Effect of a restricted diet on the in vitro glucose-induced insulin release of aging rats.

To study the effect of a sudden loss of body weight on the beta-cell function of aging rats, basal and glucose-induced insulin secretion was measured in pancreatic islets obtained from young (2-month-old), adult (12-month-old) and aging (24-month-old) rats, either fed ad libitum or fed a restricted diet (50% caloric restriction). Basal insulin secretion was similar in islets of young, adult and older rats. Glucose stimulated insulin release was significantly reduced in aging rats as compared to young animals. Animals fed a restricted diet showed a prolonged and higher secretory rate during first phase release when compared to animals fed ad libitum.     

Impaired insulin release in aging rats: Metabolic and ionic events

We investigated the effect of aging on glucose uptake, glucose-induced O₂ consumption, glucose-induced⁴⁵Ca movements, and calmodulin content to elucidate age-related impairment of glucose-induced insulin release in pancreatic islets of Wistar rats. Intact pancreatic islets from old (24-month-old) rats showed impaired glucose-induced insulin release; glucose uptake and O₂ consumption were lower in old than in young (2-month-old) or adult (12-month-old) rats. Moreover,⁴⁵Ca uptake and calmodulin content were decreased in pancreatic islets from older rats, which explained the impairment in glucose-induced insulin release in aging. No major differences between the 3 age groups in glucose-induced⁴⁵Ca efflux in pancreatic islets were observed.     

Lack of influence of vitamin D deficiency on insulin release from the isolated pancreatic islets of rats

Pancreatic islets were isolated from young (100 g) and adult (390 g), normal and vitamin D deficient male Sprague-Dawley rats. The release of insulin from leucine-stimulated or glucose-stimulated islet was not altered by vitamin D deficiency. The in vitro addition of either 25-hydroxy- or 1,25-dihydroxyvitamin-D had no effect on insulin release from either normal or vitamin D deficient islets. We conclude that the earlier report (Normal et al., Science 209 (1980) 823-825) on vitamin D deficiency depressing insulin secretion from the perfused pancreas must be related to the vitamin's effect on insulin synthesis and not the islet's release of insulin.     

Ovarian stimulation by exogenous gonadotrophins in fetal ethanol-exposed immature rats

Summary Adult pregnant rats were given either an ad libitum liquid diet containing 5% ethanol, a pair fed liquid diet or an ad libitum diet of rat chow and water administered throughout pregnancy and during the nursing period. The female offspring received either pregnant mare's serum gonadotrophin (PMSG) or PMSG followed by human chorionic gonadotrophin (hCG) at 30 days of age. The ovaries of fetal ethanol-exposed animals responded greater to the exogeneous gonadotrophins with enhanced ovarian weights, increased numbers of ova shed, greater numbers of corpora lutea and antral follicles, and higher serum progesterone levels than in animals exposed to the control diets during gestation.     

Ovarian stimulation by exogenous gonadotrophins in fetal ethanol-exposed immature rats

Adult pregnant rats were given either an ad libitum liquid diet containing 5% ethanol, a pair fed liquid diet or an ad libitum diet of rat chow and water administered throughout pregnancy and during the nursing period. The female offspring received either pregnant mare's serum gonadotrophin (PMSG) or PMSG followed by human chorionic gonadotrophin (hCG) at 30 days of age. The ovaries of fetal ethanol-exposed animals responded greater to the exogenous gonadotrophins with enhanced ovarian weights, increased numbers of ova shed, greater numbers of corpora lutea and antral follicles, and higher serum progesterone levels than in animals exposed to the control diets during gestation.     

Ethanol influence on insulin secretion from isolated rat islets

Summary This study was done to delineate the role of - and -adrenergic receptors and cyclic AMP in the mechanism of ethanol effects on insulin release from isolated islets. Rats were given an -adrenergic blocker, phentolamine, or a -adrenergic blocker, propranolol. In addition, ethanol 1 g/kg was given intragastrically 1 h prior to sacrifice. Glucose mediated insulin release from isolated islets was enhanced by phentolamine and decreased by propranolol. Ethanol treatment inhibited glucose-induced insulin release from isolated islets of control rats as well as those given phentolamine and/or propranolol. Insulin release from isolated islets in response to dibutyryl-cyclic AMP was attenuated by ethanol. Theophylline enhanced glucose mediated insulin release from control islets but ethanol treatment produced a significant inhibition of insulin response. The data suggest that the site of action of the deleterious effects of ethanol on insulin release from isolated islets in rat does not involve adrenergic system and cyclic AMP.Supported by the U.S. Veterans Administration     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion.

The role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca2+, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca2+ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 mumol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 mumol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca2+. Increasing the Ca2+ concentration to 1.26 mmol/l in TCM 199 during the 22-24 h exposure to glucose (16.7 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca2+ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Kinetics of insulin secretion in response to glucose by isolated islands of Langerhans from the fetal rat]

Isolated islets of Langerhans from 21.5 day-old foetal Rats were studied in a perifusion system in vitro. The overall dynamics of insulin release by the foetal islets in response to glucose 13.9 mM is biphasic and qualitatively similar to that obtained with islets of adult Rats. The magnitude of the initial phase of insulin secretion is similar for the foetal and adult islets. The second phase is fourfold higher in the adult than in the foetus. The response of foetal islets occurs, 45 to 60 sec. after the increase of glucose concentration in the medium and a maximum insulin release for the first phase is obtained with a lag period of 2 min. The difference between foetal and adult islets is essentially quantitative.     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion

The role of protein kinase C and Ca²⁺ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22–24h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca²⁺, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca²⁺ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 μmol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca²⁺. Increasing the Ca²⁺ concentration to 1.26 mmol/l in TCM 199 during the 22–24h exposure to glucose (16.8 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca²⁺ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Chromosomal abnormalities in starved and marginally malnourished rats and in utero upon rehabilitation

The effects of starvation and marginal malnutrition (MN) on the lymphocytes of rats were evaluated by chromosomal analysis before and after rehabilitation. The effect of parental starvation or malnutrition on chromosomal aberrations in the foetus was also studied. Wistar rats, 30–35 days old, were starved for 5 days or fed a minimally restricted or a severely restricted diet for three weeks. At the end of the period of starvation or malnutrition, lymphocytes were isolated and chromosomal analysis was performed. Starved and severely restricted rats showed significantly higher mean chromosomal aberrations than the controls. These aberrations returned to a normal level when the experimental groups were rehabilitated for a month, indicating that the damage was transient. A chromosomal aberration study done on foetal cells from rehabilitated rats which had previously been starved or fed a severely restricted diet showed significantly increased values, indicating that some damage was permanent. A low number of implantations was also recorded in these experimental groups. These observations clearly indicate that young animals exposed to conditions like starvation or chronic malnutrition are prone to permanent damage of the genetic system.     

Dynamic insulin secretion from perifused rat pancreatic islets

Insulin secretion from isolated pancreatic islets of 8- to 12-day-old rats was investigated in a dynamic in vitro (perifusion) system. The aims of the study were (i) to describe a carefully controlled in vitro method to study the mechanism of insulin secretion and to analyse the effects and dynamic interactions of bioactive compounds on isolated rat pancreatic islets, (ii) to validate the method by comparing fundamental data on the functions of the islets obtained with this method to those collected with other techniques; and (iii) to find novel features of the control of insulin secretion. The method was carefully designed to maintain the functional capacity of the explanted cells. A functional standardization system was elaborated consisting of (i) analysis of the changes in the basal hormone secretion of the cells; (ii) evaluating responses to a standard, specific stimuli (50 mM glucose for 3 min); (iii) determining the alteration of the momentary size of the hormone pool with responses to KCl; and (iv) direct determination of the total intracellular hormone content from the extract of the column. The technique provides accurate quantitative data on the dynamic responses to biologically active compounds that act directly on the pancreatic islets. The islets maintained their full responsiveness for up to 7 days, and responses as close as in 1-min intervals could be distinguished. A linear dose-response relationship was found on the glucose-induced insulin release in case of 3-min stimulation with 4 and 500 mM of glucose (lin-log graph). Utilizing this method, we showed that no desensitization to glucose-induced insulin release can be observed if the responsiveness of the cells is properly maintained and the parameters of the stimulation are carefully designed. Exposure of the explanted islets to 10 μM acetylcholine or 30 mM arginine (Arg) induced a transitory elevation of insulin release similar in shape to that experienced after glucose stimulation. Norepinephrine (NE), dopamine (DA) and somatostatin (SS) did not induce any detectable alteration on the basal insulin secretion of the islets. However, 100 nM SS given together with 50 mM glucose, 30 mM Arg or 10 μM acetylcholine significantly reduced the insulin-releasing effect of these substances (by 75.5, 71.5 and 72.5%, respectively). At the same time, SS did not alter the insulin response of the islets to 100 mM elevation of K⁺ concentration. SS also inhibited glucose-induced insulin release in a dose-dependent way (ED₅₀ = 22 nM). A similar dose-dependent inhibitory effect on glucose-induced insulin release was found with NE (ED₅₀ = 89 nM) and DA (ED₅₀ = 2.2 μM). γ-Aminobutyric acid (GABA) did not influence insulin release under similar circumstances. Received 16 January 1998; received after revision 6 May 1998; accepted 8 May 1998     

Die Rolle von Glukose im Erholungsprozess des Muskels

Summary It is well known that after exhausting muscle work glucose (or saccharose) leads to quick recovery in man. Experiments on rats have shown that in adult animals 50% of all creatine is present as creatine-phosphate (CP). In old animals above 22 months, only about 25% is present as CP. Diets with a 50% glucose content lead in old animals, in rest, to 50% CP in the muscle. Also after exhausting work on glucose diet the restitution is so complete that about 50% CP is present. The main reservoir of energy for the restitution of muscle, ADP ATP, comes from the breakdown of CP. The problem may be discussed whether high glucose diet may be damaging insulin production by exhaustion.     

Ethanol influence on insulin secretion from isolated rat islets

This study was done to delineate the role of alpha- and beta-adrenergic receptors and cyclic AMP in the mechanism of ethanol effects on insulin release from isolated islets. Rats were given an alpha-adrenergic blocker, phentolamine, or a beta-adrenergic blocker, propranolol. In addition, ethanol 1 g/kg was given intragastrically 1 h prior to sacrifice. Glucose mediated insulin release from isolated islets was enhanced by phentolamine and decreased by propranolol. Ethanol treatment inhibited glucose-induced insulin release from isolated islets of control rats as well as those given phentolamine and/or propranolol. Insulin release from isolated islets in response to dibutyryl-cyclic AMP was attenuated by ethanol. Theophylline enhanced glucose mediated insulin release from control islets but ethanol treatment produced a significant inhibition of insulin response. The data suggest that the site of action of the deleterious effects of ethanol on insulin release from isolated islets in rat does not involve adrenergic system and cyclic AMP.     

Effect of feeding cereal-legume diets to rats on in vitro absorption of45Ca

Summary The in vitro absorption of calcium from the duodenum was significantly less in a group of rats fed on a wheat diet than in a group fed a wheat and Bengal gram (7030) diet.     

Defining G protein-coupled receptor peptide ligand expressomes and signalomes in human and mouse islets

Introduction

Islets synthesise and secrete numerous peptides, some of which are known to be important regulators of islet function and glucose homeostasis. In this study, we quantified mRNAs encoding all peptide ligands of islet G protein-coupled receptors (GPCRs) in isolated human and mouse islets and carried out in vitro islet hormone secretion studies to provide functional confirmation for the species-specific role of peptide YY (PYY) in mouse islets.

Materials and methods

GPCR peptide ligand mRNAs in human and mouse islets were quantified by quantitative real-time PCR relative to the reference genes ACTB, GAPDH, PPIA, TBP and TFRC. The pathways connecting GPCR peptide ligands with their receptors were identified by manual searches in the PubMed, IUPHAR and Ingenuity databases. Distribution of PYY protein in mouse and human islets was determined by immunohistochemistry. Insulin, glucagon and somatostatin secretion from islets was measured by radioimmunoassay.

Results

We have quantified GPCR peptide ligand mRNA expression in human and mouse islets and created specific signalomes mapping the pathways by which islet peptide ligands regulate human and mouse GPCR signalling. We also identified species-specific islet expression of several GPCR ligands. In particular, PYY mRNA levels were ~ 40,000-fold higher in mouse than human islets, suggesting a more important role of locally secreted Pyy in mouse islets. This was confirmed by IHC and functional experiments measuring insulin, glucagon and somatostatin secretion.

Discussion

The detailed human and mouse islet GPCR peptide ligand atlases will allow accurate translation of mouse islet functional studies for the identification of GPCR/peptide signalling pathways relevant for human physiology, which may lead to novel treatment modalities of diabetes and metabolic disease.

     

Effects of pregnancy on the glucose-induced insulin release from cultivated pancreatic islets of the rat

Summary 7-day-cultured islets from pregnant Wistar rats released at 5.6 mM glucose significantly more insulin than islets from nonpregnant rats, whereas in vivo this heigthened glucose sensitivity is lost 48 h post partum.Investigations carried out as a part of the Forschungsprojekt Diabetes mellitus und Fettstoffwechselstörungen supported by the Ministry of Health of German Democratic Republic.     

The adhesion receptor GPR56 is activated by extracellular matrix collagen III to improve β-cell function

Aims

G-protein coupled receptor 56 (GPR56) is the most abundant islet-expressed G-protein coupled receptor, suggesting a potential role in islet function. This study evaluated islet expression of GPR56 and its endogenous ligand collagen III, and their effects on β-cell function.

Methods

GPR56 and collagen III expression in mouse and human pancreas sections was determined by fluorescence immunohistochemistry. Effects of collagen III on β-cell proliferation, apoptosis, intracellular calcium ([Ca²⁺]_i) and insulin secretion were determined by cellular BrdU incorporation, caspase 3/7 activities, microfluorimetry and radioimmunoassay, respectively. The role of GPR56 in islet vascularisation and innervation was evaluated by immunohistochemical staining for CD31 and TUJ1, respectively, in pancreases from wildtype (WT) and Gpr56^?/? mice, and the requirement of GPR56 for normal glucose homeostasis was determined by glucose tolerance tests in WT and Gpr56^?/? mice.

Results

Immunostaining of mouse and human pancreases revealed that GPR56 was expressed by islet β-cells while collagen III was confined to the peri-islet basement membrane and islet capillaries. Collagen III protected β-cells from cytokine-induced apoptosis, triggered increases in [Ca²⁺]_i and potentiated glucose-induced insulin secretion from WT islets but not from Gpr56^?/? islets. Deletion of GPR56 did not affect glucose-induced insulin secretion in vitro and it did not impair glucose tolerance in adult mice. GPR56 was not required for normal islet vascularisation or innervation.

Conclusion

We have demonstrated that collagen III improves islet function by increasing insulin secretion and protecting against apoptosis. Our data suggest that collagen III may be effective in optimising islet function to improve islet transplantation outcomes, and GPR56 may be a target for the treatment of type 2 diabetes.

     

The role of phosphoenolpyruvate in insulin secretion: the effect of L-phenylalanine

Summary Incubation of rat islets with phenylalanine increased the tissue content of phosophoenolpyruvate, both in the presence and in the absence of glucose. At the same time, L-phenylalanine neither stimulated nor inhibited insulin release. It is unlikely that insulin secretion is tightly coupled to the availability of phosphoenolpyruvate in rat islets.     

Cholesterol feeding to rats does not modulate the expression of binding sites for HDL on liver membranes

Summary The binding of HDL, Apo-E-free, was studied in rats fed a cholesterol rich diet for 2, 4 and 7 days. Plasma cholesterol increased up to 16-fold (from 55 to 900 mg/dl); liver cholesterol was also raised, from 0.5 to 16 mg/g of tissue. The HDL binding to membrane preparations was not affected while the binding of VLDL was reduced to about 50% of the controls. These data show, therefore, that liver binding sites for HDL are refractory to regulation by dietary cholesterol.     

Insulin secretion and (pro)insulin biosynthesis of cultured mice islets after glibenclamide loading in vitro

Summary Isolated islets from C57B1/6J mice exposed to 10 mmoles/1 glucose supplemented with 5 g/ml glibenclamide for 48 h released significantly less insulin in the subsequent short-time incubation thanuntreated controls (without glibenclamide), whereas insulin biosynthesis was not suppressed by glibenclamide treatment.Investigations were carried out as a part of the ForschungsprojektDiabetes mellitus und Fettstoffwechselstörungen, supported by the Ministry of Health of the GDR.