A new collagenase blend increases the number of islets isolated from mouse pancreas

Diabetes is a predominant metabolic disorder in the industrialized nations. Since pancreatic islets play a key role in type I and type II diabetes, the isolation of islets from pancreatic tissues represents an important step in diabetes research. However, to date, only a small fraction of all islets, resident within any given pancreas, are harvested by using currently available enzyme blends. This holds true for islet isolation from both the mouse and the human pancreas. In the present study, the newly developed Liberase TL Research Grade was compared to the widely used Liberase RI to investigate the effect of increased collagenase purity on islet yield. The study shows that reducing the degradation products of collagenases during Liberase production significantly increases the number of islets isolated from the mouse pancreas by 28%, and, therefore, is expected to lower the numbers of mice and resulting costs for diabetes research accordingly. Furthermore, this study also points to a possibility to increase the number and mass of islets isolated from human pancreases, for which only a limited donor pool exists.     

Inhibitory effect of kisspeptins on insulin secretion from isolated mouse islets

Islet hormone secretion is regulated by a variety of factors, and many of these signal through G protein-coupled receptors (GPCRs). A novel islet GPCR is GPR54, which couples to the Gq isoform of G proteins, which in turn signal through the phospholipase C pathway. Ligands for GPR54 are kisspeptins, which are peptides encoded in the KISS1 gene and also expressed in islet β-cells. The KISS1 gene encodes a hydrophobic 145-amino acid protein that is cleaved into a 54-amino acid protein, kisspeptin-54 or KP54. Shorter kisspeptins also exist, such as kisspeptin-10 (KP10) and kisspeptin-13 (KP13). The involvement of GPR54 and kisspeptins in the regulation of islet function is not known. To address this problem, we incubated isolated mouse islets in the presence of KP13 and KP54 for 60 min and measured insulin secretion. We found that both KP13 and KP54 at 10 nM, 100 nM and 1μM inhibited insulin secretion in the presence of 2.8 mM glucose. However, by increasing the glucose concentration, this inhibitory action of the kisspeptins vanished. Thus, at 11.1 mM glucose, KP13 and KP54 inhibited insulin secretion only at high doses, and at 16.7 mM they no longer inhibited insulin secretion in any of the doses. We conclude that kisspeptins inhibit insulin secretion at glucose concentrations below 11.1 mM. This suggests that kisspeptins are regulating insulin secretion at physiological concentrations of glucose. The mechanisms by which kisspeptins regulate islet function and insulin secretion are unknown and will be further investigated.     

Leptin increases the viability of isolated rat pancreatic islets by suppressing apoptosis

To test the hypothesis that leptin secreted from adipose tissue is a mediator linking obesity and pancreatic islet hypertrophy, we examined the effects of leptin on proliferative and apoptotic responses in rat islet cells. Rat pancreatic islets were isolated and incubated with 0, 1, 5, or 75 nM leptin for 24 h under serum-deprived conditions. Cell viability was assessed with 2,5-diphenyltetrazolium bromide and trypan blue dye exclusion tests. Cell proliferation and apoptosis were evaluated with 5-bromo-2'-deoxyuridine incorporation into DNA and DNA ladder formation, respectively. Incubation for 24 h with 1 and 5 nM leptin, the concentrations observed in obese subjects, increased the viability of isolated pancreatic islet cells. Five nanomolar concentrations of leptin did not stimulate 5-bromo-2'-deoxyuridine incorporation into incubated islet cells, indicating no influence on cell proliferation, but did inhibit DNA ladder formation, a hallmark of cell apoptosis. Moreover, 5 nM leptin reduced the triglyceride content and suppressed inducible nitric oxide synthase mRNA expression in incubated islets. These results suggest that leptin increased viable cell numbers via suppression of apoptosis in isolated pancreatic islet cells under these experimental conditions. This mechanism might account at least in part for an obesity-induced increase in pancreatic beta-cell mass.     

Diet-induced gene expression of isolated pancreatic islets from a polygenic mouse model of the metabolic syndrome

Aims/hypothesis  

Numerous new genes have recently been identified in genome-wide association studies for type 2 diabetes. Most are highly expressed in beta cells and presumably play important roles in their function. However, these genes account for only a small proportion of total risk and there are likely to be additional candidate genes not detected by current methodology. We therefore investigated islets from the polygenic New Zealand mouse (NZL) model of diet-induced beta cell dysfunction to identify novel genes and pathways that may play a role in the pathogenesis of diabetes.     

Autocrine insulin action activates Akt and increases survival of isolated human islets

Aims/hypothesis The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in promoting the survival of pancreatic beta cells. Akt becomes activated in isolated human islets following overnight culture despite significant levels of cell death. The aim of the current study was to identify the cause of the observed increase in Akt phosphorylation in isolated islets. We hypothesised that a factor secreted by the islets in culture was acting in an autocrine manner to activate Akt.Methods In order to identify the stimulus of the PI3K/Akt pathway in culture, we examined the effects of different culture conditions on Akt phosphorylation and islet survival during the immediate post-isolation period.Results We demonstrated that islet-conditioned medium induced Akt phosphorylation in freshly isolated human islets, whereas frequent medium replacement decreased Akt phosphorylation. Following overnight culture, islet-conditioned medium contained significantly elevated levels of insulin, indicating that insulin may be responsible for the observed increase in Akt phosphorylation. Indeed, treatment with an anti-insulin antibody or with inhibitors of insulin receptor/IGF receptor 1 kinase activity suppressed Akt phosphorylation, leading to decreased islet survival. In addition, dispersion of islets into single cells also suppressed Akt phosphorylation and induced islet cell death, indicating that islet integrity is also required for maximal Akt phosphorylation.Conclusions/interpretation Our findings demonstrate that insulin acts in an autocrine manner to activate Akt and mediate the survival of isolated human islets. These findings provide new information on how culturing islets prior to transplantation may be beneficial to their survival by allowing for autocrine activation of the pro-survival Akt pathway.     

Insulin release by isolated pancreatic islets of the mouse incubated in vitro

Summary The release of insulin during incubation of mouse islets of Langerhans, isolated after digestion of the pancreas with collagenase, has been studied, and the influence of various factors on the rate of release investigated. Glucose at 3.0 mg/ml (high glucose) stimulated insulin release, but had no effect at 0.6 mg/ml (low glucose). Mannoheptulose blocked the stimulation by high glucose, as did adrenaline. The adrenaline effect was abolished by phentolamine, an alpha-adrenergic blocking agent. Glucagon alone, stimulated insulin release, and also with low glucose, but not consistently with high glucose. Adrenaline abolished the stimulation by glucagon. Theophylline stimulated release with low glucose, much less so with high glucose and not at all with glucagon at either glucose concentration. Tolbutamide stimulated release with low glucose, and this effect was not inhibited by adrenaline. The suitability of this preparation for studies of islet cell metabolism and its relationship to secretion of insulin is discussed.

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Insulin-Freisetzung aus isolierten und in vitro inkubierten Pankreasinseln von Mäusen

Zusammenfassung Es wurde die Insulinausschüttung aus Langerhans'schen Inseln von Mäusen, die durch Kollagenase-Behandlung des Pankreas gewonnen worden waren, untersucht und der Einfluß verschiedener Faktoren auf die Freisetzungsgeschwindigkeit überprüft. Eine hohe Glucose-Konzentration von 3.0 mg/ml förderte die Insulin-Ausschüttung, während die niedrige Konzentration von 0.6 mg/ml keine Wirkung ergab. Mannoheptulose und Adrenalin blockierten die Stimulation durch die hohe Glucosekonzentration. Der AdrenalinEffekt konnte durch Phentolamin, eine alpha-Rezeptoren blockierende Substanz, wieder aufgehoben werden. Glucagon führte allein und in Gegenwart der niedrigen Glucosekonzentration zu einer verstärkten Insulininkretion: dies war jedoch bei Kombination mit der hohen Glucosekonzentration nicht konstant der Fall. Adrenalin hob die Stimulierung durch Glucagon auf. Theophyllin führte bei Gegenwart der niedrigen Glucosekonzentration zu einer gesteigerten Ausschüttung, dieser Effekt trat unter der höheren Glucosekonzentration in wesentlich geringerem Umfang und bei Zusatz von Glucagon und einer der beiden verwandten Glucosekonzentrationen überhaupt nicht auf. Bei niedriger Glucosekonzentration stimulierte Tolbutamid die Insulinfreisetzung; dieser Effekt ließ sich durch Adrenalin nicht aufheben. Die Aussagefähigkeit dieses Präparates für Untersuchungen des Inselzell-Stoffwechsels und seiner Beziehungen zur Insulininkretion wird diskutiert.

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Libération de l'insuline par les îlots pancréatiques isolés de la souris, incubés in vitro

Résumé La libération de l'insuline pendant l'incubation des îlots de Langerhans de la souris, isolés après digestion du pancréas par la collagénase, a été étudiée, et l'influence de divers facteurs sur la vitesse de libération a été recherchée. Le glucose à 3.0 mg/ml (glucose élevé) stimulait la libération d'insuline, mais n'avait pas d'effet à 0.6 mg/ml (taux bas). Le mannoheptulose bloquait la stimulation provoquée par du glucose élevé, comme le faisait l'adrénaline. L'effet de l'adrénaline était aboli par la phentolamine, un agent bloquant alpha adrénergique. Le glucagon seul, ainsi qu'en pre'sence d'un taux de glucose bas, stimulait la libération d'insuline, mais non de façon constante avec un taux de glucose élevé. L'adrénaline abolissait la stimulation provoquée par le glucagon. La théophylline stimulait la libération lorsque le glucose était bas, mais beaucoup moins lorsque le glucose était élevé et pas du tout avec le glucagon, quelle que soit la concentration de glucose. Le tolbutamide stimulait la libération d'insuline avec une faible concentration de glucose et cet effet n'était pas inhibé par l'adrénaline. La convenance de cette préparation pour des études sur le métabolisme des cellules insulaires et sa relation avec la sécrét ion d'insuline est discutée.

     

Ocimum sanctum leaf extracts stimulate insulin secretion from perfused pancreas, isolated islets and clonal pancreatic beta-cells

Ocimum sanctum leaves have previously been reported to reduce blood glucose when administered to rats and humans with diabetes. In the present study, the effects of ethanol extract and five partition fractions of O. sanctum leaves were studied on insulin secretion together with an evaluation of their mechanisms of action. The ethanol extract and each of the aqueous, butanol and ethylacetate fractions stimulated insulin secretion from perfused rat pancreas, isolated rat islets and a clonal rat beta-cell line in a concentration-dependent manner. The stimulatory effects of ethanol extract and each of these partition fractions were potentiated by glucose, isobutylmethylxanthine, tolbutamide and a depolarizing concentration of KCl. Inhibition of the secretory effect was observed with diazoxide, verapamil and Ca2+ removal. In contrast, the stimulatory effects of the chloroform and hexane partition fractions were associated with decreased cell viability and were unaltered by diazoxide and verapamil. The ethanol extract and the five fractions increased intracellular Ca2+ in clonal BRIN-BD11 cells, being partly attenuated by the addition of verapamil. These findings indicated that constituents of O. sanctum leaf extracts have stimulatory effects on physiological pathways of insulin secretion which may underlie its reported antidiabetic action.     

Rebaudioside A potently stimulates insulin secretion from isolated mouse islets: studies on the dose-, glucose-, and calcium-dependency

Extracts of leaves of the plant Stevia rebaudiana Bertoni (SrB), have been used for many years in traditional treatment of diabetes in South America. Stevia leaves contain diterpene glycosides, stevioside and rebaudioside A being the most abundant. Recently, it was demonstrated that stevioside stimulates the insulin secretion both in vitro and in vivo. Subsequently, we wanted to elucidate the influence of rebaudioside A on the insulin release from mouse islets using static incubations, as well as perifusion experiments. Rebaudioside A (10(-16) to 10(-6) mol/L) dose-dependently stimulated the insulin secretion in the presence of 16.7 mmol/L glucose (P < .05). The stimulation of insulin release occurs at a concentration of 10(-14) mol/L rebaudioside A, and maximal insulin response was obtained at 10(-10) mol/L (P < .01). Rebaudioside A stimulates insulin secretion in a glucose-dependent manner (3.3 to 16.7 mmol/L) and only potentiated insulin secretion at glucose > 6.6 mmol/L. The effect of rebaudioside A is critically dependent on the presence of extracellular Ca2+, ie, rebaudioside A-induced insulin stimulation at high glucose disappears in the absence of extracellular Ca2+. In conclusion, rebaudioside A possesses insulinotropic effects and may serve a potential role as treatment in type 2 diabetes mellitus.     

A method for enzymatic dissociation of cells from isolated pancreatic islets

Summary An enzymatic method for isolation of single cells from the islets of Langerhans is described. The isolated cells appeared well preserved and survived for at least 7 days when maintained in culture. The dry mass of the isolated islet cells was found to be decreased 30 min after administration of alloxan to obese-hyperglycemic mice. Isolated individual islet cells from obese-hyperglycemic mice had a higher dry mass than those from their lean litter mates. Traduzione a cura di G. U.     

Age-related decline in mitochondrial DNA copy number in isolated human pancreatic islets

AIM/HYPOTHESIS: Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity. METHODS: We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods. RESULTS: There was a significant negative correlation between mtDNA copy number and islet donor age (r = -0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged > or =50 years (n = 8) compared with those aged <50 years (n = 7) (median [interquartile range]: 418 [236-503] vs 596 [554-729] mtDNA copy number/diploid genome; p = 0.032). None of the islet preparations harboured high levels of deleted mtDNA affecting the major arc. CONCLUSION/INTERPRETATION: Given the correlation between mtDNA content and respiratory chain activity, the age-related decrease in mtDNA copy number that we observed in human pancreatic islet preparations may contribute to the age-dependent decline in pancreatic beta cell insulin secretory capacity.     

Dissociation by methylamine of insulin release from glucose-induced electrical activity in isolated mouse islets of Langerhans

The effect of methylamine on electrical activity and simultaneously measured insulin release was investigated in single perifused islets of normal mice. Methylamine, (2 mmol/L or 6 mmol/L) failed to affect beta-cell input resistance and only caused a modest and transient inhibition of electrical activity of islets exposed to 11.1 mmol/L glucose. Methylamine (2 mmol/L) inhibited insulin release evoked by a five-minute rise in glucose concentration from 5.6 to 22.2 mmol/L, even when the glucose-induced electrical activity remained unaltered. Methylamine, at 2 or 5 mmol/L, partially inhibited insulin release but failed to affect the continuous electrical activity in islets exposed throughout to 22.2 mmol/L glucose. At 10 mmol/L, methylamine reduced both insulin release and electrical activity. These data reinforce the idea that the glucose-induced changes in beta-cell membrane potential represent an early event in the process of stimulus-secretion coupling and can be dissociated from the subsequent process of insulin release.     

Exposure of mouse embryonic pancreas to metformin enhances the number of pancreatic progenitors

Aims/hypothesis

Developing beta cells are vulnerable to nutrient environmental signals. Early developmental processes that alter the number of pancreatic progenitors can determine the number of beta cells present at birth. Metformin, the most widely used oral agent for treating diabetes, alters intracellular energy status in part by increasing AMP-activated protein kinase (AMPK) signalling. This study examined the effect of metformin on developing pancreas and beta cells.

Methods

Pancreatic rudiments from CD-1 mice at embryonic day 13.0 (E13.0) were cultured with metformin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, an AMPK activator) or vehicle control in vitro. In another set of studies, pregnant C57BL/6 mice were treated with metformin throughout gestation. Embryonic (E14.0) and neonatal pancreases were then analysed for their morphometry.

Results

In vitro metformin treatment led to an increase in the proliferation and number of pancreatic duodenal homeobox 1-positive (PDX1⁺) progenitors. These results were reproduced by in vitro culture of embryonic pancreas rudiments with AICAR, suggesting that AMPK activation was involved. Similarly, metformin administration to pregnant dams induced an increase in both PDX1⁺ and neurogenin 3-positive progenitors in the embryonic pancreas at E14.0 and these changes resulted in an increased beta cell fraction in neonates.

Conclusions/interpretation

These results indicate that exposure to metformin during gestation modulates the early steps of beta cell development (prior to E14.0) towards an increase in the number of pancreatic and endocrine progenitors. These changes ultimately result in a higher beta cell fraction at birth. These findings are of clinical importance given that metformin is currently used for the treatment of gestational diabetes.     

Comparative effects of amino acids and glucose on insulin secretion from isolated rat or mouse islets

Glucose and the combination of leucine and glutamine were used to stimulate insulin secretion from rat islets during a dynamic perifusion and the responses obtained were compared with those elicited from mouse islets under identical conditions. In rat islets, glucose (15 mM) or the amino acid combination of 10 mM glutamine plus 20 mM leucine were most efficacious and peak second-phase insulin release responses were 20- to 30-fold above prestimulatory rates. In contrast to rat islet responses, sustained second-phase insulin secretory responses to the same agonists were minimally increased 1- to 2-fold from mouse islets. Parallel studies demonstrated that phospholipase C (PLC) was markedly activated in rat, but not mouse, islets by both high glucose concentrations and the amino acid combination. Additional studies documented that glucose and amino acid responses of both rat and mouse islets were amplified by carbachol or forskolin. However, wortmannin, a phosphatidylinositol 3-kinase inhibitor, amplified only the responses to glucose leaving the responses to the amino acid mixture unaltered. These observations support the concept that mitochondrial metabolism alone is minimally effective in stimulating insulin secretion from islets. The activation of the supplementary second messenger systems (PLC and/or cAMP) appears essential for the emergence of their full secretory potential. The mechanism regulating the potency and specificity of wortmannin's impact on glucose-induced secretion remains to be identified; however a unique mechanism is supported by these findings.     

Somatostatin-induced inhibition of insulin secretion by isolated pancreatic rat islets prepared by micro-dissection or collagenase digestion.

The effect of somatostatin on insulin secretion in response to a variety of stimuli was investigated in micro-dissected as well as collagenase isolated pancreatic rat islets. Somatostatin inhibited insulin secretion in both islet preparations in the presence of different initiators, but failed to affect the hormone output induced by theophylline, 1-methyl-3-isobutylxanthin (IBMX) or p-chloromercuriphenylsulfonic acid (CMBS). The inhibitory action was associated with decreased glucose metabolism by islets (measured as conversion of U-14C-glucose to 14CO2).     

Evaluation of collagenase gold plus BP protease in isolating islets from human pancreata

Selection of enzymes for optimal pancreas digestion is essential for successful human islet isolations. The aim of this study was to evaluate the efficacy and outcome of using Collagenase Gold plus BP protease (VitaCyte) (n = 8) by comparing it to two commercially available enzymes, Liberase MTF C/T (Roche) (n = 48) and Collagenase NB1/NP (Serva) (n = 15). The isolation outcomes were assessed by islet counting, viability, glucose-stimulated oxygen consumption rate (OCR), and successful graft-rate following transplantation in diabetic NOD scid mice. The pancreas donor characteristics were not significantly different between the tested enzyme groups regarding their BMI, pancreas weight, cold ischemia time (CIT) and HbA1c. The results show that digested tissue volume was not statistically significant between the VitaCyte enzyme (34.25 ± 5.4 mL) and the Roche enzyme (55.25 ± 3.42 mL, p = 0.073), however, this was significant with Serva enzyme (64.07 ± 7.95 mL, p = 0.020). Interestingly, the islet yields were not statistically different between all enzyme groups. Moreover, when islets were transplanted into NOD scid mice, the reversal rate of diabetes for the VitaCyte enzyme group was similar to all enzyme groups. In conclusion, the effectiveness of Collagenase Gold plus BP protease is comparable to the MTF C/T and the Collagenase NB1/NP enzymes; the low cost could facilitate the use of more pancreata for islet isolations.     

Spectrum effects of a new polypeptide (third hormone?) isolated from the chicken pancreas

A broad evaluation was made of the biological effects of a new polypeptide (APP), 36 amino acid residues, MW 4200, isolated from the chicken pancreas. APP is an effective hepatic glycogenolytic and plasma hypoglycerolemic agent in the absence of plasma glucose perturbation. APP (at much lower doses) is also a powerful gastric secretogogue, inducing within seconds marked increases in proventricular volume, H⁺, pepsin, and total protein release. Such proventricular response is not mediated through systemic cardiovascular alterations or the vagus nerves, and are quite opposite to those observed with beef or chicken glucagon. It is concluded that the chicken pancreas releases APP to exert a “gastrinlike” secretogogic action on the proventriculus and, at higher concentrations, a metabolic action as well.