Monica Sandberg

Endothelial cell signalling supports pancreatic beta cell function in the rat

Aims/hypothesisThe proximity of endothelial cells and beta cells in islets by necessity means that they are exposed to each other’s products. Whereas islet endothelial cells require signals from beta cells to function properly, endothelin-1, thrombospondin-1 and laminins, among others, have been identified as endothelial-derived molecules, although their full effects on beta cells have not been explored. We tested the hypothesis that islet endothelial-derived products affect beta cell function.MethodsEndothelial cells from rat islets were proliferated and purified. Endothelium-conditioned culture medium (ECCM) was obtained by maintaining the endothelial cells in culture medium. Islet function was evaluated following exposure of cultured islets to standard culture medium or ECCM. Changes in mRNA levels for key beta cell metabolic enzymes were also measured in islets after ECCM exposure.ResultsGlucose-stimulated insulin release and islet insulin content were markedly enhanced by exposure to ECCM. This was at least partly explained by improved mitochondrial function, as assessed by glucose oxidation and an upregulation of the mitochondrial gene for glycerol-3-phosphate dehydrogenase (mGpdh [also known as Gpd2]), combined with upregulation of the rate-limiting enzyme in the glycolysis, glucokinase, in the islets. The intracellular degradation of insulin was also decreased in the islets. Islet endothelial cells produced laminins, and the positive effects of islet endothelial cells were prevented by addition of a neutralising antibody to the β1-chain of laminin. Addition of exogenous laminin stimulated islet function.Conclusions/interpretationThis study provides proof of principle that endothelial cells can affect the function of beta cells in their vicinity and that this is at least partially mediated by laminins.

High Vascular Density and Oxygenation of Pancreatic Islets Transplanted in Clusters into Striated Muscle

Pancreatic islet transplantation is presently almost exclusively performed using the intraportal route for transplantation into the liver. However, islets at this site are poorly revascularized and, when also considering the poor long-term results of clinical islet transplantation, there has in recent years emerged an increased interest to evaluate alternative sites for islet transplantation. Striated muscle is easily accessible and has for decades been used for autotransplantation of parathyroid glands. Moreover, it is almost the only tissue in the adult where physiological angiogenesis occurs. The present study tested the hypothesis that striated muscle would provide good conditions for revascularization and oxygenation of transplanted islets. Because we previously have observed similar revascularization of islets implanted to the renal subcapsular site and intraportally into the liver, islets grafted to the kidney were for simplicity besides native islets used for comparison. Islets grafted into muscle were found to have three times more blood vessels than corresponding islets at the renal subcapsular site at 2 month follow-up, but still less vascular numbers than native islets. The oxygen tension in 2-month-old intramuscular islet grafts was sixfold higher than in corresponding renal subcapsular grafts, and 70% of that in native islets. However, the oxygenation of surrounding muscle was only 50% of that in renal cortex, and connective tissue constituted a larger proportion of the intramuscular than the renal subcapsular grafts, suggesting exaggerated early islet cell death at the former site. We conclude that the intramuscular site provides excellent conditions for vascular engraftment, but that interventions to improve early islet survival likely are needed before clinical application. Such could include bioengineered matrices that not only spatially disperse the islet, but also could provide local supply of oxygen carriers, growth and survival factors, strategies that are much more easily applied at the intramuscular than the intrahepatic site.

Neural crest stem cells increase beta cell proliferation and improve islet function in co-transplanted murine pancreatic islets

Aims/hypothesisLong-term graft survival after islet transplantation to patients with type 1 diabetes is insufficient, necessitating the development of new strategies to enhance transplant viability. Here we investigated whether co-transplantation of neural crest stem cells (NCSCs) with islets improves islet survival and function in normoglycaemic and diabetic mice.MethodsIslets alone or together with NCSCs were transplanted under the kidney capsule to normoglycaemic or alloxan-induced diabetic mice. Grafts were analysed for size, proliferation, apoptosis and insulin release. In diabetic recipients blood glucose levels were examined before and after graft removal.ResultsIn mixed transplants NCSCs actively migrated and extensively associated with co-transplanted pancreatic islets. Proliferation of beta cells was markedly increased and transplants displayed improved insulin release in normoglycaemic mice compared with those receiving islet-alone transplants. Mixed grafts survived successfully and partially restored normoglycaemia in alloxan-induced diabetic mice.Conclusions/interpretationCo-grafting of NCSCs with pancreatic islets improved insulin release in mixed transplants and enhanced beta cell proliferation, resulting in increased beta cell mass. This co-transplantation model offers an opportunity to restore neural–islet interactions and improve islet functions after transplantation.

Abrogation of adenosine A1 receptor signalling improves metabolic regulation in mice by modulating oxidative stress and inflammatory responses.

Aims/hypothesisAdenosine is an important regulator of metabolism; however, the role of the A1 receptor during ageing and obesity is unclear. The aim of this study was to investigate the effects of A1 signalling in modulating metabolic function during ageing.MethodsAge-matched young and aged A1 (also known as Adora1)-knockout (A1−/−) and wild-type (A1+/+) mice were used. Metabolic regulation was evaluated by body composition, and glucose and insulin tolerance tests. Isolated islets and islet arterioles were used to detect islet endocrine and vascular function. Oxidative stress and inflammation status were measured in metabolic organs and systemically.ResultsAdvanced age was associated with both reduced glucose clearance and insulin sensitivity, as well as increased visceral adipose tissue (VAT) in A1+/+ compared with A1−/− mice. Islet morphology and insulin content were similar between genotypes, but relative changes in in vitro insulin release following glucose stimulation were reduced in aged A1+/+ compared with A1−/− mice. Islet arteriolar responses to angiotensin II were stronger in aged A1+/+ mice, this being associated with increased NADPH oxidase activity. Ageing resulted in multiple changes in A1+/+ compared with A1−/− mice, including enhanced NADPH oxidase-derived O2− formation and NADPH oxidase isoform 2 (Nox2) protein expression in pancreas and VAT; elevated levels of circulating insulin, leptin and proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-12); and accumulation of CD4+ T cells in VAT. This was associated with impaired insulin signalling in VAT from aged A1+/+ mice.Conclusions/interpretationThese studies emphasise that A1 receptors regulate metabolism and islet endocrine and vascular functions during ageing, including via the modulation of oxidative stress and inflammatory responses, among other things.

Pancreatic islet blood flow and its measurement

Abstract Pancreatic islets are richly vascularized, and islet blood vessels are uniquely adapted to maintain and support the internal milieu of the islets favoring normal endocrine function. Islet blood flow is normally very high compared with that to the exocrine pancreas and is autonomously regulated through complex interactions between the nervous system, metabolites from insulin secreting β-cells, endothelium-derived mediators, and hormones. The islet blood flow is normally coupled to the needs for insulin release and is usually disturbed during glucose intolerance and overt diabetes. The present review provides a brief background on islet vascular function and especially focuses on available techniques to measure islet blood perfusion. The gold standard for islet blood flow measurements in experimental animals is the microsphere technique, and its advantages and disadvantages will be discussed. In humans there are still no methods to measure islet blood flow selectively, but new developments in radiological techniques hold great hopes for the future.

Intracellular degradation of insulin and crinophagy are maintained by nitric oxide and cyclo-oxygenase 2 activity in isolated pancreatic islets

Background information. Pancreatic β‐cells require an optimal insulin content to allow instantaneous secretion of insulin. This is maintained by insulin biosynthesis and intracellular degradation of insulin. Degradation may be effected by crinophagy, i.e. the fusion of secretory granules with lysosomes. IL‐1β (interleukin 1β) induces distinct changes of β‐cell lysosomes. To study the mechanisms for intracellular insulin degradation and crinophagy, isolated mouse pancreatic islets were exposed to IL‐1β and known pathways for IL‐1β actions were blocked. Intracellular insulin degradation was determined by following the fate of radioactively labelled insulin. Crinophagy was studied by ultrastructural analysis. The effects of blocking pathways for IL‐1β were monitored by measurements of nitrite and PGE2 (prostaglandin E2).

Steroid effects on intracellular degradation of insulin and crinophagy in isolated pancreatic islets

Under physiological conditions substantial amounts of hormone may be degraded within endocrine cells by a crinophagic process comprising fusions of secretory granules with lysosomes. Glucocorticoids may stabilise and progesterone destabilise lysosomal membranes. The effects of corticosterone and progesterone on intracellular degradation of insulin and crinophagy were determined in pancreatic beta-cells, and possible pathways mediating these effects were evaluated. Pancreatic islets were isolated from mice, intracellular degradation of insulin was measured by a pulse-chase method, and crinophagy was studied by electron microscopy. The islets were exposed to 3.3, 5.5 or 28 mM glucose with or without corticosterone, progesterone or the receptor ligands A-224817.0 and WAY-161358. Mifepristone was used to block steroid receptors and indomethacin to inhibit prostaglandin synthesis. Corticosterone caused a concentration-dependent decrease of insulin degradation at the lower glucose concentrations. Progesterone effected a concentration-dependent stimulation of insulin degradation. These results were paralleled with changes of the crinophagic activity in the beta-cells. Corticosterone decreased and progesterone increased islet production of prostaglandin E(2). Mifepristone abolished the steroid actions on insulin degradation and prostaglandin production. The effects of corticosterone were mimicked by the selective glucocorticoid receptor modulator A-224817.0, but in contrast to progesterone, the selective progesterone receptor agonist WAY-161358 had no effect on insulin degradation or prostaglandin production. Inhibition of cyclooxygenase blocked insulin degradation. The findings indicate that both corticosterone and progesterone could affect intracellular insulin degradation and crinophagy solely via the glucocorticoid receptor, and that prostaglandins may have a regulatory role in intracellular turnover of secretory material in pancreatic islet beta-cells.

Matrix Metalloproteinase-9 Is Essential for Physiological Beta Cell Function and Islet Vascularization in Adult Mice

The availability of paracrine factors in the islets of Langerhans, and the constitution of the beta cell basement membrane can both be affected by proteolytic enzymes. This study aimed to investigate the effects of the extracellular matrix-degrading enzyme gelatinase B/matrix metalloproteinase-9 (Mmp-9) on islet function in mice. Islet function of Mmp9-deficient (Mmp9(-/-)) mice and their wild-type littermates was evaluated both in vivo and in vitro. The pancreata of Mmp9(-/-) mice did not differ from wild type in islet mass or distribution. However, Mmp9(-/-) mice had an impaired response to a glucose load in vivo, with lower serum insulin levels. The glucose-stimulated insulin secretion was reduced also in vitro in isolated Mmp9(-/-) islets. The vascular density of Mmp9(-/-) islets was lower, and the capillaries had fewer fenestrations, whereas the islet blood flow was threefold higher. These alterations could partly be explained by compensatory changes in the expression of matrix-related proteins. This in-depth investigation of the effects of the loss of MMP-9 function on pancreatic islets uncovers a deteriorated beta cell function that is primarily due to a shift in the beta cell phenotype, but also due to islet vascular aberrations. This likely reflects the importance of a normal islet matrix turnover exerted by MMP-9, and concomitant release of paracrine factors sequestered on the matrix.

Activated pancreatic stellate cells can impair pancreatic islet function in mice

Abstract 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 co-cultured 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.

The use of hydrogen gas clearance for blood flow measurements in single endogenous and transplanted pancreatic islets

The blood perfusion of pancreatic islets is regulated independently from that of the exocrine pancreas, and is of importance for multiple aspects of normal islet function, and probably also during impaired glucose tolerance. Single islet blood flow has been difficult to evaluate due to technical limitations. We therefore adapted a hydrogen gas washout technique using microelectrodes to allow such measurements. Platinum micro-electrodes monitored hydrogen gas clearance from individual endogenous and transplanted islets in the pancreas of male Lewis rats and in human and mouse islets implanted under the renal capsule of male athymic mice. Both in the rat endogenous pancreatic islets as well as in the intra-pancreatically transplanted islets, the vascular conductance and blood flow values displayed a highly heterogeneous distribution, varying by factors 6-10 within the same pancreas. The blood flow of human and mouse islet grafts transplanted in athymic mice was approximately 30% lower than that in the surrounding renal parenchyma. The present technique provides unique opportunities to study the islet vascular dysfunction seen after transplantation, but also allows for investigating the effects of genetic and environmental perturbations on islet blood flow at the single islet level in vivo.