Somatostatin content and release of isolated pancreatic islets from obese-hyperglycemic mice.

The somatostatin content in pancreatic islets of obese-hyperglycemic mice was much lower than in the islets of normal mice. Also the release of somatostatin was decreased from the islets obtained from the obese-hyperglycemic mice. Tissue culture for 1 week changed neither the content of, nor the amount of somatostatin released from, the pancreatic islets.     

Hormone content of mouse pancreatic islets subjected to different in vivo and in vitro functional demands

Summary Mice treated for 4 days with tolbutamide displayed decreased serum glucose values with a concomitant decrease of their islet insulin content. Mouse islets cultured for 1 week at a low (3 mM) or a high (28 mM) glucose concentration contained less insulin than non-cultured islets and islets cultured at a medium (11 mM) glucose concentration. All groups of cultured islets contained more glucagon than non-cultured islets. The somatostatin content of high- and medium-glucose cultured islets was higher than that of freshly isolated islets.The skilled technical assistance of Astrid Nordin, Ewa Forsbeck and Eva Törnelius is gratefully acknowledged. Financial support was received from the Swedish Medical Research Council (12X-109; 12X-2297), the Nordic Insulin Fund and the Swedish Diabetes Association.     

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.     

Epilepsy in women. Oestrogen and progesterone plasma levels

Summary Comparison is made between numbers of fits and estrogen, progesterone plasma values during the menstrual cycle of women with epilepsy. All six cycles in women with ovulation had a significant positive correlation between estrogen/progesterone ratio and scores of fits. Three periods without ovulation all showed a significant correlation to estrogen variations.Acknowledgments. This work was supported by Gunvor och Josef Anérs stiftelse, Med. fac. University of Umeå, The Swedish Medical Research Council (Proj. No. 2148) and Karl Oskar Hansson's fund.     

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     

The effect of cyproheptadine on insulin biosynthesis

Summary The effect of a potent antiserotonin-antithistaminic compound, cyproheptadine (CPH) on insulin biosynthesis was studied in pancreatic islets isolated from CPH-treated rats. Though insulin content of islets was markedly reduced in CPH-treated rats, the incorporation of radiolabeled leucine into proinsulin and insulin fractions was not affected with respect to the rate and amount. It is concluded that CPH may deplete insulin content of the islets through causing the leakage of insulin.     

The effect of cyproheptadine on insulin biosynthesis

The effect of a potent antiserotonin-antihistaminic compound, cyproheptadine (CPH) on insulin biosynthesis was studied in pancreatic islets isolated from CPH-treated rats. Though insulin content of islets was markedly reduced in CPH-treated rats, the incorporation of radiolabeled leucine into proinsulin and insulin fractions was not affected with respect to the rate and amount. It is concluded that CPH may deplete insulin content of the islets through causing the leakage of insulin.     

Loss of huntingtin-associated protein 1 impairs insulin secretion from pancreatic β-cells

Hap1 was originally identified as a neuronal protein that interacts with huntingtin, the Huntington’s disease (HD) protein. Later studies revealed that Hap1 participates in intracellular trafficking in neuronal cells and that this trafficking function can be adversely affected by mutant huntingtin. Hap1 is also present in pancreatic β-cells and other endocrine cells; however, the role of Hap1 in these endocrine cells remains unknown. Using the Cre-loxP system, we generated conditional Hap1 knockout mice to selectively deplete the expression of Hap1 in mouse pancreatic β-cells. Mutant mice with Hap1 deficiency in pancreatic β-cells had impaired glucose tolerance and decreased insulin release in response to intraperitoneally injected glucose. Using cultured pancreatic β-cell lines and isolated mouse pancreatic islets, we confirmed that decreasing Hap1 could reduce glucose-mediated insulin release. Electron microscopy suggested that there was a reduced number of insulin-containing vesicles docked at the plasma membrane of pancreatic islets in Hap1 mutant mice following intraperitoneal glucose injection. Glucose treatment decreased the phosphorylation of Hap1A in cultured β-cells and in mouse pancreatic tissues. Moreover, this glucose treatment increased Hap1’s association with kinesin light chain and dynactin p150, both of which are involved in microtubule-dependent trafficking. These studies suggest that Hap1 is important for insulin release from β-cells via dephosphorylation that can regulate its intracellular trafficking function.     

Bromocriptine/SKF38393 ameliorates islet dysfunction in the diabetic (db/db) mouse

Dysfunction of pancreatic islets plays a crucial role in the etiology of type II diabetes. Chronic hyperglycaemia or hyperlipidaemia may impair islet function. Previous studies by our laboratory have demonstrated that dopaminergic agonists ameliorated hyperglycaemia and hyperlipidaemia in obese and diabetic rodents. In the present study, we investigated the effect of a treatment with the dopamine D₂ /D₁ receptor agonists (bromocriptine/SKF38393, BC/SKF) on islet dysfunction in db/db mice. Our results show that a 2-week BC/SKF treatment markedly reduced hyperglycaemia and hyperlipidaemia, and significantly improved islet dysfunction demonstrated by an increase of secretagogue-stimulated insulin release from islets of db/db mice to levels observed in islets from lean mice. There was also a fourfold increase of insulin content in the pancreas of BC/SKF-treated db/db mice compared with that in untreated controls. The effect of BC/SKF on islet function cannot be mimicked in pair-fed animals. BC/SKF had no direct stimulatory effect on islet insulin secretion, suggesting BC/SKF treatment improved islet function via an indirect mechanism. This treatment markedly improved the abnormally elevated daily levels of corticosterone, blood glucose and plasma lipids, supporting the view that BC/SKF may affect the neuroendocrine system that in turn regulates peripheral metabolism and thereby improves islet function. Received 3 April 1998; accepted 27 April 1998     

Exocrine pancreas: difference in the amylase content of the dorsal and ventral regions

Summary The amylase content of the acinar tissue is higher in the splenic region of the rat pancreas containing glucagon-rich islets than in the duodenal region harboring pancreatic polypeptide-rich islets.This work was supported by a grant from the Belgian Ministry of Scientific Policy.     

Presence of "pancreatic polypeptide" cells, and gastrin immunoreactivity in immuno-induced exocrine pancreas carcinoma]

1) In electively immuno-induced carcinomas of the exocrine pancrease in Mice, where A (glucagon) and B (insulin) endocrine cells persist, cells with a pancreatic polypeptide immunoreactivity are also detected, even in late evolution stages. These cells, like D cells, containing somatostatin, are localized only in the pancreatic remains surrounding the anaplasic carcinomatous tissue: islets, adenomatous parenchyma, and ductular epithelium. Ultrastructure of these cells shows their active elaboration of numerous chracteristic secretion granules. (2) Immunocytoenzymatic detection of gastrin is negative in the exocrine and endocrine pancreatic tissues. However one of the anti-gastrin sera used gives a positive reaction, in some carinomatous cells only. Does this immunoreactivity characterize a polypeptide specific to the pancreatic carcinomatous cell?     

A convulsant, 3-mercaptopropionic acid,decreases the level of GABA and GAD in rat pancreatic islets and brain

To investigate the properties of the gamma-aminobutyric acid (GABA) synthesizing enzyme, glutamate decarboxylase (GAD), in the brain and the pancreatic islets of the rat, GABA concentration in the brain and the pancreatic islets was measured after intraperitoneal administration of 3-mercaptopropionic acid (3-MP) at 25 mg/kg. 60 min after the administration of 3-MP, GABA concentration in the hypothalamus, the superior colliculus and the hippocampus of the brain decreased by 20–30% and in the pancreatic islets by 35%. The concentration in the pancreatic acini did not change. Western blotting showed that GAD activity in the pancreatic islets decreased after administration of 3-MP compared to the control. The activity of GAD in the pancreatic islets as well as brain can be modified by a convulsant, in this case 3-MP. These results suggest the properties of GAD may be similar in the pancreatic islets and brain.     

Pancreatic islet cell suspensions from newborn rats; different preparation procedures,viability and (pro)insulin biosynthesis

Summary Islet cell suspensions were prepared from neonatal rat pancreatic islets. While mechanical disintegration results in a higher yield, cells prepared by trypsin treatment appear to be better preserved. Trypsin treatment of pancreatic islets during the cell preparation procedure does not influence the stimulation by glucose of (pro)insulin biosynthesis in freshly isolated cells.Investigations were carried out as a part of the HFR Diabetes mellitus and Fettstoffwechselstörungen supported by the Ministry of Health of the GDR.     

Pancreatic polypeptide: A possible role in the regulation of food intake in the mouse. Hypothesis

Summary Pancreatic polypeptide (PP) is a recently identified hormone produced by pancreatic endocrine cells. The islets of genetically obese mice (ob/ob, C57 BL/6J), which are suspected to lack a circulating satiety factor, contain relatively few of the PP-producing cells. Administration of bovine pancreatic polypeptide (bPP) reduces food intake and suppresses body weight gain in the hyperphagic obese mice. It is postulated that PP participates in the regulation of food intake in a manner as yet undefined.This work was supported by grant No. 3.553.75 from Swiss National Science Foundation. We thank Mrs M. Eissler and Mr R. Cuche for their valuable help.     

Endocrine and exocrine pancreatic function after camostate-induced growth of the organ

It is well known that oral administration of camostate induces hyperplasia and hypertrophy of the rat pancreas. It is not clear, however, whether pancreatic hormone and enzyme secretion are affected by camostate treatment.In rats, daily administration of 200 mg camostate/kg b. wt for 14 days significantly increased pancreatic weight and pancreatic content of DNA, protein, amylase, lipase, trypsin and chymotrypsin, as well as the amount of insulin, glucagon and somatostatin. In the intact animal, blood glucose levels and serum concentrations of insulin and glucagon in response to an oral glucose load were not impaired after camostate treatment. In the isolated perfused pancreas, however, insulin and glucagon secretions were reduced, whereas somatostatin release was not affected. The volume of pancreatic juice produced by the unstimulated isolated perfused organ, as well as protein and enzyme secretion, were increased after camostate treatment. Likewise, the isolated perfused pancreas from camostate-treated rats secreted a larger volume of pancreatic juice and more protein in response to cholecystokinin (CCK), while enzyme secretion was affected in a non-parallel manner: amylase release was markedly reduced, lipase release was unchanged, and release of trypsin and chymotrypsin was increased.     

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.

     

Imparied bone resorption of cultured calvaria from mice with abnormal lysosomal function (the Chediak-Higashi syndrome)

Summary Spontaneous bone resorption is reduced in cultured calvarial bones from mice with the Chediak-Higashi syndrome, as indicated by decreased mobilization of calcium from the bones to the medium. Although bone resorption in calvaria from mice with this disease can be stimulated by PGE₂ and 1 (OH)D₃, the amounts of mineral released after stimulation is also decreased.This investigation was supported by grants from the Swedish Association against Rheumatic Diseases, from the Royal 80 Year Fund of King Gustav V, and from the Swedish Medical Research Council (project No 14X-05426).     

Interaction of vasoactive intestinal peptide (VIP) with cholinergic stimulation of glucagon secretion

Summary VIP-containing nerve fibers as well as cholinergic nerve fibers have a ubiquitous distribution in the body and both types of nerves have been demonstrated to innervate the pancreatic islets. The present study shows, in the intact, conscious mouse, that VIP and the cholinergic agonist carbachol stimulate glucagon secretion in a dose-dependent manner. Furthermore VIP and carbachol were found to exert potentiating interactions on glucagon secretion. These results suggest the existence of an interactive neural regulation of glucagon secretion, exerted by acetylcholine and VIP.We thank Professor V. Mutt, Karolinska Institutet, Stockholm, for giving us VIP. The skilful technical assistance of Lena K vist and Peter Okmark is gratefully acknowledged. This study was supported by grants from the Swedish Medical Research Council (14P-4289, 14X-4286) and the Medical Faculty, University of Lund, Sweden.     

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 -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.     

Interleukin-17 stimulates inducible nitric oxide synthase-dependent toxicity in mouse beta cells

The influence of the proinflammatory cytokine interleukin (IL)-17 on inducible nitric oxide (NO) synthase (iNOS)-mediated NO release was investigated in the mouse insulinoma cell line MIN6 and mouse pancreatic islets. IL-17 markedly augmented iNOS mRNA/protein expression and subsequent NO production induced in MIN6 cells or pancreatic islets by different combinations of interferon-γ, tumor necrosis factor-α, and IL-1β. The induction of iNOS by IL-17 was preceded by phosphorylation of p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK activation completely abolished IL-17-stimulated NO release. IL-17 enhanced the NO-dependent toxicity of proinflammatory cytokines toward MIN6 cells, while IL-17-specific neutralizing antibody partially reduced the NO production and rescued insulinoma cells and pancreatic islets from NO-dependent damage induced by activated T cells. Finally, a significant increase in blood IL-17 levels was observed in a multiple low-dose streptozotocin model of diabetes, suggesting that T cell-derived IL-17 might be involved in NO-dependent damage of beta cells in this disease. Received 14 June 2005; received after revision 17 September 2005; accepted 21 September 2005