Emeline Hupkens

A new role for aquaporin 7 in insulin secretion.

Bacgrouns/Aims: Several insulinotropic agents were recently reported to cause β-cell swelling. The possible participation of AQP7 to water transport was investigated in AQP7+/ + or AQP7-/- mice. Methods: Aquaporin expression, insulin secretion, cell swelling and electrical activity were investigated in pancreatic islets. Results: RT-PCR revealed the expression of AQP5 and AQP8 mRNA. Double immunofluorescent labeling indicated their presence in β-cells. Whilst basal insulin release from isolated pancreatic islets incubated at 2.8 mM D-glucose did not differ between AQP7+/ + or AQP7-/- mice, the secretion of insulin evoked by the omission of 50 mM NaCl, the substitution of 50 mM NaCl by 100 mM glycerol or a rise in D-glucose concentration to 8.3 mM and 16.7 mM was severely impaired in the islets from AQP7-/- mice. Yet, exposure of β-cells to either the hypotonic medium or a rise in D-glucose concentration caused a similar degree of swelling and comparable pattern of electrical activity in cells from AQP7+/ + and AQP7-/- mice. Both the cell swelling and change in membrane potential were only impaired in AQP7-/- cells when exposed to 50 mM glycerol. Conclusion: It is proposed, therefore, that AQP7 may, directly or indirectly, play a role at a distal site in the exocytotic pathway.

Intermittent Fasting Modulation of the Diabetic Syndrome in Streptozotocin-Injected Rats

This study investigates the effects of intermittent overnight fasting in streptozotocin-induced diabetic rats (STZ rats). Over 30 days, groups of 5-6 control or STZ rats were allowed free food access, starved overnight, or exposed to a restricted food supply comparable to that ingested by the intermittently fasting animals. Intermittent fasting improved glucose tolerance, increased plasma insulin, and lowered Homeostatis Model Assessment index. Caloric restriction failed to cause such beneficial effects. The β-cell mass, as well as individual β-cell and islet area, was higher in intermittently fasting than in nonfasting STZ rats, whilst the percentage of apoptotic β-cells appeared lower in the former than latter STZ rats. In the calorie-restricted STZ rats, comparable findings were restricted to individual islet area and percentage of apoptotic cells. Hence, it is proposed that intermittent fasting could represent a possible approach to prevent or minimize disturbances of glucose homeostasis in human subjects.

Prevention of pulmonary hypoplasia and pulmonary vascular remodeling by antenatal simvastatin treatment in nitrofen-induced congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) has a high mortality rate mainly due to lung hypoplasia and persistent pulmonary hypertension of the newborn (PPHN). Simvastatin has been shown to prevent the development of pulmonary hypertension (PH) in experimental models of PH. We, therefore, hypothesized that antenatal simvastatin would attenuate PPHN in nitrofen-induced CDH in rats. The efficacy of antenatal simvastatin was compared with antenatal sildenafil, which has already been shown to improve pathological features of PPHN in nitrofen-induced CDH. On embryonic day (E) 9.5, nitrofen or vehicle was administered to pregnant Sprague-Dawley rats. On E11, nitrofen-treated rats were randomly assigned to antenatal simvastatin (20 mg·kg(-1)·day(-1) orally), antenatal sildenafil (100 mg·kg(-1)·day(-1) orally), or placebo administration from E11 to E21. On E21, fetuses were delivered by cesarean section, killed, and checked for left-sided CDH. Lung tissue was then harvested for further pathobiological evaluation. In nitrofen-induced CDH, simvastatin failed to reduce the incidence of nitrofen-induced CDH in the offspring and to increase the body weight, but improved the lung-to-body weight ratio and lung parenchyma structure. Antenatal simvastatin restored the pulmonary vessel density and external diameter, and reduced the pulmonary arteriolar remodeling compared with nitrofen-induced CDH. This was associated with decreased lung expression of endothelin precursor, endothelin type A and B receptors, endothelial and inducible nitric oxide synthase, together with restored lung activation of apoptotic processes mainly in the epithelium. Antenatal simvastatin presented similar effects as antenatal therapy with sildenafil on nitrofen-induced CDH. Antenatal simvastatin improves pathological features of lung hypoplasia and PPHN in experimental nitrofen-induced CDH.

Perturbation of glycerol metabolism in hepatocytes from n3-PUFA-depleted rats

Second generation n3-PUFA-depleted rats represent a good animal model of metabolic syndrome as they display several features of the disease such as liver steatosis, visceral obesity and insulin resistance. The goal of our study was to investigate the influence of n3-PUFA deficiency on hepatic glycerol metabolism. Aquaglyceroporin 9 (AQP9) allows hepatic glycerol transport and consequently contributes to neoglucogenesis. AQP9 knockout mice display hypertriacyl-glycerolemia, one of the hallmarks of the metabolic syndrome. Our data show reduced AQP9 expression at the protein level in n3-PUFA-depleted rats, without any changes at the mRNA levels. [U-¹⁴C]glycerol uptake was increased in hepatocytes from n3-PUFA-depleted animal cells. The apparent discrepancy between decreased AQP9 protein expression, and increased [U-¹⁴C]glycerol uptake could be explained by an observed increase in glycerol kinase activity.

The metabolic syndrome of fructose-fed rats: Effects of long-chain polyunsaturated ω3 and ω6 fatty acids. II. Time course of changes in food intake, body weight, plasma glucose and insulin concentrations and insulin resistance

The time course for changes in food intake, body weight, plasma glucose and insulin concentrations and HOMA index was monitored over a period of 8 weeks in rats exposed from the 8th week after birth to diets containing either starch or fructose and sunflower oil. In two further groups of rats exposed to the fructose-rich diet part of the sunflower oil was substituted by either salmon oil rich in long-chain polyunsaturated ω3 fatty acids or safflower oil rich in long-chain polyunsaturated ω6 fatty acids. Despite lower food intake, the gain in body weight was higher in fructose-fed rats than in starch-fed rats. The supplementation of the fructose-rich diet by either ω3 or ω6 fatty acids lowered both food intake and body weight gain. The measurements of plasma glucose and insulin concentrations, HOMA index and insulinogenic index performed after overnight starvation were in fair agreement with those recorded at the occasion of an intraperitoneal glucose tolerance test, with higher values for plasma glucose concentration and HOMA index in the fructose-fed rats exposed to the sunflower oil (with or without enrichment with ω6 fatty acids) than in the starch-fed rats exposed to the sunflower oil or fructose-fed rats exposed to a diet enriched with ω3 fatty acids. Such was also the case for the measurements of glycated albumin at sacrifice. Moreover, the insulinogenic index was lower in the fructose-fed rats with or without dietary enrichment in ω6 fatty acids than in the fructose-fed rats with dietary enrichment in ω3 fatty acids. The elucidation of the biochemical determinants of the later difference requires further investigations in isolated pancreatic islets.

Expression and Localization of Glucose Transporters in Rodent Submandibular Salivary Glands

Background/Aim: The submandibular gland is one of the three major salivary glands, producing a mixed secretion; this saliva is hypotonic compared to plasma. It also secretes glucose, but the mechanisms responsible for this process are poorly understood. Our study addressed the question whether glucose transporters are expressed and how are they localized within specific rodent submandibular cells, in order to estimate a possible implication in salivary glucose disposal. Methods: Immunohistochemistry, RT-qPCR and Western blotting were performed to determine the presence/localization of glucose transporters in rodent submandibular glands. Results: GLUT4 was identified in the submandibular salivary gland at both mRNA and protein level. The immunohistochemical analysis revealed its localization preponderantly in the ductal cells of the gland, near to the basolateral. SGLT1 and GLUT1 were highly expressed in submandibular tissues in both acinar and ductal cells, but not GLUT2. These results were confirmed by RT-qPCR. It was also documented that insulin stimulates the net uptake of D-glucose by ductal rings prepared from submandibulary salivary glands, the relative magnitude of such an enhancing action being comparable to that found in hemidiaphragms. Conclusion: At least three major glucose transporters are expressed in the rodent submandibular glands, of which GLUT4 is specifically localized near the basolateral side of ductal structures. This points-out its possible role in regulating glucose uptake from the bloodstream, most likely to sustain ductal cellular metabolism.

The metabolic syndrome of fructose-fed rats: Effects of long-chain polyunsaturated ω3 and ω6 fatty acids. I. Intraperitoneal glucose tolerance test

The present series of experiments aim mainly at investigating the possible influence of changes in the com-position of dietary lipids (sunflower oil, salmon oil, safflower oil) upon the metabolic syndrome found in rats exposed to a fructose-rich diet. For purpose of comparison, a control group of rats received the sunflower oil diet with substitution of fructose by starch. An intraperitoneal glucose tolerance test, performed after overnight starvation fifty days after the start of the experiments at the 6th week after birth, indicated, as expected, impaired tolerance to glucose and deterioration of insulin sensitivity (HOMA index), without changes in the insulinogenic index, when comparing the fructose-fed rats to the starch-fed rats both exposed to the sunflower oil diet. In the fructose-fed rats, enrichment of the diet by long-chain polyunsaturated ω3 fatty acids supplied by salmon oil, a modest improvement of insulin sensitivity was opposed, in term of glucose homeostasis, by a decreased secretory response to glucose of insulin-producing cells. Last, in the fructose-fed rats, the partial substitution of sunflower oil by safflower oil rich in long-chain polyunsaturated ω6 fatty acids further deteriorated glucose homeostasis, with a higher mean HOMA index and a severe decrease of the insulinogenic index. These findings justify further investigations on such items as the time course for changes in metabolic and hormonal variables and both the metabolic and secretory responses of isolated pancreatic islets to selected nutrient secretagogues.

The metabolic syndrome of fructose-fed rats: Effects of long-chain polyunsaturated ω3 and ω6 fatty acids. VI. Further post-mortem investigations

The present study documents the increases in systolic arterial blood pressure, plasma leptin concentration and kidney proliferating cell nuclear antigen index, as well as the decreases in glutathione reductase, superoxide dismutase and catalase enzymatic activities in the liver, heart, kidney, soleus muscle and visceral adipose tissue homogenates of female rats exposed for 8 weeks to a diet containing 64% (w/w) D-fructose instead of 64% starch. In the fructose-fed rats, the partial substitution of sunflower oil by either safflower oil or salmon oil often opposed the fructose-induced changes in these variables. The present results, thus, extend to these functional, hormonal and enzymatic parameters the knowledge that the dietary supply of long-chain polyunsaturated ω6 fatty acids, mainly C18:2ω6, and long-chain polyunsaturated ω3 fatty acids opposes the undesirable features of the fructose-induced metabolic syndrome, with salmon oil demonstrating particular efficacy.

Leptin-Induced Endothelium-Independent Vasoconstriction in Thoracic Aorta and Pulmonary Artery of Spontaneously Hypertensive Rats: Role of Calcium Channels and Stores

Decreased leptin-induced endothelium-dependent vasodilation has been reported in spontaneously hypertensive rats (SHR). Here, we report leptin-induced vasoconstriction in endothelium-denuded pulmonary artery and thoracic aorta from SHR and sought to characterize calcium handling underlying these mechanisms. Vasoreactivity to leptin was evaluated on pulmonary artery and thoracic aorta rings from 18 weeks old male SHR with or without calcium free medium, caffeine + thapsigargin + carbonyl cyanide-4-trifluoromethoxyphenylhydrazone emptying intracellular calcium stores, nifedipine a voltage-gated calcium channel inhibitor, SKF-96365 a transient receptor potential cation channels (TRPC) inhibitor, wortmaninn, a phosphatidylinositide 3-kinases (PI3K) inhibitor, or PD98059 a mitogen-activated protein kinase kinase (MAPKK) inhibitor. Calcium imaging was performed on cultured vascular smooth muscle cells incubated with leptin in presence or not of wortmaninn or PD98059. Leptin induced vasoconstriction in denuded pulmonary artery and thoracic aorta from SHR. Response was abolished when intra- or extracellular calcium stores were emptied, after blocking TRPC or voltage-dependent calcium channels or when using MAPKK or PI3K inhibitors. In vascular smooth muscle cells, leptin increased intracellular calcium. This rise was higher in SHR and abolished by MAPKK or PI3K inhibitors. TRPC6 gene expression was upregulated in arteries from SHR. Leptin-induced vasoconstriction in denuded arteries of SHR requires intracellular stores and is TRPC- and voltage-gated calcium channels dependent. Intracellular calcium increase is more pronounced in spontaneously hypertensive rats.

A tentative model for D-glucose turnover in human saliva

OBJECTIVE The aim of the present study is to propose a tentative model for d-glucose turnover in human saliva. The whole saliva and the saliva from parotid and submandibular/sublingual glands were collected by use of the Salivette™. RESULTS The saliva glucose concentration was measured by the hexokinase method, saliva bacteria glycolysis by use of d-[5-(3)H] glucose, and the saliva ATP content by the luciferase method. The concentration of glucose amounted to 43.9±6.3 (n=29), 197.5±17.3 (n=29), 104.0±12.4 (n=27) μM in whole saliva, parotid saliva and submandibular/sublingual saliva, respectively. The rate of d-glucose utilization by oral bacteria at a physiological concentration of d-glucose in saliva (50μM) was estimated at 0.047±0.003 (n=11) nmol/min per 10(6) bacteria. Unstimulated salivary d-glucose turnover rate, as calculated from the amount of glucose secreted in saliva which comes from parotid and submandibular and sublingual glands represented 214.6±19.1%/min. In order for salivary d-glucose production to match bacterial utilization of the hexose, the total number of oral bacteria was estimated at about 2.0×10(9) bacteria, in fair agreement with previously published data. CONCLUSION This study thus provides support for a tentative model for d-glucose turnover in human saliva.