Interaction between cAMP, volume­regulated anion channels and the Na+­HCO3­­cotransporter, NBCe1, in the regulation of nutrient­ and hypotonicity­induced insulin release from isolated rat pancreatic islets and tumoral insulin­producing BRIN­BD11 cells.

Soluble adenylyl cyclase (sAC) has been hypothesized to play a role in insulin secretion. The present study aimed to investigate the interaction between adenosine 3',5'­cyclic monophosphate (cAMP), volume­regulated anion channels (VRACs) and the electrogenic sodium bicarbonate (Na+­HCO3­) cotransporter, NBCe1, in the regulation of nutrient­ and hypotonicity­induced insulin release from rat pancreatic islets and tumoral insulin­producing BRIN­BD11 cells. In the islets, 5­nitro­2­(3­phenylpropylamino)benzoic acid (NPPB) and 5­chloro­2­hydroxy­3­(thiophene­2­carbonyl)indole­1­carboxamide (tenidap) reduced glucose­stimulated insulin release, however, only NPPB suppressed the enhancing action of cAMP analogs upon such a release. Insulin output from the BRIN­BD11 cells was stimulated by 2­ketoisocaproate (KIC) or extracellular hypoosmolarity. cAMP analogs and 3­isobutyl­1­methylxanthine increased the insulin output recorded in the isotonic medium to a greater relative extent than that in the hypotonic medium. The secretory response to KIC or hypotonicity was inhibited by NPPB or tenidap, which both also opposed the enhancing action of cAMP analogs. Inhibitors of mitogen­activated protein (MAP) kinase decreased insulin output in isotonic and hypotonic media. The inhibitor of sAC, 2­hydroxyestriol, caused only a modest inhibition of insulin release, whether in the isotonic or hypotonic medium, even when tested at a concentration of 100 µM. The omission of NaHCO3 markedly decreased the secretory response to KIC or extracellular hypotonicity. The omission of Na+ suppressed the secretory response to extracellular hypotonicity. The observations of the present study do not support the hypothesis of a major role for sAC in the regulation of insulin release.

Expression of TMEM16A and SLC4A4 in human pancreatic islets.

BACKGROUND/AIMS: Stimulation of insulin release by D-glucose is accompanied by Cl(-) and HCO(3)(-) efflux from pancreatic islet cells. The efflux of these anions may involve volume-regulated anion channels, including possibly TMEM16A, and the Na(+)-HCO(3)(-)-cotransporter SLC4A4. The present study was designed to explore the expression of both TMEM16A and SLC4A4 in human pancreatic islets. METHODS: Pancreases were obtained from human cadaveric donors. Immunodetection of TMEM16A and SLC4A4 was performed by immunohistochemistry on sections of fixed pancreas, while real-time PCR for the study of corresponding gene expression was performed on RNA extracted from both total pancreatic pieces and isolated pancreatic islets. RESULTS: RT-PCR yielded lower levels of SLC4A4 in isolated islets than in the total pancreas, whilst a mirror image prevailed for TMEM16A mRNA. Immunohistochemistry of human pancreas, however, indicated comparable immunostaining of SLC4A4 in insulin-producing cells and exocrine pancreatic cells, whilst that of TMEM16A appeared less pronounced in insulin-producing cells than in exocrine cells. CONCLUSION: The present findings support the view that, in humans like in rodent, the regulation of anion fluxes in insulin-producing cells may involve both SLC4A4 and TMEM16A.

The metabolic syndrome of fructose-fed rats: effects of long-chain polyunsaturated ω3 and ω6 fatty acids. III. Secretory behaviour of isolated pancreatic islets.

In the present study, rats were exposed from the 8th week after birth and for the ensuing 8 weeks to diets containing either starch or fructose (64% w/w) and sunflower oil (5%). Two further groups of rats were exposed to the fructose-containing diet with substitution of part (1.6%) of the sunflower diet by an equal amount of either salmon oil rich in long-chain polyunsaturated ω3 fatty acids or safflower oil reach in long-chain polyunsaturated ω6 fatty acids. The insulin content of the islets and their secretory response to D-glucose (5.6, 8.3 and 16.7 mM), to the combination of D-glucose (5.6 mM) and D-fructose (10.0 mM) and to 2-ketoisocaproate (10.0 mM) were then measured. In the sunflower oil-fed rats, the dietary substitution of starch by fructose decreased basal insulin output, lowered the apparent Km for the insulinotropic action of D-glucose and altered the insulinotropic efficiency of the latter hexose relative to that of other nutrients. Some of these secretory perturbations were opposed by the enrichment of the diet in long-chain polyunsaturated fatty acids, especially ω3 fatty acids. It is proposed that these changes in B-cell secretory behaviour may be linked, in part at least, to both the apparent caloric efficiency of each diet, and hence to the regulation of the islet content in endogenous nutrients, and to alteration of insulin sensitivity considered as a major feature of the present animal model of metabolic syndrome.

The metabolic syndrome of fructose-fed rats: effects of long-chain polyunsaturated ω3 and ω6 fatty acids. IV. D-glucose metabolism by isolated pancreatic islets.

The major aim of the present study was to search for changes of D-glucose metabolism in isolated pancreatic islets possibly involved in the alteration of their secretory response to the hexose, as observed when comparing rats exposed for 8 weeks to diets containing either starch and sunflower oil or fructose and sunflower oil, as well as rats exposed to diets containing fructose, sunflower oil and either salmon oil or safflower oil. The substitution of starch by fructose in the diet affected unfavourably D-glucose phosphorylation by the isolated islets. In the fructose-fed rats, there was a close parallelism between D-[5-³H]glucose utilization and the dietary ω3/ω6 fatty acid ratio. There was little to distinguish, however, between the four groups of rats in terms of D-[U-¹4C]glucose oxidation. The paired ratio between D-[U-¹4C]glucose oxidation and D-[5-³H]glucose utilization, which always increased as the concentration of the hexose was raised from 2.8 to 8.3 and 16.7 mM, was tightly related, in the fructose-fed rats, to the HOMA index for insulin resistance.

Intermittent fasting modulation of the diabetic syndrome in sand rats. III. Post-mortem investigations.

The present report concerns several post-mortem variables examined in sand rats that were either maintained on a vegetal diet (control animals) or exposed first during a 20-day transition period to a mixed diet consisting of a fixed amount of a hypercaloric food and decreasing amounts of the vegetal food and then to a 30-day experimental period of exposure to the hypercaloric food. During the latter period, all animals were either given free access to food or fasting daily for 15 h, i.e. from 5.00 p.m. to 8.00 a.m. The body weight, liver wet weight, pancreas wet weight, plasma glucose and haemoglobin A1c concentration, plasma insulin concentration, insulinogenic index, insulin resistance HOMA, plasma cholesterol and triglyceride concentration, liver triglyceride and phospholipid content were all measured. Pancreatic islet (insulin, GLUT2) and liver (lipid droplets) histology were also examined. The main findings consisted in a lower body weight of fasting than non-fasting animals, a higher liver weight in non-diabetic and diabetic rats than in control non-fasting (but not so in fasting) animals, a decrease of pancreas weight in non-diabetic and diabetic as distinct from control animals, a fasting-induced decrease in plasma glucose, plasma insulin and insulin resistance HOMA, plasma cholesterol and triglyceride concentration and triglyceride liver content.

Expression of the electrogenic Na+-HCO3--cotransporter NBCe1 in tumoral insulin-producing BRIN-BD11 cells.

BACKGROUND/AIMS: The expression of the electrogenic Na+-HCO3--cotransporter NBCe1 was recently documented in rat pancreatic islet B-cells, it being speculated that such a protein participates in the extrusion of bicarbonate generated by the oxidative catabolism of nutrients from insulin-producing cells. Considering the prevalence of a Crabtree effect in tumoral insulin-producing cells, the possible presence of NBCe1 was now investigated in BRIN-BD11 cells, an insulin-producing cell line established by electrofusion of normal pancreatic B-cells with immortalized RINm5F cells. METHODS: The possible presence of NBCe1 in BRIN-BD11 cells was investigated by RT-PCR, western blot analysis and immunocytochemistry. The release of insulin and net uptake of 22Na+ were also measured in the BRIN-BD11 cells. RESULTS: RT-PCR, western blot analysis and immunocytochemistry documented the presence of NBCe1 in BRIN-BD11 cells. A reported inhibitor of NBCe1, i.e. tenidap, (50-100 microM), inhibited basal and hypotonicity-induced insulin release from the BRIN-BD11 cells, whilst increasing the net uptake of 22Na+ by the same cells. The latter effect was, in relative terms, more pronounced in the presence than absence of ouabain. CONCLUSION: BRIN-BD11 cells, like normal pancreatic islet B-cells, express NBCe1, with predominance of the B variant of this electrogenic Na+-HCO3--cotransporter.

The metabolic syndrome of omega3-depleted rats. I. Liver data.

Second-generation rats depleted in long-chain polyunsaturated omega3 fatty acids were recently proposed as a novel animal model for the metabolic syndrome. In the present study, a dietary deprivation of omega3 acids for 3-7 months was found sufficient to provoke in 6-week-old normal rats the same alteration of the fatty acid content and profile of liver phospholipids and triglycerides as that otherwise prevailing in the second-generation omega3-depleted rats, with emphasis on a severe decrease in their omega3 fatty acid content, alterations in the relative contribution of and ratio between selected long-chain polyunsaturated omega6 fatty acids, saturated and monodesaturated fatty acids and precursors of nervonic acid, and liver steatosis. When the omega3-depleted rats were exposed, after the first 7 months of the present experiments and for 2-4 weeks to a diet supplemented with 5% (w/w) flaxseed oil, most of these hepatic variables returned towards or beyond control values. In both the omega3-depleted rats and control animals, however, the eventual exposure to the flaxseed oil-enriched diet failed to suppress liver steatosis and, on the contrary, provoked a further increase in liver triglyceride content. It is proposed, therefore, that the present approach represents a simple and realistic animal model to study the consequences of omega3-depletion. Moreover, the results suggest that to oppose such consequences, e.g. liver steatosis, it may be necessary to combine the dietary supply of omega3 acids with a suitable control of food intake, in both qualitative and quantitative terms.

The metabolic syndrome of omega3-depleted rats. II. Body weight, adipose tissue mass and glycemic homeostasis.

Exposure of 7-week-old normal rats for 3-7 months to a diet deprived of long-chain polyunsaturated omega3 fatty acids was recently reported to induce changes in the fatty acid content and pattern of liver phospholipids and triglycerides similar to those otherwise found in second generation omega3-depleted rats. In the present study, the changes in body weight, parametrial adipose tissue mass, plasma glucose and insulin concentrations and insulin resistance index were investigated in the same control and omega3-depleted rats, which were then given access for 2 to 4-5 weeks to either a flaxseed oil-enriched diet (control and omega3-depleted rats) or a soybean oil-enriched diet (control rats). The body weight failed to differ between control and omega3-depleted rats. The latter rats, however, displayed increases in adipose tissue mass, plasma glucose and insulin concentrations, and insulin resistance index. In the control rats given access to the soybean or flaxseed oil-enriched diet, body weight and adipose tissue mass were little affected, but both the plasma glucose concentration and insulin resistance index decreased. In the omega3-depleted rats given access to the flaxseed oil-enriched diet, both body weight and adipose tissue mass underwent a rapid, pronounced and sustained increase, whilst the plasma glucose concentration and insulin resistance index decreased similarly to those in the control rats. The present design of omega3 fatty acid dietary deprivation thus reproduces the visceral obesity and insulin resistance otherwise observed in second-generation omega3-depleted rats. However, the supply of exogenous omega3 fatty acids to the omega3-depleted rats failed to oppose visceral obesity, possibly as a result of the orexigenic effects of these omega3 fatty acids.

Salivary glucose concentration and excretion in normal and diabetic subjects.

The present report aims mainly at a reevaluation of salivary glucose concentration and excretion in unstimulated and mechanically stimulated saliva in both normal and diabetic subjects. In normal subjects, a decrease in saliva glucose concentration, an increase in salivary flow, but an unchanged glucose excretion rate were recorded when comparing stimulated saliva to unstimulated saliva. In diabetic patients, an increase in salivary flow with unchanged salivary glucose concentration and glucose excretion rate were observed under the same experimental conditions. Salivary glucose concentration and excretion were much higher in diabetic patients than in control subjects, whether in unstimulated or stimulated saliva. No significant correlation between glycemia and either glucose concentration or glucose excretion rate was found in the diabetic patients, whether in unstimulated or stimulated saliva. In the latter patients, as compared to control subjects, the relative magnitude of the increase in saliva glucose concentration was comparable, however, to that of blood glucose concentration. The relationship between these two variables was also documented in normal subjects and diabetic patients undergoing an oral glucose tolerance test.

Expression of the electrogenic Na+-HCO3--cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells.

It was recently proposed that, in rat pancreatic islets, the production of bicarbonate accounts for the major fraction of the carbon dioxide generated by the oxidative catabolism of nutrient insulin secretagogues. In search of the mechanism(s) supporting the membrane transport of bicarbonate, the possible role of the electrogenic Na(+)-HCO(3) (-)-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells was investigated. Expression of NBCe1-A and NBCe1-B in rat pancreatic islet cells was documented by RT-PCR, western blotting, and immunocytochemistry. The latter procedure suggested a preferential localization of NBCe1-B in insulin-producing cells. Tenidap (3-100 microM), previously proposed as an inhibitor of NBCe1-A-mediated cotransport in proximal tubule kidney cells, caused a concentration-related inhibition of glucose-stimulated insulin secretion. It also inhibited 2-ketoisocaproate-induced insulin release and to a relatively lesser extent, the secretory response to L: -leucine. Tenidap (50-100 microM) also inhibited the metabolism of D: -glucose in isolated islets, increased (22)Na net uptake by dispersed islet cells, lowered intracellular pH and provoked hyperpolarization of plasma membrane in insulin-producing cells. This study thus reveals the expression of the electrogenic Na(+)-HCO(3) (-)-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells, and is consistent with the participation of such transporters in the process of nutrient-stimulated insulin secretion.

Long-chain fatty acyl-coenzyme A-induced inhibition of glucokinase in pancreatic islets from rats depleted in long-chain polyunsaturated omega3 fatty acids.

The metabolism of D-glucose was recently reported to be impaired in pancreatic islets from second generation rats depleted in long-chain polyunsaturated omega3 fatty acids. Considering the increased clearance of circulating non-esterified fatty acids prevailing in these rats, a possible inhibition of glucokinase in insulin-producing cells by endogenous long-chain fatty acyl-CoA was considered. The present study was mainly aimed at assessing the validity of the latter proposal. The activity of glucokinase in islet homogenates, as judged from the increase in D-glucose phosphorylation rate in response to a rise in the concentration of the hexose represented, in the omega3-depleted rats, was only 81.8 +/- 4.8% (n = 11; p < 0.005) of the paired value recorded in control animals. This coincided with the fact that the inclusion of D-glucose 6-phosphate (3.0 mM) and D-fructose 1-phosphate (1.0 mM) in the assay medium resulted in a lesser fractional decrease of D-glucose phosphorylation in omega3-depleted rats than in control animals. Moreover, whereas palmitoyl-CoA (50 microM) decreased the activity of glucokinase by 38.0 +/- 6.0% (n = 4; p < 0.01) in islet homogenates from normal rats, the CoA ester failed to affect significantly the activity of glucokinase in islet homogenates from omega3-depleted rats. These findings afford direct support for the view that glucokinase is indeed inhibited by endogenous long-chain fatty acyl-CoA in islets from omega3-depleted rats, such an inhibition probably participating to the alteration of D-glucose catabolism prevailing in these islets.