Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse β‐cell lines, human islets and CB1R‐null (CB1R^?/?) mice, we have now investigated the role of CB1Rs in modulating β‐cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP‐1‐mediated cAMP accumulation and insulin secretion as well as glucose‐stimulated insulin secretion in mouse β‐cell lines and human islets. In addition, silencing CB1R in mouse β cells resulted in an increased expression of pro‐insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in β cells lacking insulin receptor. Furthermore, CB1R^?/? mice had increased pro‐insulin, GCK and GLUT2 expression in β cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve β‐cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to β‐cell function in type 2 diabetes.     

Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet‐like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five‐step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti‐hyperglycaemic hormone‐Glp1 (glucagon‐like peptide 1) analogue Liraglutide with prolonged t_½ and Exendin 4. The differentiated islet‐like 3D clusters expressed bonafide mature and functional β‐cell markers‐PDX1 (pancreatic and duodenal homoeobox‐1), C‐peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C‐peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non‐obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long‐term in vivo functionally viable islets from hESC.     

Controlled aggregation of primary human pancreatic islet cells leads to glucose‐responsive pseudoislets comparable to native islets

Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three‐dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100–150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (<2%), comparable glucose‐responsiveness and increasing PDX1, MAFA and INSULIN gene expression with increasing aggregate size. The re‐associated human islet cells showed an a‐typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets. After transplantation of these islet cell aggregates under the kidney capsule of immunodeficient mice, human C‐peptide was detected in the serum indicating that beta cells retained their endocrine function similar to human islets. The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell–cell interactions between insuloma and/or primary islet cells.     

Chronic high glucose induced INS‐1β cell mitochondrial dysfunction: A comparative mitochondrial proteome with SILAC

As glucose‐stimulated insulin secretion of pancreatic β cell is triggered and promoted by the metabolic messengers derived from mitochondria, mitochondria take a central stage in the normal function of β cells. β cells in diabetics were chronically exposed to hyperglycemia stimulation, which have been reported to exert deleterious effects on β‐cell mitochondria. However, the mechanism of the toxic effects of hyperglycemia on β‐cell mitochondria was not clear. In this study, we characterized the biological functional changes of rat INS‐1β cells and their mitochondria with chronic exposure to hyperglycemia and created a research model of chronic hyperglycemia‐induced dysfunctional β cells with damaged mitochondria. Then, SILAC‐based quantitative proteomic approach was used to compare the mitochondrial protein expression from high glucose treated INS‐1β cells and control cells. The expression of some mitochondrial proteins was found with significant changes. Functional classification revealed most of these proteins were related with oxidative phosphorylation, mitochondrial protein biosynthesis, substances metabolism, transport, and cell death. These results presented some useful information about the effect of glucotoxicity on the β‐cell mitochondria.     

Enhanced islet expansion by β‐cell proliferation in young diabetes‐prone rats fed a protective diet

Type 1 diabetes is inhibited in diabetes‐prone BioBreeding (BBdp) rats fed a low‐antigen hydrolyzed casein (HC) diet. In cereal‐fed BBdp rats, islet expansion is defective accompanied by a futile upregulation of islet neogenesis without increased islet mass, due to a subtle blockage in islet cell cycle. We hypothesized that islet growth is enhanced before insulitis in HC‐fed young BBdp rats and that islet neogenesis could be stimulated by a trophic factor, islet neogenesis‐associated protein (INGAP). β‐Cell homeostasis was analyzed using immunohistochemistry, morphometry, laser capture microdissection and RT‐PCR in BBdp rats fed HC or cereal diets. β‐cell proliferation in small and medium islets, and the number and area fraction of medium and large islets were increased in HC‐fed animals. In situ islet cell cycle analysis revealed an increased proportion of proliferating S + G2 cells in medium and large islets of 25–45 day HC‐fed rats. Expression of the cell cycle inhibitor, p16^INK4a correlated with islet size and the percentage of p16^INK4a+ β‐cells increased in HC‐fed BBdp rats, likely reflecting an increase in large islet area fraction. In HC‐fed rats, extra‐islet insulin⁺ clusters (EIC), insulin⁺ duct cells, large islet area fraction, and β‐cell mass were increased. Neurogenin‐3 and Pdx‐1, markers of β‐cell progenitors, were increased in EIC of weanling HC‐fed rats. Daily injection of INGAP (30–45 days) increased the number of small islets, total islets, and insulin⁺ cells in small ducts. Thus, in BBdp rats fed a protective HC diet, β‐cell expansion is enhanced through increased β‐cell proliferation and stimulation of islet neogenesis. J. Cell. Physiol. 224: 501–508, 2010. © 2010 Wiley‐Liss, Inc.     

Immunolocalization of Glucagon and Insulin Cells in Endocrine Pancreas and Cyclic Disparity of Plasma Glucose in the Lizard Psammophilus dorsalis

Reptiles are an interesting group to study the physiology of reproduction. As information on mobilisation of glucose and associated endocrine alterations in the pancreas are not known, a study on the lizard Psammophilus dorsalis was undertaken using immunohistochemistry. Glucagon-immunoreactive (IR) and insulin-IR cells never formed islets instead spread throughout the pancreas in P. dorsalis. Glucagon-IR cells appeared to release the secretory granules from all over the surface, insulin-IR cells are unidirectional in their secretion. Both cell types often have no direct contact with capillaries. The morphometric analysis showed that the glucagon-IR cells were the most abundant amounting approximately 63 % as against insulin-IR cells, amounting approximately to 37 % of the endocrine pancreatic mass in the present study. On an average P. dorsalis exhibited 288.3 ± 25.28 mg% of plasma glucose, the abdominal fat underwent clearance as there was energy demand during reproductive and recrudescent periods. Glycogen granules were evident in liver and this was inversely related with plasma glucose.     

Role and impact of the extracellular matrix on integrin‐mediated pancreatic β‐cell functions

Understanding the organisation and role of the extracellular matrix (ECM) in islets of Langerhans is critical for maintaining pancreatic β‐cells, and to recognise and revert the physiopathology of diabetes. Indeed, integrin‐mediated adhesion signalling in response to the pancreatic ECM plays crucial roles in β‐cell survival and insulin secretion, two major functions, which are affected in diabetes. Here, we would like to present an update on the major components of the pancreatic ECM, their role during integrin‐mediated cell‐matrix adhesions and how they are affected during diabetes. To treat diabetes, a promising approach consists in replacing β‐cells by transplantation. However, efficiency is low, because β‐cells suffer of anoikis, due to enzymatic digestion of the pancreatic ECM, which affects the survival of insulin‐secreting β‐cells. The strategy of adding ECM components during transplantation, to reproduce the pancreatic microenvironment, is a challenging task, as many of the regulatory mechanisms that control ECM deposition and turnover are not sufficiently understood. A better comprehension of the impact of the ECM on the adhesion and integrin‐dependent signalling in β‐cells is primordial to improve the healthy state of islets to prevent the onset of diabetes as well as for enhancing the efficiency of the islet transplantation therapy.     

Generation of glucose-responsive functional islets with a three-dimensional structure from mouse fetal pancreatic cells and iPS cells in vitro

Islets of Langerhans are a pancreatic endocrine compartment consisting of insulin-producing β cells together with several other hormone-producing cells. While some insulin-producing cells or immature pancreatic cells have been generated in vitro from ES and iPS cells, islets with proper functions and a three-dimensional (3D) structure have never been successfully produced. To test whether islets can be formed in vitro, we first examined the potential of mouse fetal pancreatic cells. We found that E16.5 pancreatic cells, just before forming islets, were able to develop cell aggregates consisting of β cells surrounded by glucagon-producing α cells, a structure similar to murine adult islets. Moreover, the transplantation of these cells improved blood glucose levels in hyperglycemic mice. These results indicate that functional islets are formed in vitro from fetal pancreatic cells at a specific developmental stage. By adopting these culture conditions to the differentiation of mouse iPS cells, we developed a two-step system to generate islets, i.e. immature pancreatic cells were first produced from iPS cells, and then transferred to culture conditions that allowed the formation of islets from fetal pancreatic cells. The islets exhibited distinct 3D structural features similar to adult pancreatic islets and secreted insulin in response to glucose concentrations. Transplantation of the islets improved blood glucose levels in hyperglycemic mice. In conclusion, the two-step culture system allows the generation of functional islets with a 3D structure from iPS cells.     

Deletion of pancreatic β‐cell adenosine kinase improves glucose homeostasis in young mice and ameliorates streptozotocin‐induced hyperglycaemia

Severe reduction in the β‐cell number (collectively known as the β‐cell mass) contributes to the development of both type 1 and type 2 diabetes. Recent pharmacological studies have suggested that increased pancreatic β‐cell proliferation could be due to specific inhibition of adenosine kinase (ADK). However, genetic evidence for the function of pancreatic β‐cell ADK under physiological conditions or in a pathological context is still lacking. In this study, we crossed mice carrying LoxP‐flanked Adk gene with Ins2‐Cre mice to acquire pancreatic β ‐cell ADK deficiency (Ins2‐Cre^±Adk^fl/fl) mice. Our results revealed that Ins2‐Cre^+/‐Adk^fl/fl mice showed improved glucose metabolism and β‐cell mass in younger mice, but showed normal activity in adult mice. Moreover, Ins2‐Cre^±Adk^fl/fl mice were more resistant to streptozotocin (STZ) induced hyperglycaemia and pancreatic β‐cell damage in adult mice. In conclusion, we found that ADK negatively regulates β‐cell replication in young mice as well as under pathological conditions, such as STZ induced pancreatic β‐cell damage. Our study provided genetic evidence that specific inhibition of pancreatic β‐cell ADK has potential for anti‐diabetic therapy.     

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.     

N‐cadherin is dispensable for pancreas development but required for β‐cell granule turnover

The cadherin family of cell adhesion molecules mediates adhesive interactions that are required for the formation and maintenance of tissues. Previously, we demonstrated that N‐cadherin, which is required for numerous morphogenetic processes, is expressed in the pancreatic epithelium at E9.5, but later becomes restricted to endocrine aggregates in mice. To study the role of N‐cadherin during pancreas formation and function we generated a tissue‐specific knockout of N‐cadherin in the early pancreatic epithelium by inter‐crossing N‐cadherin‐floxed mice with Pdx1Cre mice. Analysis of pancreas‐specific ablation of N‐cadherin demonstrates that N‐cadherin is dispensable for pancreatic development, but required for β‐cell granule turnover. The number of insulin secretory granules is significantly reduced in N‐cadherin‐deficient β‐cells, and as a consequence insulin secretion is decreased. genesis 48:374–381, 2010. © 2010 Wiley‐Liss, Inc.     

Gimmicks of gamma‐aminobutyric acid (GABA) in pancreatic β‐cell regeneration through transdifferentiation of pancreatic α‐ to β‐cells

In vivo regeneration of lost or dysfunctional islet β cells can fulfill the promise of improved therapy for diabetic patients. To achieve this, many mitogenic factors have been attempted, including gamma‐aminobutyric acid (GABA). GABA remarkably affects pancreatic islet cells’ (α cells and β cells) function through paracrine and/or autocrine binding to its membrane receptors on these cells. GABA has also been studied for promoting the transformation of α cells to β cells. Nonetheless, the gimmickry of GABA‐induced α‐cell transformation to β cells has two different perspectives. On the one hand, GABA was found to induce α‐cell transformation to β cells in vivo and insulin‐secreting β‐like cells in vitro. On the other hand, GABA treatment showed that it has no α‐ to β‐cell transformation response. Here, we will summarize the physiological effects of GABA on pancreatic islet β cells with an emphasis on its regenerative effects for transdifferentiation of islet α cells to β cells. We will also critically discuss the controversial results about GABA‐mediated transdifferentiation of α cells to β cells.     

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox‐sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin‐secreting Rin‐m5f β‐cells and its role in autocrine and paracrine MP‐mediated cell crosstalk under conditions of oxidative stress. MP from aPC‐treated primary endothelial (EC) or β‐cells were applied to H₂O₂‐treated Rin‐m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26‐stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR‐1 expression in EC and to a lesser extent in β‐cells. MP from aPC‐treated EC (eM_aPC) exhibited high EPCR and annexin A1 content, protected β‐cells, restored insulin secretion and were captured by 80% of β cells in a phosphatidylserine and ANXA1‐dependent mechanism. eMP activated EPCR/PAR‐1 and ANXA1/FPR2‐dependent pathways and up‐regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H₂O₂‐treated rat islets with increased viability (62% versus 48% H₂O₂), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets.     

Pancreatic β-cell regeneration: From molecular mechanisms to therapy

Pancreatic β cells are a type of cells that are present in the islets of Langerhans. These cells are highly specialized for the secretion of insulin in response to low increasing of blood glucose levels. Hence, pancreatic β cells could contribute to maintaining systemic glucose homeostasis. Increasing evidence has revealed that a variety of internal (ie, genetic and epigenetic factors) and external factors (ie, radical-oxidative stress) are involved in the protection and/or regeneration of pancreatic β cells. The pathways regulating β-cell replication have been intensely investigated. Glucose has an important role in cell cycle entry of quiescent β cells, which exerts its effect via glucose metabolism and unfolded proteins. A variety of growth factors, hormones, and signaling pathways (ie, calcium-calcineurin nuclear factor of activated T cells) are others factors that could affect β-cell replication under different conditions. Therefore, a greater understanding of the underlying pathways involved in the regeneration and protection of pancreatic β cells could lead to finding and developing new therapeutic approaches. Utilization of stem cells and various phytochemical agents have provided new aspects for preventing β-cell degeneration and stimulating the endogenous regeneration of islets. Thus, these therapeutic platforms could be used as potential therapies in the treatment of insulin-dependent diabetes mellitus. Here, we summarized the various mechanisms involved in pancreatic β-cell regeneration. Moreover, we highlighted different therapeutic approaches which could be used for the regeneration of pancreatic β cells.