Sustained Enhancement of Ca2+ Influx by Glibenclamide Induces Apoptosis in RINm5F Cells

doi:10.1006/bbrc.2000.2616

Biochemical and Biophysical Research Communications

Volume 271, Issue 2, 10 May 2000, Pages 422-428

https://doi.org/10.1006/bbrc.2000.2616 Get rights and content

Abstract

Cytosolic Ca²⁺ elevations are known to be involved in triggering apoptosis in many tissues, but the effect of sustained enhancement of Ca²⁺ influx on apoptosis in β cells remains unknown. We have found that the viability of RINm5F cells is decreased dose-dependently by continuous exposure to glibenclamide at concentrations from 10⁻⁷ to 10⁻⁴ M, and that this effect is partially ameliorated by pretreatment with cycloheximide. Electrophoresis of the cells exposed to glibenclamide revealed ladder-like fragmentation characteristic of apoptosis, and which also is suppressed by cycloheximide pretreatment. By using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, we detected increased DNA fragmentation in the nuclei of the cells exposed to glibenclamide, and staining with Hoechst 33342 and propidium iodide showed a dose-dependent increase in the number of cells with the chromatin condensation and fragmentation in their nuclei that is characteristic of apoptosis. The effects of glibenclamide on cell viability and apoptotic cell death were partially inhibited by treatment with Ca²⁺ channel blocker, and by reducing the extracellular Ca²⁺ concentration during glibenclamide exposure, suggesting that they may be derived from increased Ca²⁺ influx. Furthermore, only the percentage of apoptotic cells, and not that of necrotic cells, increased with the increasing intracellular Ca²⁺ concentration during glibenclamide exposure. In conclusion, we have demonstrated that the sustained enhancement of Ca²⁺ influx caused by glibenclamide exposure can induce apoptotic cell death in a pure β cell line.

References (34)

A. Dunger et al.
J. Autoimmun.
(1996)
I.B. Efanova et al.
J. Biol. Chem.
(1998)
G. Muller et al.
Biochim. Biophys. Acta
(1994)
H. Schmid-Antomarchi et al.
J. Biol. Chem.
(1987)
H. Schmid-Antomarchi et al.
Biochem. Biophys. Res. Commun.
(1987)
A.H. Wyllie et al.
Int. Rev. Cytol.
(1980)
G.H. Hur et al.
Chem.-Biol. Interact.
(1998)
L. Hebert et al.
Exp. Cell. Res.
(1994)
B.A. O'Brien et al.
Diabetes
(1997)
M.O. Kurrer et al.
Proc. Natl. Acad. Sci. USA
(1997)

C.A. Delaney et al.

Endocrinology

(1997)

H. Kaneto et al.

Diabetes

(1995)

O.H. Bar et al.

Pancreas

(1999)

J. Janson et al.

Diabetes

(1999)

D.J. McConey et al.

J. Leukocyte Biol.

(1995)

E. Bonfoco et al.

J. Neurochem.

(1996)

Cited by (55)

A potential role of calpains in sulfonylureas (SUs) –mediated death of human pancreatic cancer cells (1.2B4)
2021, Toxicology in Vitro
Sulfonylureas (SUs) are suggested to accelerate the pancreatic β-cells mass loss via apoptosis. However, little is known whether calpains mediate this process. The aim of the present study is to evaluate the involvement of calpains in SUs-induced death of human pancreatic cancer (PC) cell line 1.2B4. The cells were exposed to: glibenclamide, glimepiride and gliclazide for 72 h. The expression analysis of caspase-3 (CASP-3), TP53, calpain 1 (CAPN-1), calpain 2 (CAPN-2) and calpain 10 (CAPN-10) was detected using RT-PCR method. Intracellular Ca²⁺ concentrations, CASP-3 activity and total calpain activity were also evaluated. Our results have shown that glibenclamide and glimepiride decrease 1.2B4 cells viability with accompanied increase in intracellular Ca²⁺ concentration and increased expression of apoptosis-related CASP-3 and TP53. Gliclazide did not affect 1.2B4 cell viability and Ca²⁺ concentration, however, it downregulated CASP-3 and upregulated TP53. Interestingly, 50 μM glimepiride increased expression of CAPN-1, CAPN-2 and CAPN-10 whereas 50 μM glibenclamide solely upregulated CAPN-2 expression. We have shown that 10 μM and 50 μM glibenclamide and glimepiride increased the activity of CASP-3, but decreased total calpain activity. Our results suggest that calpains may be involved in glibenclamide- and glimepiride-induced death of PC cells. However, further investigation is required to confirm the engagement of calpains in SUs-mediated death of PC cells, especially studies on protein level of particular isoforms of calpains should be conducted.
Multi-Tissue Acceleration of the Mitochondrial Phosphoenolpyruvate Cycle Improves Whole-Body Metabolic Health
2020, Cell Metabolism
Citation Excerpt :
An aspect of the current consensus model of beta cell glucose sensing is that glucokinase (GK)-determined oxidative phosphorylation (OxPhos) lowers ADP to control plasma membrane depolarization needed for insulin secretion. Further based on the current model, pharmacological targeting of glucose entry (e.g., with GK activators, GKa) and depolarization of the beta cell membrane (e.g., with sulphonylureas, SU) stimulate insulin secretion, but such approaches have had incrementally diminished long-term therapeutic success (De Ceuninck et al., 2013; Efanova et al., 1998; Erion et al., 2014; Iwakura et al., 2000). Successful targeting of the intermediate steps in the canonical pathway by other approaches, including enhancing OxPhos, have also been of limited success, with no agents currently approved for use in humans.
The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2^−/− mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator in vivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.
Effect of mesenchymal stem cells on induced skeletal muscle chemodenervation atrophy in adult male albino rats
2017, International Journal of Biochemistry and Cell Biology
The present research was conducted to evaluate the effect of bone marrow derived mesenchymal stem cells (BM-MSCs) as a potential therapeutic tool for improvement of skeletal muscle recovery after induced chemodenervation atrophy by repeated local injection of botulinum toxin-A in the right tibialis anterior muscle of adult male albino rats. Forty five adult Wistar male albino rats were classified into control and experimental groups. Experimental group was further subdivided into 3 equal subgroups; induced atrophy, BM-MSCs treated and recovery groups. Biochemical analysis of serum LDH, CK and Real-time PCR for Bcl-2, caspase 3 and caspase 9 was measured. Skeletal muscle sections were stained with H and E, Mallory trichrome, and Immunohistochemical reaction for Bax and CD34. Improvement in the skeletal muscle histological structure was noticed in BM-MSCs treated group, however, in the recovery group, some sections showed apparent transverse striations and others still affected. Immunohistochemical reaction of Bax protein showed strong positive immunoreaction in the cytoplasm of muscle fibers in the induced atrophy group. BM-MSCs treated group showed weak positive reaction while the recovery group showed moderate reaction in the cytoplasm of muscle fibers. Immunohistochemical reaction for CD34 revealed occasional positive CD34 stained cells in the induced atrophy group. In BM-MSCs treated group, multiple positive CD34 stained cells were detected. However, recovery group showed some positive CD34 stained cells at the periphery of the muscle fibers. Marked improvement in the regenerative capacity of skeletal muscles after BM-MSCs therapy. Hence, stem cell therapy provides a new hope for patients suffering from myopathies and severe injuries.
Nuciferine stimulates insulin secretion from beta cells - An in vitro comparison with glibenclamide
2012, Journal of Ethnopharmacology
Several Asian plants are known for their anti-diabetic properties and produce alkaloids and flavonoids that may stimulate insulin secretion.
Using Vietnamese plants (Nelumbo nucifera, Gynostemma pentaphyllum, Smilax glabra, and Stemona tuberosa), we extracted two alkaloids (neotuberostemonine, nuciferine) and four flavonoids (astilbin, engeletin, smitilbin, and 3,5,3′-trihydroxy-7,4′-dimethoxyflavone), and studied their insulin stimulatory effects.
Nuciferine, extracted from Nelumbo nucifera, stimulated both phases of insulin secretion in isolated islets, whereas the other compounds had no effect. The effect of nuciferine was totally abolished by diazoxide and nimodipine, and diminished by protein kinase A and protein kinase C inhibition. Nuciferine and potassium had additive effects on insulin secretion. Nuciferine also stimulated insulin secretion in INS-1E cells at both 3.3 and 16.7 mM glucose concentrations. Compared with glibenclamide, nuciferine had a stronger effect on insulin secretion and less beta-cell toxicity. However, nuciferine did not compete with glibenclamide for binding to the sulfonylurea receptor.
Among several compounds extracted from anti-diabetic plants, nuciferine was found to stimulate insulin secretion by closing potassium-adenosine triphosphate channels, explaining anti-diabetic effects of Nelumbo nucifera.
Exendin-4 protects against sulfonylurea-induced β-cell apoptosis
2012, Journal of Pharmacological Sciences
Sulfonylurea is one of the commonly used anti-diabetic drugs that stimulate insulin secretion from β-cells. Despite their glucose lowering effects in type 2 diabetes mellitus, long-term treatment brought on secondary failure characterized by β-cell exhaustion and apoptosis. ER stress induced by Ca²⁺ depletion in endoplasmic reticulum (ER) is speculated be one of the causes of secondary failure, but it remains unclear. Glucagon like peptide-1 (GLP-1) has anti-apoptotic effects in β-cells after the induction of oxidative and ER stress. In this study, we examined the antiapoptotic action of a GLP-1 analogue in β-cell lines and islets against ER stress induced by chronic treatment of sulfonylurea. HIT-T15 and dispersed islet cells were exposed to glibenclamide for 48 h, and apoptosis was evaluated using Annexin/PI flow cytometry. Expression of the ER stress–related molecules and sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) 2/3 was determined by real-time PCR and western blot analysis. Chronic exposure to glibenclamide increased apoptosis by depletion of ER Ca²⁺ concentration through reduced expression of SERCA 2/3. Pretreatment with Exendin-4 had an anti-apoptotic role through ER stress modulation and ER Ca²⁺ replenishing by SERCA restoration. These findings will further the understanding of one cause of glibenclamide-induced β-cell loss and therapeutic availability of GLP-1–based drugs in secondary failure by sulfonylurea during treatment of diabetes.
Initial combination therapy for type 2 diabetes mellitus: Is it ready for prime time?
2011, American Journal of Medicine
Citation Excerpt :
It has been suggested that, rather than ameliorating the decline in β-cell function characteristic of type 2 diabetes, sulfonylurea therapy may actually accelerate this process by chronic overstimulation of insulin release. A preclinical study has demonstrated that sulfonylureas may deplete β-cell insulin stores101 or induce apoptosis,102-104 although it should be noted that other data do not support the view that sulfonylureas exert long-term negative effects on β-cell function or survival.105,106 Given the potential adverse effects on weight and hypoglycemia, the use of initial early combination therapy with a sulfonylurea and metformin does not appear to be particularly attractive.
The increased prevalence of type 2 diabetes mellitus is primarily being driven by the increasing global rates of overweight/obesity. Given the magnitude of this epidemic, we can expect these metabolic abnormalities to play an increasing role in the development of cardiovascular disease. In a pathophysiologic sense, type 2 diabetes is a multiorgan, multifactorial condition, characterized by β-cell dysfunction, insulin resistance in peripheral tissues and the liver, defective incretin activity, and elevated levels of free fatty acids and proinflammatory mediators. Despite the considerable burden of disease associated with type 2 diabetes, most patients are not at, or are unable to achieve, recommended glycemic control guideline targets. In part, this is because of the relentlessly progressive nature of the disease, but it may also be attributable to the current diabetes treatment paradigm, which is characterized by ineffective lifestyle interventions, followed by monotherapy and frequent early treatment failure with prolonged periods of elevated glucose as a consequence of clinical inertia. Thus, it is most appropriate to rethink the current treatment paradigm for type 2 diabetes in the context of a more aggressive initial therapy; specifically with early initiation of combination therapy. Our current understanding of the complex pathophysiology of the disease and the progressive deterioration in glycemic control over time supports the philosophy of earlier intervention with a more comprehensive initial therapy. Thus, while control of hyperglycemia remains the paramount goal, focusing on the underlying pathophysiology of type 2 diabetes is increasingly becoming the therapeutic strategy, with the aim of potentially providing disease modification. Although this is a logical approach, it remains to be demonstrated that early combination therapy will result in disease modification in a clinical setting. Not surprisingly, the incretin-based therapies have gained a great deal of attention in the context of being a component of initial combination therapy, given their potential beneficial effects on β-cell function with lowered risk of weight gain and hypoglycemia.

View all citing articles on Scopus

View full text

Regular ArticleSustained Enhancement of Ca2+ Influx by Glibenclamide Induces Apoptosis in RINm5F Cells

Abstract

J. Autoimmun.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Int. Rev. Cytol.

Chem.-Biol. Interact.

Exp. Cell. Res.

Diabetes

Proc. Natl. Acad. Sci. USA

Endocrinology

Diabetes

Pancreas

Diabetes

J. Leukocyte Biol.

J. Neurochem.

Regular Article
Sustained Enhancement of Ca²⁺ Influx by Glibenclamide Induces Apoptosis in RINm5F Cells