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Hormones and neurotransmitters are released from (neuro)endocrine cells by regulated exocytosis of secretory granules. During exocytosis, the granule membrane fuses with the plasma membrane, which allows release of the stored content into the bloodstream or the surrounding tissue. Here, we give a detailed description of two complementary methods to observe and quantify exocytosis in single cells: high-resolution TIRF microscopy and patch-clamp capacitance recordings. Precise stimulation of exocytosis is achieved by local pressure application or voltage-clamp depolarizations. While the chapter is focused on insulin-secreting cells as an accessible and disease-relevant model system, the methodology is applicable to a wide variety of secretory cells including chromaffin and PC12 cells.

Pancreatic islet transplantation has not yet succeeded as an overall treatment for type 1 diabetes because of limited access to donor islets, as well as low efficacy and poor reproducibility of the current procedure. Herein, a method to create islets-like composite clusters (coclusters) from dispersed endocrine cells and supportive cells is described, attempting to improve compatibility with the recipient and more efficiently make use of the donor-derived material. To mimic the extracellular matrix environment, recombinant,spider silk functionalized with cell binding motifs are used as 3D support for the coclusters. A cell binding motif derived from fibronectin (FN) was found superior in promoting cell adherence, while a plain RGD-motif incorporated in the repetitive part of the silk protein (2R) increased the mobility and cluster formation of endocrine cells. Self-assembly of a mixture of FN/2R silk is utilized to integrate endocrine cells together with endothelial and mesenchymal cells into islet-like coclusters. Both xenogenic and allogenic versions of these coclusters were found to be viable and were able to respond to dynamic glucose stimulation with insulin release. Moreover, the endothelial cells were found to be colocalized with the endocrine cells, showing that the silk combined with supportive cells may promote vascularization. This method to engineer combined islet-like coclusters allows donor-derived endocrine cells to be surrounded by supportive cells from the recipient, which have the potential to further promote engraftment in the host and considerably reduce risk of rejection.

Pancreatic islet transplantation has not yet succeeded as an overall treatment for type 1 diabetes because of limited access to donor islets, as well as low efficacy and poor reproducibility of the current procedure. Herein, a method to create islets-like composite clusters (coclusters) from dispersed endocrine cells and supportive cells is described, attempting to improve compatibility with the recipient and more efficiently make use of the donor-derived material. To mimic the extracellular matrix environment, recombinant,spider silk functionalized with cell binding motifs are used as 3D support for the coclusters. A cell binding motif derived from fibronectin (FN) was found superior in promoting cell adherence, while a plain RGD-motif incorporated in the repetitive part of the silk protein (2R) increased the mobility and cluster formation of endocrine cells. Self-assembly of a mixture of FN/2R silk is utilized to integrate endocrine cells together with endothelial and mesenchymal cells into islet-like coclusters. Both xenogenic and allogenic versions of these coclusters were found to be viable and were able to respond to dynamic glucose stimulation with insulin release. Moreover, the endothelial cells were found to be colocalized with the endocrine cells, showing that the silk combined with supportive cells may promote vascularization. This method to engineer combined islet-like coclusters allows donor-derived endocrine cells to be surrounded by supportive cells from the recipient, which have the potential to further promote engraftment in the host and considerably reduce risk of rejection., QC 20200325

Pancreatic islet transplantation has not yet succeeded as an overall treatment for type 1 diabetes because of limited access to donor islets, as well as low efficacy and poor reproducibility of the current procedure. Herein, a method to create islets-like composite clusters (coclusters) from dispersed endocrine cells and supportive cells is described, attempting to improve compatibility with the recipient and more efficiently make use of the donor-derived material. To mimic the extracellular matrix environment, recombinant,spider silk functionalized with cell binding motifs are used as 3D support for the coclusters. A cell binding motif derived from fibronectin (FN) was found superior in promoting cell adherence, while a plain RGD-motif incorporated in the repetitive part of the silk protein (2R) increased the mobility and cluster formation of endocrine cells. Self-assembly of a mixture of FN/2R silk is utilized to integrate endocrine cells together with endothelial and mesenchymal cells into islet-like coclusters. Both xenogenic and allogenic versions of these coclusters were found to be viable and were able to respond to dynamic glucose stimulation with insulin release. Moreover, the endothelial cells were found to be colocalized with the endocrine cells, showing that the silk combined with supportive cells may promote vascularization. This method to engineer combined islet-like coclusters allows donor-derived endocrine cells to be surrounded by supportive cells from the recipient, which have the potential to further promote engraftment in the host and considerably reduce risk of rejection.

The naturally occurring quassinoid compound brusatol improves the survival of insulin-producing cells when exposed to the proinflammatory cytokines IL-1b and IFN-g in vitro. The aim of the present study was to investigatewhetherbrusatol also promotes beneficial effects inmice fed a high-fat diet (HFD), and if so, to study the mechanisms by which brusatol acts. In vivo, we observed that the impaired glucose tolerance of HFD-fed male C57BL/ 6micewas counteracted by a 2wk treatmentwith brusatol. Brusatol treatment improvedbothb-cell function and peripheral insulin sensitivity of HFD-fed mice. In vitro, brusatol inhibited b-cell total protein and proinsulin biosynthesis, withanED50 of 40nM. In linewith this, brusatol blocked cytokine-inducediNOSprotein expression via inhibition of iNOS mRNA translation. Brusatol may have affected protein synthesis, at least in part, via inhibition of eukaryotic initiation factor 5A (eIF5A) hypusination, as eIF5A spermidine association and hypusinationin RIN-5AHcellswas reducedinadose-andtime-dependentmanner. The eIF5AhypusinationinhibitorGC7 promoted a similar effect. Both brusatol and GC7 protected rat RIN-5AH cells against cytokine-induced cell death. Brusatol reduced eIF5A hypusination and cytokine-induced cell death in EndoC-bH1 cells as well. Finally, hypusinated eIF5A was reduced in vivo by brusatol in islet endocrine and endothelial islet cells of mice fed anHFD. The results of the present study suggest that brusatol improves glucose intolerance in mice fed an HFD, possibly by inhibiting protein biosynthesis and eIF5A hypusination.-Turpaev, K., Krizhanovskii, C., Wang, X., Sargsyan, E., Bergsten, P., Welsh, N. The protein synthesis inhibitor brusatol normalizes high-fat diet-induced glucose intolerance in male C57BL/ 6 mice: role of translation factor eIF5A hypusination. FASEB J. 33, 3510-3522 (2019). www.fasebj.org

Emerging evidence suggests a role for the gut microbiota in glucose metabolism and diabetes. Few studies have examined the associations between the faecal microbiome and insulin sensitivity and secretion using gold-standard methods in high-risk populations prior to diabetes onset. We investigated the relationships between faecal microbiota composition (16S rRNA sequencing) and gold-standard measures of insulin sensitivity (hyperinsulinaemic-euglycaemic clamp) and insulin secretion (intravenous glucose tolerance test) in 38 overweight or obese otherwise healthy individuals. Genus Clostridium was positively associated with insulin sensitivity, and genera Dialister and Phascolarctobacterium were related to both insulin sensitivity and secretion. Insulin sensitivity was associated with a higher abundance of Phascolarctobacterium and lower abundance of Dialister. Those with higher insulin secretion had a higher abundance of Dialister and lower abundance of Bifidobacterium, compared to those with lower insulin secretion. Body mass index (BMI) was positively correlated with Streptococcus abundance whereas Coprococcus abundance was negatively correlated to BMI and percent body fat. These results suggest that faecal microbiota is related to insulin sensitivity and secretion in overweight or obese adults. These correlations are distinct although partially overlapping, suggesting different pathophysiological pathways. Our findings can inform future trials aiming to manipulate gut microbiome to improve insulin sensitivity and secretion and prevent type 2 diabetes.Copyright © 2019 by the authors. Licensee MDPI, Basel, Switzerland.

Emerging evidence suggests a role for the gut microbiota in glucose metabolism and diabetes. Few studies have examined the associations between the faecal microbiome and insulin sensitivity and secretion using gold-standard methods in high-risk populations prior to diabetes onset. We investigated the relationships between faecal microbiota composition (16S rRNA sequencing) and gold-standard measures of insulin sensitivity (hyperinsulinaemic-euglycaemic clamp) and insulin secretion (intravenous glucose tolerance test) in 38 overweight or obese otherwise healthy individuals. Genus Clostridium was positively associated with insulin sensitivity, and genera Dialister and Phascolarctobacterium were related to both insulin sensitivity and secretion. Insulin sensitivity was associated with a higher abundance of Phascolarctobacterium and lower abundance of Dialister. Those with higher insulin secretion had a higher abundance of Dialister and lower abundance of Bifidobacterium, compared to those with lower insulin secretion. Body mass index (BMI) was positively correlated with Streptococcus abundance whereas Coprococcus abundance was negatively correlated to BMI and percent body fat. These results suggest that faecal microbiota is related to insulin sensitivity and secretion in overweight or obese adults. These correlations are distinct although partially overlapping, suggesting different pathophysiological pathways. Our findings can inform future trials aiming to manipulate gut microbiome to improve insulin sensitivity and secretion and prevent type 2 diabetes.Copyright © 2019 by the authors. Licensee MDPI, Basel, Switzerland.

Background: Secretory recombinant protein production with Pichia (syn. Komagataella) pastoris is commonly associated with the induction of an unfolded protein response (UPR) usually apparent through increased intracellular levels of endoplasmic reticulum (ER) resident chaperones such as Kar2/Bip. During methanol-induced secretory production of an insulin precursor (IP) under industrially relevant fed-batch conditions the initially high level of intracellular Kar2/Bip after batch growth on glycerol unexpectedly declined in the following methanol fed-batch phase misleadingly suggesting that IP production had a low impact on UPR activation. Results: Analysis of the protein production independent level of Kar2/Bip revealed that high Kar2/Bip levels were reached in the exponential growth phase of glycerol batch cultures followed by a strong decline of Kar2/Bip during entry into stationary phase. Ultra-structural cell morphology studies revealed autophagic processes (e.g. ER phagy) at the end of the glycerol batch phase most likely responsible for the degradation of ER resident chaperones such as Kar2/Bip. The pre-induction level of Kar2/Bip did not affect the IP secretion efficiency in the subsequent methanol-induced IP production phase. During growth on methanol intracellular Kar2/Bip levels declined in IP producing and non-producing host cells. However, extracellular accumulation of Kar2/Bip was observed in IP-producing cultures but not in non-producing controls. Most importantly, the majority of the extracellular Kar2/Bip accumulated in the culture supernatant of IP producing cells as truncated protein (approx. 35 kDa). Conclusions: Rapid growth leads to higher basal levels of the major UPR marker protein Kar2/Bip independent of recombinant protein production. Entry into stationary phase or slower growth on poorer substrate, e.g. methanol, leads to a lower basal Kar2/Bip level. Methanol-induced secretory IP production elicits a strong UPR activation which counteracts

Background: Vitamin D supplementation has been proposed as a potential strategy to prevent type 2 diabetes. Existing clinical trials have been limited by short duration, low doses of Vitamin D, variability in participants' Vitamin D-deficiency status, and the use of surrogate measures of body composition, insulin sensitivity, and insulin secretion. Objective(s): To address existing knowledge gaps, we conducted a double-blind, randomized, placebo-controlled trial to investigate whether Vitamin D supplementation that is provided in a sufficient dose and duration to Vitamin D-deficient individuals would improve insulin sensitivity or secretion as measured with the use of goldstandard methods. We hypothesized that Vitamin D supplementation would improve insulin sensitivity and secretion compared with placebo. Design(s): Sixty-five overweight or obese, Vitamin D-deficient (25-hydroxyVitamin D [25(OH)D] concentration <=50 nmol/L) adults were randomly assigned to receive either a bolus oral dose of 100,000 IU cholecalciferol followed by 4000 IU cholecalciferol/d or a matching placebo for 16 wk. Before and after the intervention, participants received gold-standard assessments of body composition (via dual X-ray absorptiometry), insulin sensitivity (via hyperinsulinemic-euglycemic clamps), and insulin secretion [via intravenous-glucose-tolerance tests (IVGTTs)]. Result(s): Fifty-four participants completed the study [35 men and 19 women; mean +/- SD age: 31.9 +/- 8.5 y; body mass index (in kg/m2): 30.9 +/- 4.4]. 25(OH)D increased with Vitamin D supplementation compared with placebo (57.0 +/- 21.3 compared with 1.9 +/- 15.1 nmol/L, respectively; P = 0.02). Vitamin D and placebo groups did not differ in change in insulin sensitivity (0.02 +/- 2.0 compared with 20.03 +/- 2.8 mg * kg-1 * min-1, respectively; P = 0.9) or firstphase insulin secretion (-21 +/- 212 compared with 24 +/- 184 mU/L, respectively; P = 0.9). Results remained nonsignificant after adjustment for age, sex

Introduction There is conflicting evidence regarding the efficacy of high dose salicylates in improving cardiometabolic risk in healthy and type 2 diabetes patients. We aimed to determine whether treatment with salicylates at an anti-inflammatory dose (>= 1 g daily) would improve cardiometabolic risk in healthy individuals and type 2 diabetes patients, compared to placebo. Methods Medline, Medline-in-process, Embase, and all EBM databases were searched for studies published up to December 2016. Twenty-eight articles from 24 studies comprising 1591 participants were included. Two reviewers independently assessed the risk of bias and extracted data from included studies. Meta-analyses using random-effects model were used to analyze the data. Results High dose salicylates (>= 3 g/d) decreased fasting glucose (MD -0.4 mmol/l, 95% CI - 0.54, - 0.27) and glucose area under the curve (MD -0.41 mmol/l, 95% CI - 0.81, - 0.01). Salicylates (>= 3 g/d) also increased fasting insulin (MD 2.4 muU/ml, 95% CI 0.3, 4.4), 2-h insulin (MD 25.4 muU/ml, 95% CI 8.2, 42.6), insulin secretion (MD 79.2, 95% CI 35, 123) but decreased fasting C-peptide (MD -0.11 nmol/l, 95% CI - 0.2, - 0.04), insulin clearance (MD -0.26 l/min, 95% CI - 0.36, - 0.16) and triglycerides (MD -0.36 mmol/l, 95% CI - 0.51, - 0.21) and increased total adiponectin (MD 1.97 mug/ml, 95% CI 0.99, 2.95). A lower salicylate dose (1-2.9 g) did not change any cardiometabolic parameters (p > 0.1). No significant difference was observed between those receiving salicylates and placebo following withdrawal due to adverse events. Conclusions High dose salicylates appear to improve cardiometabolic risk factors in healthy individuals and type 2 diabetes patients. PROSPERO registration number CRD42015029826.Copyright © 2017 Elsevier Inc.

Background: Vitamin D supplementation has been proposed as a potential strategy to prevent type 2 diabetes. Existing clinical trials have been limited by short duration, low doses of Vitamin D, variability in participants' Vitamin D-deficiency status, and the use of surrogate measures of body composition, insulin sensitivity, and insulin secretion. Objective(s): To address existing knowledge gaps, we conducted a double-blind, randomized, placebo-controlled trial to investigate whether Vitamin D supplementation that is provided in a sufficient dose and duration to Vitamin D-deficient individuals would improve insulin sensitivity or secretion as measured with the use of goldstandard methods. We hypothesized that Vitamin D supplementation would improve insulin sensitivity and secretion compared with placebo. Design(s): Sixty-five overweight or obese, Vitamin D-deficient (25-hydroxyVitamin D [25(OH)D] concentration <=50 nmol/L) adults were randomly assigned to receive either a bolus oral dose of 100,000 IU cholecalciferol followed by 4000 IU cholecalciferol/d or a matching placebo for 16 wk. Before and after the intervention, participants received gold-standard assessments of body composition (via dual X-ray absorptiometry), insulin sensitivity (via hyperinsulinemic-euglycemic clamps), and insulin secretion [via intravenous-glucose-tolerance tests (IVGTTs)]. Result(s): Fifty-four participants completed the study [35 men and 19 women; mean +/- SD age: 31.9 +/- 8.5 y; body mass index (in kg/m2): 30.9 +/- 4.4]. 25(OH)D increased with Vitamin D supplementation compared with placebo (57.0 +/- 21.3 compared with 1.9 +/- 15.1 nmol/L, respectively; P = 0.02). Vitamin D and placebo groups did not differ in change in insulin sensitivity (0.02 +/- 2.0 compared with 20.03 +/- 2.8 mg * kg-1 * min-1, respectively; P = 0.9) or firstphase insulin secretion (-21 +/- 212 compared with 24 +/- 184 mU/L, respectively; P = 0.9). Results remained nonsignificant after adjustment for age, sex

Introduction There is conflicting evidence regarding the efficacy of high dose salicylates in improving cardiometabolic risk in healthy and type 2 diabetes patients. We aimed to determine whether treatment with salicylates at an anti-inflammatory dose (>= 1 g daily) would improve cardiometabolic risk in healthy individuals and type 2 diabetes patients, compared to placebo. Methods Medline, Medline-in-process, Embase, and all EBM databases were searched for studies published up to December 2016. Twenty-eight articles from 24 studies comprising 1591 participants were included. Two reviewers independently assessed the risk of bias and extracted data from included studies. Meta-analyses using random-effects model were used to analyze the data. Results High dose salicylates (>= 3 g/d) decreased fasting glucose (MD -0.4 mmol/l, 95% CI - 0.54, - 0.27) and glucose area under the curve (MD -0.41 mmol/l, 95% CI - 0.81, - 0.01). Salicylates (>= 3 g/d) also increased fasting insulin (MD 2.4 muU/ml, 95% CI 0.3, 4.4), 2-h insulin (MD 25.4 muU/ml, 95% CI 8.2, 42.6), insulin secretion (MD 79.2, 95% CI 35, 123) but decreased fasting C-peptide (MD -0.11 nmol/l, 95% CI - 0.2, - 0.04), insulin clearance (MD -0.26 l/min, 95% CI - 0.36, - 0.16) and triglycerides (MD -0.36 mmol/l, 95% CI - 0.51, - 0.21) and increased total adiponectin (MD 1.97 mug/ml, 95% CI 0.99, 2.95). A lower salicylate dose (1-2.9 g) did not change any cardiometabolic parameters (p > 0.1). No significant difference was observed between those receiving salicylates and placebo following withdrawal due to adverse events. Conclusions High dose salicylates appear to improve cardiometabolic risk factors in healthy individuals and type 2 diabetes patients. PROSPERO registration number CRD42015029826.Copyright © 2017 Elsevier Inc.

Type 2 diabetes occurs when an insufficient amount of insulin is released in the bloodstream, thus failing to maintain normal glycaemia. Pancreatic beta-cells are the main cell type residing in the pancreatic islets and are responsible for the release of insulin. beta-cells are excitable and express several voltage-gated ion channels and among them, voltage-gated Ca2+ channels are critical for the release of insulin. In fact, once a pancreatic beta-cell is depolarized, high voltage-gated Ca2+ channels activate, leading to Ca2+ influx and raise in cytosolic Ca2+ concentrations. The subsequent Ca2+ increase triggers the exocytic machinery and thus the release of insulin. Voltage-gated Ca2+ channels are composed by the pore-forming alpha1 subunit and by auxiliary subunits beta and alpha2delta. alpha2delta subunits regulate the membrane targeting of the alpha1 subunit and its current kinetics and they have shown their importance in a variety of neuronal and non-neuronal cell types. However, the importance of alpha2delta subunits for the release of insulin has never been previously reported. In this thesis, we investigated the role of the alpha2delta-1 subunit in pancreatic beta-cells. We have found that alpha2delta-1 is the main alpha2delta isoform expressed in pancreatic islets. Moreover, mice lacking the alpha2delta-1 subunits develop diabetes in a gender-dependent fashion. alpha2delta-1-/- male mice show a severe glucose intolerance, while alpha2delta-1-/- female mice are pre-diabetic, with a milder glucose intolerance. Pancreatic beta-cells from both alpha2delta-1-/- male and female mice have reduced Ca2+ influx. Nonetheless, only pancreatic islets from male alpha2delta-1-/- mice show reduced and delayed Ca2+ transients, while islets from female alpha2delta-1-/- mice have unaffected Ca2+ increase, however still delayed. Moreover, islets from alpha2delta-1+/+ female mice have reduced Ca2+ increase, compared to islets from alpha2delta-1+/+ male mice. We have shown tha, Type 2 diabetes occurs when an insufficient amount of insulin is released in the bloodstream, thus failing to maintain normal glycaemia. Pancreatic beta-cells are the main cell type residing in the pancreatic islets and are responsible for the release of insulin. beta-cells are excitable and express several voltage-gated ion channels and among them, voltage-gated Ca2+ channels are critical for the release of insulin. In fact, once a pancreatic beta-cell is depolarized, high voltage-gated Ca2+ channels activate, leading to Ca2+ influx and raise in cytosolic Ca2+ concentrations. The subsequent Ca2+ increase triggers the exocytic machinery and thus the release of insulin. Voltage-gated Ca2+ channels are composed by the pore-forming alpha1 subunit and by auxiliary subunits beta and alpha2delta. alpha2delta subunits regulate the membrane targeting of the alpha1 subunit and its current kinetics and they have shown their importance in a variety of neuronal and non-neuronal cell types. However, the importance of alpha2delta subunits for the release of insulin has never been previously reported. In this thesis, we investigated the role of the alpha2delta-1 subunit in pancreatic beta-cells. We have found that alpha2delta-1 is the main alpha2delta isoform expressed in pancreatic islets. Moreover, mice lacking the alpha2delta-1 subunits develop diabetes in a gender-dependent fashion. alpha2delta-1-/- male mice show a severe glucose intolerance, while alpha2delta-1-/- female mice are pre-diabetic, with a milder glucose intolerance. Pancreatic beta-cells from both alpha2delta-1-/- male and female mice have reduced Ca2+ influx. Nonetheless, only pancreatic islets from male alpha2delta-1-/- mice show reduced and delayed Ca2+ transients, while islets from female alpha2delta-1-/- mice have unaffected Ca2+ increase, however, Mag. Vincenzo Mastrolia, Im Titel 2+ hochgestellt und 2 tiefgestellt, Medical University of Innsbruck, Dissertation, 2017, (VLID)1744707

La cellule ß pancréatique sécrète l’insuline, la seule hormone capable de diminuer la concentration en glucose plasmatique. L'insuline est sécrétée de manière régulée sous l’influence de divers stimuli dont avant tout l’élévation de la glycémie, celle-ci augmentant lors de chaque repas. Elle est donc essentielle à la préservation de l'homéostasie du glucose. Son importance est attestée par le développement du diabète sucré et de ses complications sévères et parfois mortelles lorsque sa sécrétion est supprimée ou insuffisante et/ou lorsqu'il existe un défaut de son action.La cellule ß est une cellule excitable, c'est à dire capable de moduler son potentiel membranaire très rapidement en variant certaines conductances ioniques (Dean & Matthews 1968; 1970a; 1970b). En réponse à une élévation du glucose extracellulaire, la cellule ß se dépolarise et génère des potentiels d’action qui aboutissent à une augmentation du calcium cytoplasmique induisant la libération d’insuline par exocytose. La succession des différentes étapes menant à cette sécrétion régulée d'insuline est complexe, souvent désignée sous le vocable de couplage stimulus-sécrétion. Elle commence par une augmentation de l’entrée du glucose et de son métabolisme dans la cellule ß. L’augmentation de la concentration en ATP (ou du rapport de concentrations ATP/ADP) qui en résulte, entraîne la fermeture des canaux potassiques ATP-sensibles (KATP) avec une dépolarisation partielle de la membrane. Toutefois la fermeture de ces canaux ne semble pas suffisante pour expliquer la dépolarisation membranaire observée sous l’effet du glucose et une dépolarisation supplémentaire est nécessaire pour arriver au seuil d’activation des canaux calciques voltage-dépendants ou Cav (Gilon & Rorsman, 2009). Un efflux de Cl- de la cellule ß est déclenché lors d’une stimulation au glucose (Sehlin 1978; Sehlin 1987), proportionnellement à la concentration de glucose entre 5 et 20 mM (Malaisse et coll. 2004). Une diminution de la conc, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

Ex vivo expansion of endocrine cells constitutes an interesting alternative to be able to match the unmet need of transplantable pancreatic islets. However, endocrine cells become fragile once removed from their extracellular matrix (ECM) and typically become senescent and loose insulin expression during conventional 2D culture. Herein we develop a protocol where 3D silk matrices functionalized with ECM derived motifs are used for generation of insulin-secreting islet-like clusters from mouse and human primary cells. The obtained clusters were shown to attain an islet-like spheroid shape and to maintain functional insulin release upon glucose stimulation in vitro. Furthermore, in vivo imaging of transplanted murine clusters showed engraftment with increasing vessel formation during time. There was no sign of cell death and the clusters maintained or increased in size throughout the period, thus suggesting a suitable cluster size for transplantation., QC 20160520

Objective Carnosine is a naturally present dipeptide in humans and an over-the counter food additive. Evidence from animal studies supports the role for carnosine in the prevention and treatment of diabetes and cardiovascular disease, yet there is limited human data. This study investigated whether carnosine supplementation in individuals with overweight or obesity improves diabetes and cardiovascular risk factors. Methods In a double-blind randomized pilot trial in nondiabetic individuals with overweight and obesity (age 43 +/- 8 years; body mass index 31 +/- 4 kg/m2), 15 individuals were randomly assigned to 2 g carnosine daily and 15 individuals to placebo for 12 weeks. Insulin sensitivity and secretion, glucose tolerance (oral glucose tolerance test), blood pressure, plasma lipid profile, skeletal muscle (1H-MRS), and urinary carnosine levels were measured. Results Carnosine concentrations increased in urine after supplementation (P < 0.05). An increase in fasting insulin and insulin resistance was hampered in individuals receiving carnosine compared to placebo, and this remained significant after adjustment for age, sex, and change in body weight (P = 0.02, P = 0.04, respectively). Two-hour glucose and insulin were both lower after carnosine supplementation compared to placebo in individuals with impaired glucose tolerance (P < 0.05). Conclusions These pilot intervention data suggest that carnosine supplementation may be an effective strategy for prevention of type 2 diabetes.Copyright © 2016 The Obesity Society.

In this study, insulin-containing nanoparticles were loaded into pellet cores and orally administered to diabetic rats. Polyethylene imine-based nanoparticles, either placebo or loaded with insulin, were incorporated by extrusion and spheronization technology into cores that were subsequently coated with three overlapping layers and a gastroresistant film. The starting and coated systems were evaluated in vitro for their physico-technololgical characteristics, as well as disintegration and release performance. Nanoparticles-loaded cores showed homogeneous particle size distribution and shape. When a superdisintegrant and a soluble diluent were included in the composition enhanced disintegration and release performance were observed. The selected formulations, coated either with enteric or three-layer films, showed gastroresistant and release delayed behavior in vitro, respectively. The most promising formulations were finally tested for their hypoglycemic effect in diabetic rats. Only the nanoformulations loaded into the three-layer pellets were able to induce a significant hypoglycemic activity in diabetic rats. Our results suggest that this efficient activity could be attributed to a retarded release of insulin into the distal intestine, characterized by relatively low proteolytic activity and optimal absorption.

Objective Carnosine is a naturally present dipeptide in humans and an over-the counter food additive. Evidence from animal studies supports the role for carnosine in the prevention and treatment of diabetes and cardiovascular disease, yet there is limited human data. This study investigated whether carnosine supplementation in individuals with overweight or obesity improves diabetes and cardiovascular risk factors. Methods In a double-blind randomized pilot trial in nondiabetic individuals with overweight and obesity (age 43 +/- 8 years; body mass index 31 +/- 4 kg/m2), 15 individuals were randomly assigned to 2 g carnosine daily and 15 individuals to placebo for 12 weeks. Insulin sensitivity and secretion, glucose tolerance (oral glucose tolerance test), blood pressure, plasma lipid profile, skeletal muscle (1H-MRS), and urinary carnosine levels were measured. Results Carnosine concentrations increased in urine after supplementation (P < 0.05). An increase in fasting insulin and insulin resistance was hampered in individuals receiving carnosine compared to placebo, and this remained significant after adjustment for age, sex, and change in body weight (P = 0.02, P = 0.04, respectively). Two-hour glucose and insulin were both lower after carnosine supplementation compared to placebo in individuals with impaired glucose tolerance (P < 0.05). Conclusions These pilot intervention data suggest that carnosine supplementation may be an effective strategy for prevention of type 2 diabetes.Copyright © 2016 The Obesity Society.

In this study, insulin-containing nanoparticles were loaded into pellet cores and orally administered to diabetic rats. Polyethylene imine-based nanoparticles, either placebo or loaded with insulin, were incorporated by extrusion and spheronization technology into cores that were subsequently coated with three overlapping layers and a gastroresistant film. The starting and coated systems were evaluated in vitro for their physico-technololgical characteristics, as well as disintegration and release performance. Nanoparticles-loaded cores showed homogeneous particle size distribution and shape. When a superdisintegrant and a soluble diluent were included in the composition enhanced disintegration and release performance were observed. The selected formulations, coated either with enteric or three-layer films, showed gastroresistant and release delayed behavior in vitro, respectively. The most promising formulations were finally tested for their hypoglycemic effect in diabetic rats. Only the nanoformulations loaded into the three-layer pellets were able to induce a significant hypoglycemic activity in diabetic rats. Our results suggest that this efficient activity could be attributed to a retarded release of insulin into the distal intestine, characterized by relatively low proteolytic activity and optimal absorption.

Ex vivo expansion of endocrine cells constitutes an interesting alternative to be able to match the unmet need of transplantable pancreatic islets. However, endocrine cells become fragile once removed from their extracellular matrix (ECM) and typically become senescent and loose insulin expression during conventional 2D culture. Herein we develop a protocol where 3D silk matrices functionalized with ECM-derived motifs are used for generation of insulin-secreting islet-like clusters from mouse and human primary cells. The obtained clusters were shown to attain an islet-like spheroid shape and to maintain functional insulin release upon glucose stimulation in vitro. Furthermore, in vivo imaging of transplanted murine clusters showed engraftment with increasing vessel formation during time. There was no sign of cell death and the clusters maintained or increased in size throughout the period, thus suggesting a suitable cluster size for transplantation.

Background. Pancreatic or islet fibrosis is often associated with activated pancreatic stellate cells (PSCs). PSCs are considered not only to promote fibrosis, but also to be associated with glucose intolerance in some diseases. We therefore evaluated morphological and functional relationships between islets and PSCs in the normal mouse pancreas and transplanted islets. Methods. Immunohistochemistry was used to map the presence of PSCs in the normal mouse pancreas and islets implanted under the renal capsule. We isolated and cultured mouse PSCs and characterized them morphologically by immunofluorescence staining. Furthermore, we measured their cytokine production and determined their effects on insulin release from simultaneously cultured islets. Results. PSCs were scattered throughout the pancreas, with occasional cells within the islets, particularly in the islet capsule. In islet transplants they were found mainly in the graft periphery. Cultured PSCs became functionally activated and produced several cytokines. Throughout the culture period they linearly increased their production of interleukin-6 and mammalian keratinocyte-derived chemokine. PSC cytokine production was not affected by acute hyperglycemia. Syngeneic islets cocultured with PSCs for 24-48 h increased their insulin release and lowered their insulin content. However, short-term insulin release in batch-type incubations was unaffected after 48 h of co-culture. Increased islet cell caspase-3 activation and a decreased islet cell replication were consistently observed after co-culture for 2 or 7 days. Conclusion. Activated PSCs may contribute to impaired islet endocrine function seen in exocrine pancreatitis and in islet fibrosis associated with some cases of type 2 diabetes.

Background: Despite Australia's sunny climate, low vitamin D levels are increasingly prevalent. Sun exposure is limited by long working hours, an increase in time spent indoors, and sun protection practices, and there is limited dietary vitamin D fortification. While the importance of vitamin D for bone mineralization is well known, its role as a protective agent against chronic diseases, such as type 2 diabetes and cardiovascular disease, is less understood. Observational and limited intervention studies suggest that vitamin D might improve insulin sensitivity and secretion, mainly via its anti-inflammatory properties, thereby decreasing the risk of development and progression of type 2 diabetes. The primary aim of this trial is to investigate whether improved plasma concentrations of 25-hydroxyvitamin D (25(OH)D), obtained through vitamin D supplementation, will increase insulin sensitivity and insulin secretion. A secondary aim is to determine whether these relationships are mediated by a reduction in underlying subclinical inflammation associated with obesity. Methods/Design: Fifty overweight but otherwise healthy nondiabetic adults between 18 and 60 years old, with low vitamin D levels (25(OH)D < 50 nmol/l), will be randomly assigned to intervention or placebo. At baseline, participants will undergo a medical review and anthropometric measurements, including dual X-ray absorptiometry, an intravenous glucose tolerance test, muscle and fat biopsies, a hyperinsulinemic euglycemic clamp, and questionnaires assessing diet, physical activity, sun exposure, back and knee pain, and depression. The intervention group will receive a first dose of 100,000 IU followed by 4,000 IU vitamin D (cholecalciferol) daily, while the placebo group will receive apparently identical capsules, both for a period of 16 weeks. All measurements will be repeated at follow-up, with the primary outcome measure expressed as a change from baseline in insulin sensitivity and secretion for the in

Background: Despite Australia's sunny climate, low vitamin D levels are increasingly prevalent. Sun exposure is limited by long working hours, an increase in time spent indoors, and sun protection practices, and there is limited dietary vitamin D fortification. While the importance of vitamin D for bone mineralization is well known, its role as a protective agent against chronic diseases, such as type 2 diabetes and cardiovascular disease, is less understood. Observational and limited intervention studies suggest that vitamin D might improve insulin sensitivity and secretion, mainly via its anti-inflammatory properties, thereby decreasing the risk of development and progression of type 2 diabetes. The primary aim of this trial is to investigate whether improved plasma concentrations of 25-hydroxyvitamin D (25(OH)D), obtained through vitamin D supplementation, will increase insulin sensitivity and insulin secretion. A secondary aim is to determine whether these relationships are mediated by a reduction in underlying subclinical inflammation associated with obesity. Methods/Design: Fifty overweight but otherwise healthy nondiabetic adults between 18 and 60 years old, with low vitamin D levels (25(OH)D < 50 nmol/l), will be randomly assigned to intervention or placebo. At baseline, participants will undergo a medical review and anthropometric measurements, including dual X-ray absorptiometry, an intravenous glucose tolerance test, muscle and fat biopsies, a hyperinsulinemic euglycemic clamp, and questionnaires assessing diet, physical activity, sun exposure, back and knee pain, and depression. The intervention group will receive a first dose of 100,000 IU followed by 4,000 IU vitamin D (cholecalciferol) daily, while the placebo group will receive apparently identical capsules, both for a period of 16 weeks. All measurements will be repeated at follow-up, with the primary outcome measure expressed as a change from baseline in insulin sensitivity and secretion for the in

Objectives. To examine the effects of inhibition of cyclooxygenase (COX) on islet hormone secretion in vitro and on pancreatic islet blood flow in vivo. Methods. Insulin release was measured in a static incubation system of islets isolated from Wistar-F rats after inhibition of COX-1 and COX-2 with SC 560 (COX-1), FR 122047 (COX-1), rofecoxib (COX-2), or indomethacin (both COX-1 and COX-2). In other rats organ blood flow values were measured with a microsphere technique during both normo- and hyperglycemia after administration of these enzyme inhibitors. Results. Serum insulin values were lower after pretreatment with a COX-1 inhibitor or a non-selective COX inhibitor in both control and glucose-injected rats in vivo, whereas COX-2 inhibition had no such effects. However, inhibition of COX had only minor effects on insulin release in vitro. Inhibition of COX affected neither total pancreatic nor islet blood flow in normoglycemic rats. Hyperglycemia caused an increase in both these flow values and in the duodenum. The increase in total pancreatic and duodenal blood flow was prevented by inhibition of COX-2 or non-selective COX inhibition. However, no effects on islet blood flow were seen after COX inhibition. Conclusion. Inhibition of COX affects insulin release and blood glucose concentrations in vivo. However, COX inhibition has only minor effects on pancreatic islet blood flow, but prevents the glucose-induced increase in total pancreatic blood flow.

Glucagon-like peptide 1 (GLP-1) and related peptide agonists have been extensively investigated for glycaemic control in Type 2 diabetes, and may also have therapeutic applications for other diseases. Due to the short half-life (t1/2<2 min) of the endogenous peptide, caused by proteolytic degradation and renal clearance, different strategies for half-life extension and sustained release have been explored. In the present study, conjugates between a GLP-1 analogue and a 5 kDa albumin-binding domain (ABD) derived from streptococcal protein G have been chemically synthesized and evaluated. ABD binds with high affinity to human serum albumin, which is highly abundant in plasma and functions as a drug carrier in the circulation. Three different GLP-1-ABD conjugates, with the two peptides connected by linkers of two, four, and six PEG units, respectively, were synthesized and tested in mouse pancreatic islets at high (11 mM) and low (3 mM) glucose concentration. Insulin release upon stimulation was shown to be glucose-dependent, showing no significant difference between the three different GLP-1-ABD conjugates and unconjugated GLP-1 analogue. The biological activity, in combination with the high affinity binding to albumin, make the GLP-1-ABD conjugates promising GLP-1 receptor agonists expected to show extended in vivo half-life., Updated from Manuscript to Journal. QC 20140711

It is currently believed that the stimulation of insulin release by nutrient secretagogues reflects their capacity to act as fuel in pancreatic islet beta cells. In this review, it is proposed that such a fuel concept is not incompatible with a receptor hypothesis postulating the participation of cell-surface receptors in the recognition of selected nutrients as insulinotropic agents. Pursuant to this, attention is drawn to such matters as the anomeric specificity of the beta cell secretory response to d-glucose and its perturbation in diabetes mellitus, the insulinotropic action of artificial sweeteners, the possible role of bitter taste receptors in the stimulation of insulin secretion by l-glucose pentaacetate, the recently documented presence of cell-surface sweet taste receptors in insulin-producing cells, the multimodal signalling process resulting from the activation of these latter receptors, and the presence in beta cells of a sweet taste receptor mediating the fructose-induced potentiation of glucose-stimulated insulin secretion. © 2014 Springer-Verlag., SCOPUS: re.j, info:eu-repo/semantics/published

It is currently believed that the stimulation of insulin release by nutrient secretagogues reflects their capacity to act as fuel in pancreatic islet beta cells. In this review, it is proposed that such a fuel concept is not incompatible with a receptor hypothesis postulating the participation of cell-surface receptors in the recognition of selected nutrients as insulinotropic agents. Pursuant to this, attention is drawn to such matters as the anomeric specificity of the beta cell secretory response to d-glucose and its perturbation in diabetes mellitus, the insulinotropic action of artificial sweeteners, the possible role of bitter taste receptors in the stimulation of insulin secretion by l-glucose pentaacetate, the recently documented presence of cell-surface sweet taste receptors in insulin-producing cells, the multimodal signalling process resulting from the activation of these latter receptors, and the presence in beta cells of a sweet taste receptor mediating the fructose-induced potentiation of glucose-stimulated insulin secretion. © 2014 Springer-Verlag., SCOPUS: re.j, info:eu-repo/semantics/published

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), volume-regulated anion channels (VRACs) and the electrogenic sodium bicarbonate (Na+- HCO3-) cotransporter, NBCe1, in the regulation of nutrient-and hypotonicity-induced insulin release from rat pancreatic islets and tumoral insulin-producing BRIN-BD11 cells. In the islets, 5-nitro-2-(3- phenylpropylamino)benzoic acid (NPPB) and 5-chloro-2-hydroxy-3-(thiophene-2- carbonyl)indole-1-carboxamide (tenidap) reduced glucose-stimulated insulin release, however, only NPPB suppressed the enhancing action of cAMP analogs upon such a release. Insulin output from the BRIN-BD11 cells was stimulated by 2-ketoisocaproate (KIC) or extracellular hypoosmolarity. cAMP analogs and 3-isobutyl-1-methylxanthine 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 mitogen-activated protein (MAP) kinase decreased insulin output in isotonic and hypotonic media. The inhibitor of sAC, 2-hydroxyestriol, 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., SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction. Morphological evidence for reinnervation of pancreatic islet grafts is plentiful. However, to what extent intra-graft nerves influence the endocrine functions of the islet transplant is largely unknown. We therefore aimed to directly stimulate nerves leading to islet grafts with electrodes and measure insulin secretion in response to this. Methods. We implanted syngeneic islets under the renal capsule of rats, and examined them 1 or 7-9 months later. In anesthetized rats blood samples were collected from the renal vein and femoral artery, respectively, during electrode stimulation of the nerves leading to the islet grafts. Results. As expected, nerve stimulation decreased renal blood flow. However, serum insulin concentrations in samples derived from the renal vein or femoral artery changed in concert with one another, both during normoglycemia and acute hyperglycemia. Conclusion. Reinnervation which occurs after islet transplantation under the renal capsule has minor effects on graft endocrine function.

Insulin-like peptides (ILPs) and growth factors (IGFs) not only regulate development, growth, reproduction, metabolism, stress resistance, and lifespan, but also certain behaviors and cognitive functions. ILPs, IGFs, their tyrosine kinase receptors and downstream signaling components have been largely conserved over animal evolution. Eight ILPs have been identified in Drosophila (DILP1-8) and they display cell and stage-specific expression patterns. Only one insulin receptor, dInR, is known in Drosophila and most other invertebrates. Nevertheless, the different DILPs are independently regulated transcriptionally and appear to have distinct functions, although some functional redundancy has been revealed. This review summarizes what is known about regulation of production and release of DILPs in Drosophila with focus on insulin signaling in the daily life of the fly. Under what conditions are DILP-producing cells (IPCs) activated and which factors have been identified in control of IPC activity in larvae and adult flies? The brain IPCs that produce DILP2, 3 and 5 are indirectly targeted by DILP6 and a leptin-like factor from the fat body, as well as directly by a few neurotransmitters and neuropeptides. Serotonin, octopamine, GABA, short neuropeptide F (sNPF), corazonin and tachykinin-related peptide have been identified in Drosophila as regulators of IPCs. The GABAergic cells that inhibit IPCs and DILP release are in turn targeted by a leptin-like peptide (unpaired 2) from the fat body, and the IPC-stimulating corazonin/sNPF neurons may be targeted by gut-derived peptides. We also discuss physiological conditions under which IPC activity may be regulated, including nutritional states, stress and diapause induction.

Prenatal stress might increase cardiometabolic disease risk. We measured prenatal stress due to an ice storm in 1998, and measured glucose tolerance among a subsample of 32 exposed adolescents in 2011. Severity of stress was positively associated with insulin secretion, suggesting that prenatal stress independently predicts metabolic outcomes in adolescence.

The ageing process in men is marked by changes in body composition (loss of fat-free mass (FFM) and skeletal muscle, and gain in fat mass (FM)) and is associated with a decline in serum testosterone. Correlations between these aspects of ageing and the acknowledged role of exogenous testosterone in reversing the loss of FFM and gain in FM seen in adult men with congenital or acquired hypoandrogenism have led to the hypothesis that testosterone therapy in ageing men will result in favourable changes in body composition and may improve metabolic status and/or cardiovascular risk. Data from randomized controlled trials of testosterone therapy in ageing men addressing the endpoints of body composition and components of the metabolic syndrome and cardiovascular risk factors are reviewed, and the impact of the increasing prevalence of obesity on these relationships is considered. © 2007 Nature Publishing Group All rights reserved.

Transplantation of pancreatic islets has become a promising clinical option to treat patients with type 1 diabetes, alternative to the standard therapy with insulin injections. Islet transplantation is a minimally invasive therapeutic approach, and it allows a better metabolic control and a long-term insulin independence in more than 80% of patients (Ryan et al., 2002). However this therapeutic treatment has some side effects, such as the poor yield of pancreatic islet explants and even more the immune graft rejection, which have as a consequence the very limited lifespan of transplanted pancreatic islets. To avoid these side effects several strategies have been proposed and, besides the treatment with immunosuppressive drugs, promising results have been obtained with the use of Mesenchymal Stem cells (MSCs), already known in literature to be able to support the survival of many cell types (Scuteri et al., 2006). Several in vivo studies have demonstrated that the concurrent transplantation of pancreatic islets with MSCs reduces the number of islets required to achieve glycemic control in diabetic rats, but the mechanisms of these encouraging results are still unknown (Figliuzzi et al., 2009). For these reasons in this in vitro study we characterized the effect of co-culture of rat MSC on survival and functioning of rat pancreatic islets, by evaluating for 4 weeks: i) MSC adhesion to pancreatic islets; ii) viability of pancreatic islets co-cultured with MSCs; iii) the expression of insulin after co-culture; iv) the ability of co-cultured pancreatic islets to correctly adjust insulin release after variation of glucose concentration. Our results demonstrated that MSCs are able to adhere to pancreatic islets, but to increase only partly the pancreatic islet survival, which retain the ability to express and correctly release insulin after glucose variation in medium culture. Noteworthy that the insulin level in the medium of co-cultured pancreatic islets is always higher

SCOPUS: ar.j, info:eu-repo/semantics/published

Background Development of type 2 diabetes mellitus (T2DM) is characterized by deranged insulin secretory patterns, where elevated insulin levels at non-stimulatory basal conditions and reduced hormonal levels at stimulatory conditions are major components. To delineate mechanisms responsible for these alterations we cultured INS-1E cells for 48 hours at 20 mM glucose in absence or presence of 0.5 mM palmitate, when stimulatory release was reduced or basal secretion was elevated, respectively. Results After culture, cells were protein profiled by SELDI-TOF MS and 2D-PAGE. Differentially expressed proteins were discovered and identified by peptide mass fingerprinting. Complimentary protein profiles were obtained by the two approaches with SELDI-TOF MS being more efficient in separating proteins in the low molecular range and 2D-PAGE in the high molecular range. Identified proteins included alpha glucosidase, calmodulin, gars, glucose-6-phosphate dehydrogenase, heterogenous nuclear ribonucleoprotein A3, lon peptidase, nicotineamide adenine dinucleotide hydrogen (NADH) dehydrogenase, phosphoglycerate kinase, proteasome p45, rab2, pyruvate kinase and t-complex protein. The observed glucose-induced differential protein expression pattern indicates enhanced glucose metabolism, defense against reactive oxygen species, enhanced protein translation, folding and degradation and decreased insulin granular formation and trafficking. Palmitate-induced changes were related to altered exocytosis. Conclusions The identified altered proteins indicate mechanism important for deranged b-cell function in T2DM.

Transplanting pancreatic islets into the liver through the portal vein is currently the most common procedure in clinical islet transplantations for treating patients with brittle type 1 diabetes. However, most islet grafts fail within a 5-year period necessitating retransplantation. The vascular connections are disrupted at islet isolation and implanted islets depend on diffusion of oxygen and nutrients in the immediate posttransplantation period. Rapid and efficient revascularization is of utmost importance for the survival and long-term function of transplanted islets. In this thesis, the influence of the implantation microenvironment for islet engraftment and function was studied. Islets were transplanted into the liver, the renal subcapsular site or the pancreas. Islets implanted into the liver contained fewer glucagon-positive cells than islets implanted to the kidney and endogenous islets. Intraportally transplanted islets responded with insulin and glucagon release to secretagogues, but only when stimulated through the hepatic artery. Thus, the intrahepatic grafts were selectively revascularized from the hepatic artery. The vascular density in human islets transplanted into the liver of athymic mice was markedly lower when compared to human islets grafted to the kidney. Islets implanted into their physiological environment, the pancreas, were markedly better revascularized. Insulin content, glucose-stimulated insulin release, (pro)insulin biosynthesis and glucose oxidation rate were markedly decreased in transplanted islets retrieved from the liver, both when compared to endogenous and transplanted islets retrieved from the pancreas. Only minor changes in metabolic functions were observed in islets implanted into the pancreas when compared to endogenous islets. The present findings demonstrate that the microenvironment has a major impact on the engraftment of transplanted islets. Elucidating the beneficial factors that promote engraftment would improve the s

Transplanting pancreatic islets into the liver through the portal vein is currently the most common procedure in clinical islet transplantations for treating patients with brittle type 1 diabetes. However, most islet grafts fail within a 5-year period necessitating retransplantation. The vascular connections are disrupted at islet isolation and implanted islets depend on diffusion of oxygen and nutrients in the immediate posttransplantation period. Rapid and efficient revascularization is of utmost importance for the survival and long-term function of transplanted islets. In this thesis, the influence of the implantation microenvironment for islet engraftment and function was studied. Islets were transplanted into the liver, the renal subcapsular site or the pancreas. Islets implanted into the liver contained fewer glucagon-positive cells than islets implanted to the kidney and endogenous islets. Intraportally transplanted islets responded with insulin and glucagon release to secretagogues, but only when stimulated through the hepatic artery. Thus, the intrahepatic grafts were selectively revascularized from the hepatic artery. The vascular density in human islets transplanted into the liver of athymic mice was markedly lower when compared to human islets grafted to the kidney. Islets implanted into their physiological environment, the pancreas, were markedly better revascularized. Insulin content, glucose-stimulated insulin release, (pro)insulin biosynthesis and glucose oxidation rate were markedly decreased in transplanted islets retrieved from the liver, both when compared to endogenous and transplanted islets retrieved from the pancreas. Only minor changes in metabolic functions were observed in islets implanted into the pancreas when compared to endogenous islets. The present findings demonstrate that the microenvironment has a major impact on the engraftment of transplanted islets. Elucidating the beneficial factors that promote engraftment would improve the s

Transplanting pancreatic islets into the liver through the portal vein is currently the most common procedure in clinical islet transplantations for treating patients with brittle type 1 diabetes. However, most islet grafts fail within a 5-year period necessitating retransplantation. The vascular connections are disrupted at islet isolation and implanted islets depend on diffusion of oxygen and nutrients in the immediate posttransplantation period. Rapid and efficient revascularization is of utmost importance for the survival and long-term function of transplanted islets. In this thesis, the influence of the implantation microenvironment for islet engraftment and function was studied. Islets were transplanted into the liver, the renal subcapsular site or the pancreas. Islets implanted into the liver contained fewer glucagon-positive cells than islets implanted to the kidney and endogenous islets. Intraportally transplanted islets responded with insulin and glucagon release to secretagogues, but only when stimulated through the hepatic artery. Thus, the intrahepatic grafts were selectively revascularized from the hepatic artery. The vascular density in human islets transplanted into the liver of athymic mice was markedly lower when compared to human islets grafted to the kidney. Islets implanted into their physiological environment, the pancreas, were markedly better revascularized. Insulin content, glucose-stimulated insulin release, (pro)insulin biosynthesis and glucose oxidation rate were markedly decreased in transplanted islets retrieved from the liver, both when compared to endogenous and transplanted islets retrieved from the pancreas. Only minor changes in metabolic functions were observed in islets implanted into the pancreas when compared to endogenous islets. The present findings demonstrate that the microenvironment has a major impact on the engraftment of transplanted islets. Elucidating the beneficial factors that promote engraftment would improve the s

Transplanting pancreatic islets into the liver through the portal vein is currently the most common procedure in clinical islet transplantations for treating patients with brittle type 1 diabetes. However, most islet grafts fail within a 5-year period necessitating retransplantation. The vascular connections are disrupted at islet isolation and implanted islets depend on diffusion of oxygen and nutrients in the immediate posttransplantation period. Rapid and efficient revascularization is of utmost importance for the survival and long-term function of transplanted islets. In this thesis, the influence of the implantation microenvironment for islet engraftment and function was studied. Islets were transplanted into the liver, the renal subcapsular site or the pancreas. Islets implanted into the liver contained fewer glucagon-positive cells than islets implanted to the kidney and endogenous islets. Intraportally transplanted islets responded with insulin and glucagon release to secretagogues, but only when stimulated through the hepatic artery. Thus, the intrahepatic grafts were selectively revascularized from the hepatic artery. The vascular density in human islets transplanted into the liver of athymic mice was markedly lower when compared to human islets grafted to the kidney. Islets implanted into their physiological environment, the pancreas, were markedly better revascularized. Insulin content, glucose-stimulated insulin release, (pro)insulin biosynthesis and glucose oxidation rate were markedly decreased in transplanted islets retrieved from the liver, both when compared to endogenous and transplanted islets retrieved from the pancreas. Only minor changes in metabolic functions were observed in islets implanted into the pancreas when compared to endogenous islets. The present findings demonstrate that the microenvironment has a major impact on the engraftment of transplanted islets. Elucidating the beneficial factors that promote engraftment would improve the s

A number of metabolic, ionic and secretory variables were recently found to be affected in pancreatic islets obtained from second generation rats depleted in long-chain polyunsaturated omega 3 fatty acids (omega 3 rats). The present study further documents three sets of anomalies in such islets. First, after 90 min exposure to D-glucose (8.3 mM), the release of insulin from perifused islets, prelabelled with 45Ca, is lower in omega 3 rats than in control animals, despite comparable 45Ca fractional outflow rate. Second, over 15 min exposure to carbamylcholine (0.1 mM), in the presence of D: -glucose, the cytosolic concentration of Ca2+ is increased to a greater relative extent in dispersed islet cells from omega 3 rats, as compared to control animals. This coincides with a greater relative increase in insulin output from perifused islets during the second phase of the secretory response to the cholinergic agent. Last, the increase provoked by ouabain (1.0 mM) in cytosolic Ca2+ concentration, 45Ca fractional outflow rate and insulin release are all delayed in the omega 3 rats. Taking into account the decreased activity of Na+, K+-ATPase in the islets of omega 3 rats, these findings are interpreted as reflecting an impaired priming of insulin-producing cells when first exposed for 105 min to a physiological postprandial concentration of D-glucose., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Aims/hypothesis: The few patients subjected to autotransplantation of pancreatic islets after pancreatectomy usually become normoglycaemic after using islets from the resected organ only, whereas allogeneic recipients usually require at least two grafts to retain normoglycaemia. Previous experimental studies have demonstrated that islets transplanted to non-pancreatectomised recipients acquire a markedly decreased blood vessel density, which leads to a hypoxic microenvironment. The aim of the present study was to test the hypothesis that autotransplanted islets have better vascular engraftment and function as a result of the pancreatic surgery involved. Materials and methods: In the present study, athymic mice and inbred rats were subjected to a 60% pancreatectomy and transplanted with human or rat islets, respectively, 4 days later. Control animals underwent sham surgery. Blood flow, oxygen tension, vascular density and endocrine volume in the islet grafts were measured 1 month after transplantation. Separate grafts were used for perfusion experiments and for assessment of beta cell proliferation and endocrine cellular apoptosis at different time periods after transplantation. Results: Islet grafts in partially pancreatectomised recipients had an increased blood flow, oxygen tension, blood vessel density and endocrine mass 1 month post-transplantation compared with control animals. They also exhibited increased insulin release in perfusion experiments performed 1 month post-transplantation, and decreased cellular apoptosis early after transplantation. Conclusions/interpretation: The present study shows that the pancreatectomy procedure itself has beneficial effects on the engraftment of transplanted human and rat islets. Our results provide an additional explanation, besides diminished immunological responses, of the much better outcome of islet autotransplantations compared with allogeneic transplantations in the clinic.

Bank voles (Clethrionomys glareolus) kept in captivity develop diabetes mellitus to a significant extent. Also in wild bank voles, elevated blood glucose has been observed. A newly isolated picornavirus named Ljungan virus (LV) has been found in the pancreas of these bank voles. Moreover, LV infection in combination with environmental factors may cause glucose intolerance/diabetes (GINT/D) in normal mice. The aim of the present study was to investigate the functional characteristics of pancreatic islets, isolated from bank voles, bred in the laboratory but considered LV infected. About 20% of all males and females were classified as GINT/D following a glucose tolerance test. Of these animals the majority had become diabetic by 20 weeks of age, with a tendency towards an earlier onset in the males. GINT/D animals had increased serum insulin levels. Islets were tested on the day of isolation (day 0) and after 1 week of culture for their insulin content and their capacity to synthesize (pro)insulin, secrete insulin and metabolize glucose. Functional differences could be observed between normal and GINT/D animals as well as between genders. An elevated basal insulin secretion was observed on day 0 indicating β-cell dysfunction among islets isolated from diabetic males. In vitro culture could reverse some functional changes. The increased serum insulin level and the increased basal islet insulin secretion may suggest that the animals had developed a type 2 diabetes-like condition. It is likely that the putative stress imposed in the laboratory, maybe in combination with LV infection, can lead to an increased functional demand on the β-cells.

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Mice were subjected to gastrectomy (GX) or sham operation (controls). Four to six weeks later the pancreatic islets were isolated and analysed for cAMP or alternatively incubated in a Krebs-Ringer based medium in an effort to study insulin secretion and cAMP accumulation in response to glucose or the adenylate cyclase activator forskolin. Freshly isolated islets from GX mice had higher cAMP content than islets from control mice, a difference that persisted after incubation for I h at a glucose concentration of 4 mmol/l. Addition of forskolin to this medium induced much greater cAMP and insulin responses in islets from GX mice than in islets from control mice. In contrast, the insulin response to high glucose (16.7 mmol/l) was much weaker in GX islets than in control islets. Glucose-induced insulin release was associated with a 2-fold rise in the cAMP content in control islets. Surprisingly no rise in cAMP was noted in GX islets incubated at high glucose. Capacitance measurements conducted on isolated insulin cells from GX mice revealed a much lower exocytotic response to a single 500 ms depolarisation (from -70 mV to zero) than in control insulin cells. Addition of cAMP to the cytosol enhanced the exocytotic response in insulin cells from control mice but not from GX mice. The depolarisation-triggered inward Ca2+ current in insulin cells from GX mice did not differ from that in control mice, and hence the reduced exocytotic response following GX cannot be ascribed to a decreased Ca2+ influx. Experiments involving a train of ten 500 ms depolarisations revealed that the exocytotic response was prominent in control insulin cells but modest in GX insulin cells. It seems that cAMP is capable of eliciting insulin release from insulin cells of GX mice only when cAMP is generated in a specific microdomain conceivably through the intervention of membrane-associated adenylate cyclases that can be activated by forskolin. The GX-evoked impairment of depolarisation-induced exocy

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Glucose induces insulin secretion (IS) and also potentiates the insulin-releasing action of secretagogues such as arginine and sulfonylureas. This potentiating effect is known to be impaired in type 2 diabetic patients, but its cellular mechanisms are unclear. IS and cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured in mouse islets during perifusion with 3-15 mmol/l glucose (G3-G15, respectively) and pulse or stepwise stimulation with 1-10 mmol/l arginine or 5-250 micromol/l tolbutamide. In G3, arginine induced small increases in [Ca(2+)](i) but no IS. G7 alone only slightly increased [Ca(2+)](i) and IS but markedly potentiated arginine effects on [Ca(2+)](i), which resulted in significant IS (already at 1 mmol/l). For each arginine concentration, both responses further increased at G10 and G15, but the relative change was distinctly larger for IS than [Ca(2+)](i). At all glucose concentrations, tolbutamide dose dependently increased [Ca(2+)](i) and IS with thresholds of 25 micromol/l for [Ca(2+)](i) and 100 micromol/l for IS at G3 and of 5 micromol/l for both at G7 and above. Between G7 and G15, the effect of tolbutamide on [Ca(2+)](i) increased only slightly, whereas that on IS was strongly potentiated. The linear relationship between IS and [Ca(2+)](i) at increasing arginine or tolbutamide concentrations became steeper as the glucose concentration was raised. Thus glucose augmented more the effect of each agent on IS than that on [Ca(2+)](i). In conclusion, glucose potentiation of arginine- or tolbutamide-induced IS involves increases in both the rise of [Ca(2+)](i) and the action of Ca(2+) on exocytosis. This dual mechanism must be borne in mind to interpret the alterations of the potentiating action of glucose in type 2 diabetic patients.