Anne Virsolvy

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

BACKGROUND AND PURPOSE 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 (H2O2). 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.

Terlipressin, a provasopressin drug exhibits direct vasoconstrictor properties: consequences on heart perfusion and performance.

Objective:Terlipressin has been proposed as an alternative treatment to catecholamines to restore blood pressure in septic shock. Terlipressin is considered as a vasopressin prodrug capable of releasing small but sustained amounts of [Lysine8] vasopressin (LVP) and to provide prolonged biological effect. However, terlipressin may act as a direct vasopressor beyond its conversion into LVP. We investigated terlipressin direct vasoconstrictive properties and consequences on myocardial perfusion and performance. Design:Experimental studies. Settings:National Research Institute Laboratories. Subjects:Rat aorta and heart, human uterine artery. Interventions:Studies of vasoconstriction on isolated vascular rings obtained either from rat aorta or human uterine artery, and of coronary flow, ventricular performance, and heart rhythm on rat hearts using a modified Langendorff heart apparatus. Measurements and Main Results:Terlipressin induced a rapid, saturable, and dose-dependent contraction of rat aortas and human uterine arteries. Although the maximal terlipressin-induced vasoconstriction observed on rat arteries was weaker than LVP, or arginine-vasopressin, pharmacologic properties on human arteries, such as full agonism and strong maximal effect (900% of the maximal response obtained with phenylephrine), suggest a high potential of terlipressin to directly vasoconstrict human vessels. Similarly, terlipressin induced a saturable and dose-dependent vasoconstriction of coronary arteries that was reversible and antagonized by selective V1a antagonists. Maximum rates of left ventricle pressure rise (dP/dtmax) and fall (dP/dtmin) decreased both only in proportion to the decrease in coronary flow. Conclusions:Besides long lasting effect through slow conversion into LVP, terlipressin is a fast acting vasopressor peptide per se that has an impact on coronary circulation and myocardial function.

Direct Action of Endothelin-1 on Podocytes Promotes Diabetic Glomerulosclerosis

The endothelin system has emerged as a novel target for the treatment of diabetic nephropathy. Endothelin-1 promotes mesangial cell proliferation and sclerosis. However, no direct pathogenic effect of endothelin-1 on podocytes has been shown in vivo and endothelin-1 signaling in podocytes has not been investigated. This study investigated endothelin effects in podocytes during experimental diabetic nephropathy. Stimulation of primary mouse podocytes with endothelin-1 elicited rapid calcium transients mediated by endothelin type A receptors (ETARs) and endothelin type B receptors (ETBRs). We then generated mice with a podocyte-specific double deletion of ETAR and ETBR (NPHS2-Cre×Ednra(lox/lox)×Ednrb(lox/lox) [Pod-ETRKO]). In vitro, treatment with endothelin-1 increased total β-catenin and phospho-NF-κB expression in wild-type glomeruli, but this effect was attenuated in Pod-ETRKO glomeruli. After streptozotocin injection to induce diabetes, wild-type mice developed mild diabetic nephropathy with microalbuminuria, mesangial matrix expansion, glomerular basement membrane thickening, and podocyte loss, whereas Pod-ETRKO mice presented less albuminuria and were completely protected from glomerulosclerosis and podocyte loss, even when uninephrectomized. Moreover, glomeruli from normal and diabetic Pod-ETRKO mice expressed substantially less total β-catenin and phospho-NF-κB compared with glomeruli from counterpart wild-type mice. This evidence suggests that endothelin-1 drives development of glomerulosclerosis and podocyte loss through direct activation of endothelin receptors and NF-κB and β-catenin pathways in podocytes. Notably, both the expression and function of the ETBR subtype were found to be important. Furthermore, these results indicate that activation of the endothelin-1 pathways selectively in podocytes mediates pathophysiologic crosstalk that influences mesangial architecture and sclerosis.

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

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 ([Ca2+]i) in beta cells, in the absence of any co‐stimulating factor.

Protection against hypoxic-ischemic injury in transgenic mice overexpressing Kir6.2 channel pore in forebrain

The role of the K-ATP channel pore-forming subunit Kir6.2 on protection from cerebral hypoxic-ischemic injury was assessed in transgenic mice overexpressing normal Kir6.2 or a dominant negative form (AFA) of this subunit in the forebrain. The resulting mice overexpress either the Kir6.2 or the AFA transgene mainly in the cerebral cortex and hippocampus. The Kir6.2 transgenic mice are resistant to hypoxic-ischemic injury showing a decreased region of cortical damage as compared to the dominant negative AFA and the wild-type mice. Moreover, the overexpression of Kir6.2 allowed an important silencing of the neurons present in forebrain regions thus protecting them from ischemic injury. Interestingly, the phenotype observed in Kir6.2 transgenic mice was observed without increased sulfonylurea binding. Taken together, these results indicate that the transgenic overexpression of Kir6.2 in forebrain significantly protects mice from hypoxic-ischemic injury and neuronal damage seen in stroke.

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 β cells

1 The MIN6 cell line derived from in vivo immortalized insulin‐secreting pancreatic β 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 drived from it, might be the basis for a new pharmacological approach to the mangement of type II (non insulin‐dependent) diabetes.

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

Abstract. ATP-dependent potassium (KATP) channels occupy a key position in the control of insulin release from the pancreatic β 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 β 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, α-endosulfine, and discuss its possible role in the physiology of the β cell as well as in pathology.

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

The MIN6 cell line derived from in vivo immortalized insulin‐secreting pancreatic β 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. S‐22068, was able to release insulin from MIN6 cells in a dose‐dependent manner with a half‐maximal stimulation at 100 μM. 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. Similarly to sulphonylureas and classical imidazolines, S‐22068 blocked KATP channels and, in turn, opened nifedipine‐sensitive voltage‐dependent Ca2+ channels, triggering Ca2+ entry. Similarly to other imidazolines, S‐22068 induced a closure of cloned KATP channels injected to Xenopus oocytes by interacting with the pore‐forming Kir6.2 moiety. S‐22068 did not interact with the sulphonylurea binding site nor with the non‐I1 and non‐I2 imidazoline site evidenced in the β cells that is recognized by the imidazoline compounds efaroxan, phentolamine and RX821002. 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 β cell KATP channels.

Block of Ca2+-channels by alpha-endosulphine inhibits insulin release

α‐Endosulphine, isolated as an endogenous equivalent for sulphonylureas, is a 121‐amino acids protein of 19 kDa apparent molecular mass, member of a cyclic AMP‐regulated phosphoprotein family. We have previously shown that α‐endosulphine inhibits sulphonylurea binding and KATP channel activity, thereby stimulating basal insulin secretion. We now describe that in the perfused rat pancreas, no stimulation was detected and that α‐endosulphine inhibited glucose stimulated insulin release. This inhibition was dose‐dependent and affected both phases of insulin secretion. This inhibitory effect of α‐endosulphine also occurred on MIN6 β‐cells when insulin release was stimulated either by glucose, sulphonylureas or a high K+ depolarization. Inhibition was concentration‐dependent with a half‐maximal inhibition at 0.5 μM and was mirrored by inhibition of calcium influx. Electrophysiological experiments demonstrated, in comparison to the effects of the sulphonylurea tolbutamide, that these inhibitory effects were linked to a direct inhibition of L‐type Ca2+‐channels and were independent from a regulation of KATP channels. Although α‐endosulphine is able to stimulate insulin release under specific conditions acting via modulation of KATP channel activity, the present study suggests that, under physiological conditions, the peptide mainly acts to block voltage‐gated Ca2+‐channels. This block leads to the inhibition of calcium influx and triggers inhibition of insulin release. We conclude that α‐endosulphine is not exclusively an endogenous equivalent for sulphonylureas and not solely a KATP channel regulator.

Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine

Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na+ current (INa), and is known to reduce the Na+-dependent Ca2+ overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na+ channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca2+ calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine.