Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells.

BACKGROUND AND PURPOSE: Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca(2+)]i) in beta cells, in the absence of any co-stimulating factor. EXPERIMENTAL APPROACH: Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca(2+)]i were measured using the ratiometric fluorescent Ca(2+) indicator Fura-2. Ca(2+) channel currents were recorded with the whole-cell patch-clamp technique. KEY RESULTS: Quercetin concentration-dependently increased insulin secretion and elevated [Ca(2+)]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 µmol·L(-1)), but were nearly abolished by the L-type Ca(2+) channel antagonist nifedipine (1 µmol·L(-1)). Similar to the L-type Ca(2+) channel agonist Bay K 8644, quercetin enhanced the L-type Ca(2+) current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca(2+)]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 µmol·L(-1)), with the two drugs having cumulative effects on [Ca(2+)]i. CONCLUSIONS AND IMPLICATIONS: Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca(2+) influx through an interaction with L-type Ca(2+) channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion.

Quercetin potentiates insulin secretion and protects INS-1 pancreatic ß-cells against oxidative damage via the ERK1/2 pathway.

BACKGROUND AND PURPOSE: Quercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic ß-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting ß-cells. Using the INS-1 ß-cell line, the effects of quercetin were determined on glucose- or glibenclamide-induced insulin secretion and on ß-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. EXPERIMENTAL APPROACH: 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 ß-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. CONCLUSION AND IMPLICATIONS: Quercetin potentiated glucose and glibenclamide-induced insulin secretion and protected ß-cells against oxidative damage. Our study suggested that ERK1/2 played a major role in those effects. The potential of quercetin in preventing ß-cell dysfunction associated with diabetes deserves further investigation.

[Molecular effects of new calcium antagonists: is the principle of parcimony out of place?]. / Effets moléculaires de nouveaux antagonistes calciques: le principe de parcimonie est-il toujours de mise ?

The calcium (Ca2+) channel antagonists (CCA) are used successfully in the treatment of hypertension and angina pectoris. Their mode of action is to decrease Ca2+ entry in the vascular smooth muscle cells. Their molecular targets are voltage activated Ca2+ channels (VACC), especially the L-type (VACC-L). This review examines the role of the VACC-L and of the T-type (VACC-T) in vascular physiology and hypertension. The molecular mechanisms at the base of the vascular selectivity of CCA are presented with, in filigree, the concern of trying to understand the effect of recently developed molecules. In particular, we will examine the ideas having recently emerged concerning the mode of action of last generation dihydropyridines (DHPs) stripped of some of the undesirable effects of prototypes AC considered as highly specific of the VACC-L. These properties could result, in particular, from their effects on the VACC-T, which could occur in addition to those classically observed on the VACC-L.

New perspectives on the key role of calcium in the progression of heart disease.

The heart continuously adapts to adjust its output to a continuum of pathophysiological situations ensuring adequate blood distribution. These situations range from high performance in well-trained athletes to failure in a variety of cardiac syndromes. Changes in the concentration of intracellular Ca2+ ([Ca2+]i) are crucial. They have immediate and late effects that can be oversimplified as follows. Immediate effects result from abrupt and large variations in [Ca2+]i triggering contraction after binding to the contractile proteins. These variations are involved in the process known to as excitation-contraction (EC) coupling. In contrast, the late effects involve a process that is, by analogy, referred to as excitation-transcription (ET) coupling. This process involves activation of gene expression by Ca2+. In this scheme, specific and localised elevations of Ca2+ can be converted into changes in gene expression with long-term effects on the adaptation of the heart to a sustained stimulus. There is emerging evidence of an extraordinary diversity of responses, depending on the location, intensity, and duration of Ca2+ signals that can be activated during pathology. Whereas alterations of cellular and molecular mechanisms underlying chronic pathology are relatively well defined, the initial changes and their hierarchy are unknown. However, the actual picture suggests promising perspectives for new therapeutic interventions on old targets or new strategies. Some of these aspects are reviewed here.

Localization of alpha-endosulphine in pancreatic somatostatin delta cells and expression during rat pancreas development.

AIMS/HYPOTHESIS: alpha-Endosulphine, a protein that belongs to the cAMP-regulated-phosphoprotein family, has been reported to modulate insulin secretion in vitro through interaction with the pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channel. In this study, we analysed the tissue distribution of alpha-endosulphine and determined its pancreatic cellular localization. METHODS: Quantitative tissue distribution of alpha-endosulphine was studied by RIA on tissue extracts and cellular/subcellular localization was done using immunocytochemistry, morphometry and western blot analysis. alpha-Endosulphine and somatostatin release from RINT-3 somatostatin-secreting cells was quantified by RIA. RESULTS: alpha-Endosulphine, concentrated particularly in the central nervous system, was also detected in a wide variety of tissues including the pancreas. Immunohistochemistry analysis of adult rat pancreatic sections showed that alpha-endosulphine localized in somatostatin delta cells, where its expression increased during post-natal development. Immunoreactive cells were detected from foetal age E19, and the number of somatostatin cells co-expressing alpha-endosulphine increased with developmental age from E19 until adult. alpha-Endosulphine, highly expressed in the cytoplasm of RINT3 somatostatin-secreting cell line, was recovered in the particulate fraction of RINT3 cell extracts but was not co-secreted with somatostatin. CONCLUSION/INTERPRETATION: alpha-Endosulphine is expressed in all tissues tested including pancreas and is also detected in plasma. Pancreatic alpha-endosulphine is specifically localized in somatostatin delta cells. This cytosolic protein is not co-secreted with somatostatin and could be physically associated with particulate components of the cells. These findings are not in favour of an endocrine/paracrine effect of alpha-endosulphine on the beta-cell K(ATP) channel.

In vitro mechanism of action on insulin release of S-22068, a new putative antidiabetic compound.

1. The MIN6 cell line derived from in vivo immortalized insulin-secreting pancreatic beta cells was used to study the insulin-releasing capacity and the cellular mode of action of S-22068, a newly synthesized imidazoline compound known for its antidiabetic effect in vivo. 2. S-22068, was able to release insulin from MIN6 cells in a dose-dependent manner with a half-maximal stimulation at 100 micronM. Its efficacy (8 fold over the basal value), which did not differ whatever the glucose concentration (stimulatory or not), was intermediate between that of sulphonylurea and that of efaroxan. 3. Similarly to sulphonylureas and classical imidazolines, S-22068 blocked K(ATP) channels and, in turn, opened nifedipine-sensitive voltage-dependent Ca2+ channels, triggering Ca2+ entry. 4. Similarly to other imidazolines, S-22068 induced a closure of cloned K(ATP) channels injected to Xenopus oocytes by interacting with the pore-forming Kir6.2 moiety. 5. S-22068 did not interact with the sulphonylurea binding site nor with the non-I1 and non-I2 imidazoline site evidenced in the beta cells that is recognized by the imidazoline compounds efaroxan, phentolamine and RX821002. 6. We conclude that S-22068 is a novel imidazoline compound which stimulates insulin release via interaction with an original site present on the Kir6.2 moiety of the beta cell K(ATP) channels.

Isolation, characterization, and chromosomal localization of the human ENSA gene that encodes alpha-endosulfine, a regulator of beta-cell K(ATP) channels.

Human alpha-endosulfine is an endogenous regulator of the beta-cell K(ATP) channels. The recombinant alpha-endosulfine inhibits sulfonylurea binding to beta-cell membranes, reduces cloned K(ATP) channel currents, and stimulates insulin secretion from beta-cells. These properties led us to study the human ENSA gene that encodes alpha-endosulfine. Here, we describe the isolation, the partial characterization, and the chromosomal localization of the ENSA gene. The ENSA gene appears to be a 1.8-kb-long sequence that contains the transcription initiation site located 528 bp upstream of the initiation codon. The ENSA gene is intronless, and a single copy gene seems to be present in the genome. Finally, the ENSA gene co-localizes on human chromosome 14 (14q24.3-q31) with a locus for susceptibility to type 1 diabetes called IDDM11; thus, the ENSA gene represents an IDDM11 candidate.

Characterization of low-affinity binding sites for glibenclamide on the Kir6.2 subunit of the beta-cell KATP channel.

The ATP-sensitive K+ channel, an octameric complex of two structurally unrelated types of subunits, SUR1 and Kir6.2, plays a central role in the physiological regulation of insulin secretion. The sulfonylurea glibenclamide, which trigger insulin secretion by blocking the ATP-sensitive K+ channel, interacts with both high and low affinity binding sites present on beta-cells. The high affinity binding site has been localized on SUR1 but the molecular nature of the low affinity site is still uncertain. In this study, we analyzed the pharmacology of glibenclamide in a transformed COS-7 cell line expressing the rat Kir6.2 cDNA and compared with that of the MIN6 beta cell line expressing natively both the Kir6.2 and the SUR1 subunits. Binding studies and Scatchard analysis revealed the presence of a single class of low affinity binding sites for glibenclamide on the COS/Kir6.2 cells with characteristics similar to that observed for the low affinity site of the MIN6 beta cells.

alpha-Endosulfine, a new entity in the control of insulin secretion.

ATP-dependent potassium (K ATP) channels occupy a key position in the control of insulin release from the pancreatic beta cell since they couple cell polarity to metabolism. These channels close when more ATP is produced via glucose metabolism. They are also controlled by sulfonylureas, a class of drugs used in type 2 diabetic patients for triggering insulin secretion from beta cells that have lost part of their sensitivity to glucose. We have demonstrated the existence of endogenous counterparts to sulfonylureas which we have called 'endosulfines.' In this review, we describe the discovery, isolation, cloning, and biological features of the high-molecular-mass form, alpha-endosulfine, and discuss its possible role in the physiology of the beta cell as well as in pathology.

Human alpha-endosulfine, a possible regulator of sulfonylurea-sensitive KATP channel: molecular cloning, expression and biological properties.

Sulfonylureas are a class of drugs commonly used in the management of non-insulin-dependent diabetes mellitus. Their therapeutic action results primarily from their ability to inhibit ATP-sensitive potassium (KATP) channels in the plasma membrane of pancreatic beta cells and thereby stimulate insulin release. A key question is whether an endogenous ligand for the KATP channel exists that is able to mimic the inhibitory effects of sulfonylureas. We describe here the cloning of the cDNA encoding human alpha-endosulfine, a 13-kDa peptide that is a putative candidate for such a role. alpha-Endosulfine is expressed in a wide range of tissues including muscle, brain, and endocrine tissues. The recombinant protein displaces binding of the sulfonylurea [3H]glibenclamide to beta cell membranes, inhibits cloned KATP channel currents, and stimulates insulin secretion. We propose that endosulfine is an endogenous regulator of the KATP channel, which has a key role in the control of insulin release and, more generally, couples cell metabolism to electrical activity.

Stimulation of insulin release from the MIN6 cell line by a new imidazoline compound, S-21663: evidence for the existence of a novel imidazoline site in beta cells.

1. The MIN6 cell line derived from in vivo immortalized insulin-secreting pancreatic beta cells was used to study the insulin-releasing capacity and the cellular mode of action of S-21663, a newly synthesized imadizoline compound known for its antidiabetic effect in vivo and its ability to release insulin from perfused pancreas. 2. S-21663, at concentrations ranging from 10(-5) M to 10(-3) M was able to release insulin from MIN6 cells; its activity peaked at 10(-4) M, a drop in the stimulant factor being noted between 10(-4) and 10(-3) M. Its efficacy, which did not differ whatever the glucose concentration (stimulant or not), was higher than that of the other secretagogues tested, glucose, sulphonylureas or the peptide tGLP-1. 3. In contrast to tGLP-1, S-21663 did not change the cyclic AMP content, whereas it increased Ca2+ influx via verapamil- and nifedipine-sensitive voltage-dependent calcium channels, the insulin release being a direct consequence of this Ca2+ entry. The S-21663-induced Ca2+ influx appears to be essentially the consequence of closure of K+ channels which differ from the ATP-dependent K+ (K-ATP) channels as determined by measurement of 86Rb efflux and use of a K-ATP channel opener. 4. Comparison of the effects of S-21663 to that of efaroxan, another imidazoline compound shown to act on insulin release in a glucose-dependent way via binding sites distinct from the imidazoline I1 and I2 sites, suggested that S-21663 acts through a novel site which displays a remarkably stable expression along the cell culture. 5. It is concluded that S-21663 is a very efficient, glucose-independent insulin secretagogue acting through a novel imidazoline site, linked to K+ channels, distinct from the I1, I2 and 'efaroxan' binding sites. In vitro and in vivo features of S-21663 indicate that this compound, or new drugs derived from it, might be the basis for a new pharmacological approach to the mangement of type II (non insulin-dependent) diabetes.