure after long-term therapy by these hypoglycemic agents, and should have important clinical implications regarding pharmacotherapy for type 2 diabetes.

First Phase of Glucose-Stimulated Insulin Secretion From MIN 6 Cells Does Not Always Require Extracellular Calcium Influx

To demonstrate an involvement of ATP-sensitive potassium (K(ATP)) channel-independent pathways in the first phase of glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells, the time course of GSIS from MIN6 cells was analyzed at 30-s sample intervals. GSIS was biphasic with the first phase being observed 120 to 390 s after glucose addition, peaking at 180 s, and with a shoulder at 240 to 330 s. Both 10 microM diazoxide and 3 microM verapamil completely inhibited tolbutamide- or glibenclamide-induced insulin secretion and suppressed the peak of the first phase of GSIS, but did not result in complete suppression. The shoulder following the peak was suppressed by 1 muM dantrolene. The peak, but not shoulder, disappeared under the extracellular Ca2+-free condition. A significant amount of insulin secretion remained even in the combined presence of verapamil and dantrolene. The Na+ channel blocker tetrodotoxin (30 nM) nearly completely inhibited the first phase release. These results suggest that the first phase of GSIS from MIN6 cells depends on both Ca2+-dependent and -independent mechanisms. The former mechanism includes the extracellular Ca2+ influx via L-type voltage-dependent calcium channel and intracellular Ca2+ release from endoplasmic reticulum via ryanodine receptors, and the latter mechanism involves the pathways associated with Na+ channels.

Decreased glibenclamide uptake in hepatocytes of hepatocyte nuclear factor-1alpha-deficient mice: a mechanism for hypersensitivity to sulfonylurea therapy in patients with maturity-onset diabetes of the young, type 3 (MODY3).

Diabetes in subjects with hepatocyte nuclear factor (HNF)-1alpha gene mutations (maturity-onset diabetes of the young [MODY]-3) is characterized by impaired insulin secretion. Surprisingly, MODY3 patients exhibit hypersensitivity to the hypoglycemic actions of sulfonylurea therapy. To study the pharmacogenetic mechanism(s), we have investigated glibenclamide-induced insulin secretion, glibenclamide clearance from the blood, and glibenclamide metabolism in wild-type and Hnf-1alpha-deficient mice. We show that despite a profound defect in glucose-stimulated insulin secretion, diabetic Hnf-1alpha(-/-) mice have a robust glibenclamide-induced insulin secretory response. We demonstrate that the half-life (t(1/2)) of glibenclamide in the blood is increased in Hnf-1alpha(-/-) mice compared with wild-type littermates (3.9 +/- 1.3 vs. 1.5 +/- 1.8 min, P <or= 0.05). The clearance of glibenclamide from the blood during the first hours after intravenous administration was reduced approximately fourfold in Hnf-1alpha(-/-) mice compared with Hnf-1alpha(+/+) littermates. Glibenclamide uptake into hepatocytes was dramatically decreased in vivo and in vitro. To study the metabolism of glibenclamide in Hnf-1alpha(-/-) animals, we analyzed liver extracts from [(3)H]glibenclamide-injected animals by reverse-phase chromatography. We found that the ratio of the concentrations of glibenclamide and its metabolites was moderately increased in livers of Hnf-1alpha(-/-) mice, suggesting that hepatic glibenclamide metabolism was not impaired in animals with Hnf-1alpha deficiency. Our data demonstrate that high serum glibenclamide concentrations and an increased t(1/2) of glibenclamide in the blood of Hnf-1alpha(-/-) mice are caused by a defect in hepatic uptake of glibenclamide. This suggests that hypersensitivity to sulfonylureas in MODY3 patients may be due to impaired hepatic clearance and elevated plasma concentrations of the drug.

Unresponsiveness to glibenclamide during chronic treatment induced by reduction of ATP-sensitive K+ channel activity.

The insulin response to the sulfonylurea glibenclamide was markedly impaired in pancreatic beta-cell line MIN6 cells with chronic glibenclamide treatment (MIN6-Glib). The intracellular calcium concentration increased only slightly in response to glibenclamide in MIN6-Glib. While the properties of the voltage-dependent calcium channels were not altered, the conductance of the K(ATP) channels, the primary target of glibenclamide, was significantly reduced in MIN6-Glib. The ATP-sensitive K+ (K(ATP)) channels in MIN6 cells comprise inwardly rectifying K+ channel member Kir6.2 subunits and sulfonylurea receptor (SUR) 1 subunits. MIN6 cells have both high- and low-affinity binding sites for glibenclamide. The binding affinities at these two sites were unchanged, but the maximum binding capacities at both sites were similarly increased by chronic glibenclamide treatment. Both SUR1 and Kir6.2 mRNA levels were not altered, but SUR1 protein was rather increased in MIN6-Glib. In addition, electron microscopic examination revealed a majority of the SUR1 to be present in a cluster near the plasma membrane in control MIN6, while it tends to be distributed in the cytoplasm in MIN6-Glib. These data suggest that chronic glibenclamide treatment causes the defect in acute glibenclamide-induced insulin secretion by reducing the number of functional K(ATP) channels on the plasma membrane of the beta-cells.

Inhibitory effect of ODN, a naturally occurring processing product of diazepam binding inhibitor, on secretagogues-induced insulin secretion

Diazepam binding inhibitor (DBI 1–86) is a peptide that is present in large amounts in the intestine and pancreas and which inhibits glucose-stimulated insulin release from both perfused pancreas and isolated islets in low nanomolar concentrations. Here, DBI 33–50 (also known as ODN, octadecaneuropeptide), one of the naturally occurring processing products of DBI 1–86, and certain synthetic modified derivatives, have been shown to inhibit glucose and glibenclamide-stimulated insulin secretion from isolated rat islets and glibenclamide-stimulated insulin secretion from hamster-insulinoma (HIT-T15) β-cell line. DBI 17–50 (TTN; triakontatetraneuropeptide), another prominent processing product of DBI, had no effect. The 50% inhibitory concentration (IC 50) for the effect of ODN on insulin secretion induced by 8.3 or 16.7 mM glucose was approximately the same: 5 to 6 nM. Moreover, ODN inhibited insulin release induced by 0.01 or 1 μM glibenclamide with a similar IC 50 (8 to 10 nM) in both isolated pancreatic islets and in HIT-T15 β-cells. At concentration up to 1 μM, ODN had no effect on insulin secretion induced by PACAP (pituitary adenylate cyclase polypeptide), BAYK 8644 (methyl-(1,4-dihydro-2,6-dimethyl-3-nitro-4,2-trifluoromethylphenyl)pyridine-5-carboxylate), and only marginally it affected IBMX-(isobutylmethylxanthine) induced insulin secretion. This indicates that ODN does not act directly on ATP-regulated K + channels, voltage dependent Ca 2+ channels or cAMP production. In contrast, ODN inhibited insulin secretion induced by sodium nitroprussiate in a manner that is independent from the presence of extracellular Ca 2+. These results suggest that ODN or ODN-like peptide fragments of DBI, may inhibit glucose or glibenclamide-induced insulin secretion via a signaling pathway that regulate the cytoplasmic free Ca 2+ concentration.

Difference in calcium dependency of insulin, glucagon and somatostatin secretion in response to glibenclamide in perfused rat pancreas.

The extracellular calcium requirements for insulin, glucagon and somatostatin release induced by 1 microgram/ml of glibenclamide have been compared in the perfused, isolated rat pancreas. In the absence of glucose, the drug evoked insulin release equally well at physiological (2.6 mmol/l) and low (0.25 mmol/l) levels of total calcium. In contrast, glibenclamide evoked somatostatin release at 2.6 but not at 0.25 mmol/l of calcium. At 2.6 mmol/l of calcium, glibenclamide evoked bimodal effects (stimulation followed by inhibition) on glucagon secretion. At 0.25 mmol/l of calcium, basal secretory rates of glucagon were elevated and a small stimulatory effect of glibenclamide was seen. Addition of 0.5 mmol/l of EGTA to media with low calcium concentrations uniformly abolished the A, B and D cell secretory responses to glibenclamide. The possible modulation of calcium dependency by a non-stimulatory concentration of glucose was tested by its addition at 3.3 mmol/l to the perfusion media. Glucose enhanced glibenclamide-induced insulin secretion, both at 0.25 and 2.6 mmol/l of calcium. However, at 0.25 mmol/l of calcium, the enhancing effect of glucose was more pronounced than at 2.6 mmol/l. At 2.6 mmol/l of calcium, glucose diminished the somatostatin and abolished the glucagon response to glibenclamide. At 0.25 mmol/l of calcium, glucose did not influence somatostatin release while the presence of the sugar diminished basal and glibenclamide-induced glucagon secretion. The present data confirm the requirement of extracellular calcium for A, B and D cell secretion, demonstrating different calcium dependencies for the cell types and indicate that this dependency can, in part, be modulated by glucose.

Effects of glucagon and pentagastrin on glibenclamide-induced insulin release.

It was recently reported that administration of sulphonylureas may lead to a stimulation of the release of glucagon and a gastrin-like peptide. These peptides may then eventually influence the insulin releasing action of the sulphonylureas. Therefore, the effects of glucagon and pentagastrin on basal and glibenclamide-induced insulin secretion were studied. It was found that at a dose of 4.25 nmol/kg the two peptides induced a significant stimulation of basal insulin secretion. However, pentagastrin at the dose of 4.25 nmol/kg did not influence the insulin release induced by glibenclamide, whereas an equimolar dose of glucagon potentiated the insulin secretory response to this sulphonylurea drug by about 40%. Glucagon in contrast to pentagastrin thus positively modulates the insulin secretory pathway stimulated by the sulphonylurea drug glibenclamide.

Studies on the dynamics and mechanism of glibenclamide-induced insulin secretion.

Sustained, 60-minute perfusion of glibenclamide (0.5, 1.5 and 10 mug/ml) elicits a one-phase insulin release profile, formed by a rapid secretion peak followed by a second peak with lower insulin levels than the former. Basal insulin secretion values are observed during the period comprised between 13 and 60 minutes of perfusion. Concurrent stimulation with glucose (100, 150, 200 and 300 mg%) plus glibenclamide (1 mug/ml) causes a marked rise in both phases of insulin secretion. The addition of glibenclamide does not modify the biphasic secretion pattern caused by maximal glucose concentration (400 mg%). The maximal values of both phases of secretion in the dose-response curve elicited by different glucose concentrations shift to the left when glibenclamide is added to the perfusate. The increase in insulin secretion caused by glibenclamide is not inhibited by puromycin. Both theophylline and phentolamine modify and increase the glibenclamide-induced insulin release pattern. Propranolol and imidazole inhibit glibenclamide-induced insulin release. Our results suggest that: 1. Glibenclamide increases beta cell sensitivity to glucose stimulation. 2. Glibenclamide and glucose induce secretion of insulin originating in the same compartment. 3. Modification of alpha and beta adrenergic receptors may modify glibodulate the beta cell response to glibenclamide.