Beta-cell Proliferation Rate Research Articles

Irisin administration restores beta-cell functional mass in a mouse model of type 2 diabetes

Aim: Irisin is a hormone secreted by skeletal muscle able to improve metabolic homeostasis. Serum irisin levels are reduced in type 2 diabetes (T2D), while exogenous irisin administration improves glycemic control in diabetic mice. We have previously demonstrated that irisin promotes beta-cell survival and function both in vitro and in vivo in healthy wild type mice. We have also demonstrated that irisin restores the defective glucose-stimulated insulin secretion (GSIS) and reduces apoptosis in human pancreatic islets from patients with T2D. Nevertheless, the beta-cellular effects of in vivo irisin administration to T2D mice are still unknown. Methods: C57Bl/6 mice (n = 8) were fed a high-fat diet (HFD, 60% of energy deriving from fat) for 10 weeks and then intraperitoneally injected with streptozotocin (STZ, 100 mg/kg) to induce diabetes. Four standard diet (SD)-fed mice were used as control. HFD/STZ mice were treated with 0.5 μg/g irisin (n = 4) or vehicle (n = 4), for 14 days. Fasting glycemia, insulinemia, body weight, glucose tolerance, and pancreatic islet function were assessed. Pancreatic islet architecture was also evaluated through immunofluorescence analyses. Results: Compared to SD mice, HFD/STZ mice showed higher fasting glycemia and body weight, glucose intolerance, and reduced GSIS; in addition, HFD/STZ mice showed reduced islet volume (-78%), beta-cell area (-35%), and insulin content (-60%), and increased alpha-cell area (+54%). Irisin administration significantly restored glycemia (-31%), body weight (-13%), glucose tolerance (-27%), GSIS (+23%), islet volume (+61%), beta-cell area (+34%) and alpha-cell area (-49%), and insulin content (+36%). Of note, irisin induced a 9-fold increase in beta-cell proliferation rate. Conclusions: These results show that irisin improves glycemic homeostasis and restores the functional beta-cell mass when administered in vivo to diabetic mice, probably by promoting beta-cell proliferation.

\u03b2-Arrestin-1 is required for adaptive \u03b2-cell mass expansion during obesity

Obesity is the key driver of peripheral insulin resistance, one of the key features of type 2 diabetes (T2D). In insulin-resistant individuals, the expansion of beta-cell mass is able to delay or even prevent the onset of overt T2D. Here, we report that beta-arrestin-1 (barr1), an intracellular protein known to regulate signaling through G protein-coupled receptors, is essential for beta-cell replication and function in insulin-resistant mice maintained on an obesogenic diet. Specifically, insulin-resistant beta-cell-specific barr1 knockout mice display marked reductions in beta-cell mass and the rate of beta-cell proliferation, associated with pronounced impairments in glucose homeostasis. Mechanistic studies suggest that the observed metabolic deficits are due to reduced Pdx1 expression levels caused by beta-cell barr1 deficiency. These findings indicate that strategies aimed at enhancing barr1 activity and/or expression in beta-cells may prove useful to restore proper glucose homeostasis in T2D.

38-OR: Beta-Arrestin-1 Regulates Pancreatic Beta-Cell Function and Plays a Key Role in Maintaining Whole Body Glucose Homeostasis

Beta-arrestin-1 and -2 (barr1 and barr2, respectively) are major GPCR-associated signaling molecules that are widely expressed throughout the body. Studies with whole body barr1 and barr2 KO mice have shown that beta-arrestins play important roles in key metabolic functions. In this study, we inactivated the barr1 gene in a conditional fashion specifically in beta-cells of adult mice (beta-barr1-KO mice). In vivo studies showed that beta-barr1-KO mice exhibited a pronounced impairment in glucose tolerance and glucose-stimulated insulin secretion (GSIS), when mice were maintained on high-fat diet (HFD). Studies with isolated perifused pancreatic islets prepared from HFD beta-barr1-KO mice confirmed that barr1 deletion in beta-cells led to a significant decrease in GSIS. Interestingly, HFD beta-barr1-KO mice displayed pronounced reductions in beta-cell mass and the rate of beta-cell proliferation, suggesting that barr1 is required for the expansion of beta-cell mass that occurs during HFD feeding. To test whether enhanced barr1 signaling in beta-cells might ameliorate the metabolic deficits associated with the consumption of a HFD, we also generated transgenic mice over-expressing barr1 in beta-cells (RIPII-barr1 mice). These transgenic mice displayed metabolic phenotypes that were opposite to those observed with the beta-barr1-KO mice, including improved glucose tolerance and GSIS in vivo as well as increased beta-cell mass. This is the first study demonstrating that barr1 is critical for the proper function of pancreatic beta-cells and for maintaining whole body glucose homeostasis. Identification of the cellular mechanisms through which barr1 signaling promotes beta-cell function and proliferation may lead to the development of novel drugs useful for the treatment of type 2 diabetes. Disclosure L.F. Barella: None. M. Rossi: None. S. Pydi: None. S. Jain: None. L. Zhu: None. W. Chen: None. J. Wess: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases

Oligofructose as an adjunct in treatment of diabetes in NOD mice.

In type 1 diabetes, restoration of normoglycemia can be achieved if the autoimmune attack on beta cells ceases and insulin requirement is met by the residual beta cells. We hypothesize that an adjunctive therapy that reduces insulin demand by increasing insulin sensitivity will improve the efficacy of an immunotherapy in reversing diabetes. We tested the gut microbiota-modulating prebiotic, oligofructose (OFS), as the adjunctive therapy. We treated non-obese diabetic mice with an immunotherapy, monoclonal anti-CD3 antibody (aCD3), with or without concurrent dietary supplement of OFS. After 8 weeks of OFS supplement, the group that received both aCD3 and OFS (aCD3 + OFS) had a higher diabetes remission rate than the group that received aCD3 alone. The aCD3 + OFS group had higher insulin sensitivity accompanied by reduced lymphocytic infiltrate into the pancreatic islets, higher beta-cell proliferation rate, higher pancreatic insulin content, and secreted more insulin in response to glucose. The addition of OFS also caused a change in gut microbiota, with a higher level of Bifidobacterium and lower Clostridium leptum. Hence, our results suggest that OFS can potentially be an effective therapeutic adjunct in the treatment of type 1 diabetes by improving insulin sensitivity and beta-cell function, leading to improved glycemic control.

The subcellular distribution of cyclin-D1 and cyclin-D3 within human islet cells varies according to the status of the pancreas donor.

In humans, the rate of beta cell proliferation declines rapidly during the postnatal period and remains low throughout adult life. Recent studies suggest that this may reflect the distribution of cell cycle regulators which, unusually, are located in the cytosolic compartment of beta cells in islets isolated from adults. In the present work, we examined whether the localisation of cyclin-D molecules is also cytosolic in the islet cells of pancreatic samples studied in situ. Immunohistochemical approaches were employed to examine the subcellular localisation of cyclin-D1, -D2 and -D3 in human pancreatic samples recovered either from heart-beating donors or post mortem. Immunofluorescence methods were used to reveal the cellular localisation of cyclin-D1 and -D3. The distribution of cyclin-D2 was invariably cytosolic in islet cells, whereas the localisation of cyclin-D1 and -D3 varied according to the status of the donor. In pancreatic sections from heart-beating donors these molecules were primarily nuclear. By contrast, in samples collected post mortem, they were mainly cytosolic. Cyclin-D1 was detected only in beta cells whereas cyclin-D3 was detected in both alpha and beta cells. The proportion of donors who were immunopositive for cyclin-D1 declined from 71% in controls to 30% in those with type 1 diabetes. Cyclin-D3 was present in the islets of the majority of donors in both groups. The subcellular localisation of cyclin-D molecules varies according to the status of the donor. Both cyclin-D1 and -D3 can be found in the nuclei of human islet cells in situ.

Mouse Insulin Cells Expressing an Inducible RIPCre Transgene Are Functionally Impaired

We used cre-lox technology to test whether the inducible expression of Cre minimize the deleterious effect of the enzyme on beta cell function. We studied mice in which Cre is linked to a modified estrogen receptor (ER), and its expression is controlled by the rat insulin promoter (RIP). Following the injection of tamoxifen (TM), CreER- migrates to the nucleus and promotes the appearance of a reporter protein, enhanced yellow fluorescent protein (EYFP), in cells. Immunocytochemical analysis indicated that 46.6 ± 2.1% insulin cells of adult RIPCreER- EYFP expressed EYFP. RIPCreER-EYFP (+TM) mice were normoglycemic throughout the study, and their glucose tolerance test results were similar to control CD-1 mice. However, an extended exposure to reagents that stimulate insulin synthesis was detrimental to the survival of IN+EYFP+cells. The administration of an inhibitor of the enzyme dipeptidyl-peptidase (DPP4i), which prevents the cleavage of glucagon-like peptide (GLP-1), to adult RIPCreER-EYFP mice lead to a decrease in the percentage of IN+EYFP+ to 17.5 ± 1.73 and a significant increase in apoptotic cells in islets. Similarly, a 2-week administration of the GLP-1 analog exendin 4 (ex-4) induced an almost complete ablation of IN+ expressing a different reporter protein and a significant decrease in the beta cell mass and rate of beta cell proliferation. Since normal beta cells do not die when induced to increase insulin synthesis, our observations indicate that insulin cells expressing an inducible RIPCre transgene are functionally deficient. Studies employing these mice should carefully consider the pitfalls of the Cre-Lox technique.

The dark side of islet vasculature

Pancreatic islets are highly vascularised and contain about five times more capillaries than exocrine pancreatic tissue [1, 2]. In addition, the islets receive 5–15% of the entire pancreatic blood supply, even though they represent only 1–2% of the pancreatic mass [3]. Moreover, as determined by beta cellor pancreas-specific deletion of vascular endothelial growth factor-A (VEGF-A), the islet microvasculature is required for normal insulin release and physiological blood glucose levels [4, 5], indicating that a dense islet vasculature is needed for proper islet function. In addition, VEGF-A is necessary for proper islet revascularisation following islet transplantation [5, 6]. Interestingly, Carlsson and colleagues recently suggested that there are two populations of islets within a healthy rodent pancreas: (1) a large islet population that is well bloodperfused and oxygenated, contains a dense vascular network and has a high beta cell proliferation rate and strong secretory function, and (2) a small population of islets representing about 20–25% of all islets within a rat pancreas that are not well perfused or well oxygenated, contain only very few proliferating beta cells and have a low secretory function [7, 8]. The authors employ fluorescent microspheres that they inject into the arterial blood stream to discriminate between microsphere-containing, well-perfused islets and non-perfused islets harbouring no microspheres [9]. Importantly, the latter islets can be recruited on demand into the well-perfused and functional islet population, as evidenced by the conversion of all islets into well-perfused ones following partial pancreatectomy [7]. The correlation between vascular density and islet function can be partially explained by the fact that islet beta cells metabolise glucose almost exclusively via aerobic glycolysis, making them heavily dependent on a high and continuous supply of oxygen [10]. Moreover, the blood vessels within the islets also contribute to islet function by supplying paracrine factors, such as components of the vascular basement membrane or hepatocyte growth factor [11, 12]. In their present study, reported in this issue ofDiabetologia [13], Ullsten and colleagues report that highly perfused islets are more susceptible to cell death induced by the inflammatory cytokines TNFα, IFNγ and IL-1β or hypoxia in vitro. In addition, despite being better vascularised and oxygenated in the host, the highly perfused, microparticle-containing islets were found to be more prone to cell death and fibrosis. Therefore, the findings reveal for the first time the other side of the vascularisation coin: strongly vascularised islets seem to be more susceptible to cell death following islet transplantation. This finding could be explained by the highly perfused islets being more accessible to inflammatory cytokines and immune cells. Alternatively, or additionally, these islets are metabolically more active and therefore more prone to the induction of cell death. A high glycolytic flux in islets that are more metabolically active, in combination with the relatively limited capability of islet cells to remove reactive oxygen P. In’t Veld (*) Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium e-mail: intveld@vub.ac.be

Glucagon-like peptide-1 receptor agonist treatment reduces beta cell mass in normoglycaemic mice

Incretin-based therapies improve glycaemic control in patients with type 2 diabetes. In animal models of diabetes, glucagon-like peptide-1 receptor agonists (GLP-1RAs) increase beta cell mass. GLP-1RAs are also evaluated in non-diabetic individuals with obesity and cardiovascular disease. However, their effect on beta cell mass in normoglycaemic conditions is not clear. Here, we investigate the effects of the GLP-1RA liraglutide on beta cell mass and function in normoglycaemic mice. C57BL/6J mice were treated with the GLP-1RA liraglutide or PBS and fed a control or high-fat diet (HFD) for 1 or 6 weeks. Glucose and insulin tolerance tests were performed after 6 weeks. BrdU was given to label proliferating cells 1 week before the animals were killed. The pancreas was taken for either histology or islet isolation followed by a glucose-induced insulin-secretion test. Treatment with liraglutide for 6 weeks led to increased insulin sensitivity and attenuation of HFD-induced insulin resistance. A reduction in beta cell mass was observed in liraglutide-treated control and HFD-fed mice at 6 weeks, and was associated with a lower beta cell proliferation rate after 1 week of treatment. A similar reduction in alpha cell mass occurred, resulting in an unchanged alpha to beta cell ratio. In contrast, acinar cell proliferation was increased. Finally, islets isolated from liraglutide-treated control mice had enhanced glucose-induced insulin secretion. Our data show that GLP-1RA treatment in normoglycaemic mice leads to increases in insulin sensitivity and beta cell function that are associated with reduced beta cell mass to maintain normoglycaemia.

Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin.

In rodents and humans, the rate of beta cell proliferation declines rapidly after birth; formation of the islets of Langerhans begins perinatally and continues after birth. Here, we tested the hypothesis that increasing levels of E-cadherin during islet formation mediate the decline in beta cell proliferation rate by contributing to a reduction of nuclear β-catenin and D-cyclins. We examined E-cadherin, nuclear β-catenin, and D-cyclin levels, as well as cell proliferation during in vitro and in vivo formation of islet cell aggregates, using β-TC6 cells and transgenic mice with green fluorescent protein (GFP)-labelled beta cells, respectively. We tested the role of E-cadherin using antisense-mediated reductions of E-cadherin in β-TC6 cells, and mice segregating for a beta cell-specific E-cadherin knockout (Ecad [also known as Cdh1] βKO). In vitro, pseudo-islets of β-TC6 cells displayed increased E-cadherin but decreased nuclear β-catenin and cyclin D2, and reduced rates of cell proliferation, compared with monolayers. Antisense knockdown of E-cadherin increased cell proliferation and levels of cyclins D1 and D2. After birth, beta cells showed increased levels of E-cadherin, but decreased levels of D-cyclin, whereas islets of Ecad βKO mice showed increased levels of D-cyclins and nuclear β-catenin, as well as increased beta cell proliferation. These islets were significantly larger than those of control mice and displayed reduced levels of connexin 36. These changes correlated with reduced insulin response to ambient glucose, both in vitro and in vivo. The findings support our hypothesis by indicating an important role of E-cadherin in the control of beta cell mass and function.

Superior beta cell proliferation, function and gene expression in a subpopulation of rat islets identified by high blood perfusion

The blood perfusion of individual pancreatic islets is highly variable, with a subgroup of islets having high perfusion and blood vessels responsive to further blood flow increase induced by glucose. This study tested the hypothesis that there is heterogeneity between islets with regard to beta cell proliferation, function and gene expression based on differences in their blood perfusion. Fluorescent microspheres were injected into the ascending aorta, and then microsphere-containing and non-microsphere-containing pancreatic islets were isolated for investigation. By this procedure, the 5% of islets with the greatest blood perfusion were identified for study. Islet endothelial cells were isolated separately to investigate the role of improved vascular support in the observed differences. The vascular network was found to be more dense and tortuous in microsphere-containing than other islets. The most highly blood-perfused islets also had a higher rate of beta cell proliferation, superior beta cell function and a markedly different gene expression from other islets. Cultured islets exposed to islet endothelial cell products had a similarly increased beta cell proliferation rate, yet significantly fewer changes in gene expression than observed in the most highly blood-perfused islets. A novel heterogeneity between islets was observed, with superior beta cell proliferation, function and gene expression in a subpopulation of islets identified by high blood perfusion. In contrast with a previously described population of low-oxygenated, sleeping islets, which are recruited into functionality when needed, the presently described heterogeneity is shown to remain in vitro after islet isolation.

Chronic treatment with a glucokinase activator delays the onset of hyperglycaemia and preserves beta cell mass in the Zucker diabetic fatty rat

Glucokinase activators (GKAs) are currently being developed as new therapies for type 2 diabetes and have been shown to enhance beta cell survival and proliferation in vitro. Here, we report the effects of chronic GKA treatment on the development of hyperglycaemia and beta cell loss in the male Zucker diabetic fatty (ZDF) rat, a model of type 2 diabetes with severe obesity. Cell protection by GKA was studied in MIN6 and INS-1 cells exposed to hydrogen peroxide. Glucose homeostasis and beta cell mass were evaluated in ZDF rats dosed for 41days with Cpd-C (a GKA) or glipizide (a sulfonylurea) as food admixtures at doses of approximately 3 and 10mgkg(-1)day(-1). Incubation of MIN6 and INS-1 832/3 insulinoma cell cultures with GKA significantly reduced cell death and impairment of intracellular NADH production caused by exposure to hydrogen peroxide. Progression from prediabetes (normoglycaemia and hyperinsulinaemia) to overt diabetes (hyperglycaemia and hypoinsulinaemia) was significantly delayed in male ZDF rats by in-feed treatment with Cpd-C, but not glipizide. Glucose tolerance, tested in the fifth week of treatment, was also significantly improved by Cpd-C, as was pancreatic insulin content and beta cell area. In a limited immunohistochemical analysis, Cpd-C modestly and significantly enhanced the rate of beta cell proliferation, but not rates of beta cell apoptosis relative to untreated ZDF rats. These findings suggest that chronic activation of glucokinase preserves beta cell mass and delays disease in the ZDF rat, a model of insulin resistance and progressive beta cell failure.

A mouse model for sleeve gastrectomy: Applications for diabetes research

Discovery of enhanced glucose tolerance following bariatric surgery has sparked renewed interest in the investigation of unchartered underlying pathways of glucose homeostasis. Delineation of this pathway may ultimately be the first step in the creation of a novel therapy for type II diabetes. Nevertheless, the technical complexity and formidable nature of these surgeries coupled with the fragile nature of small rodents has made the creation of a mouse model to study these effects incredibly challenging. We have created a simplified sleeve gastrectomy mouse model to study the effects of bariatric surgery on glucose tolerance and beta cell proliferation. Nineteen mice were randomized to undergo either sleeve gastrectomy (SG) (9) or sham operation (SH) (10). Weight and serum glucose were measured three times weekly and serum insulin measurements and pancreatic harvest were performed at the time of sacrifice. Five mice from each group were sacrificed after one week and the remainder sacrificed after one month. Survival of mice was 100% for both groups. The SG group demonstrated an initial drop in weight and serum glucose as compared to SH, which normalized by one month following surgery. Serum insulin levels and rate of beta cell proliferation were similar in both groups after one week and one month. The simplified sleeve gastrectomy is a technically straightforward, low-mortality technique for creating a bariatric mouse model which most faithfully replicates bariatric surgery performed in humans. This model can be a valuable tool to investigate the glucose tolerance and beta cell effects of bariatric surgery.

Blood Glucose Levels Regulate Pancreatic β-Cell Proliferation during Experimentally-Induced and Spontaneous Autoimmune Diabetes in Mice

BackgroundType 1 diabetes mellitus is caused by immune-mediated destruction of pancreatic β-cells leading to insulin deficiency, impaired intermediary metabolism, and elevated blood glucose concentrations. While at autoimmune diabetes onset a limited number of β-cells persist, the cells' regenerative potential and its regulation have remained largely unexplored. Using two mouse autoimmune diabetes models, this study examined the proliferation of pancreatic islet ß-cells and other endocrine and non-endocrine subsets, and the factors regulating that proliferation.Methodology and Principal FindingsWe adapted multi-parameter flow cytometry techniques (including DNA-content measurements and 5′-bromo-2′-deoxyuridine [BrdU] incorporation) to study pancreatic islet single cell suspensions. These studies demonstrate that β-cell proliferation rapidly increases at diabetes onset, and that this proliferation is closely correlated with the diabetic animals' elevated blood glucose levels. For instance, we show that when normoglycemia is restored by exogenous insulin or islet transplantation, the β-cell proliferation rate returns towards low levels found in control animals, yet surges when hyperglycemia recurs. In contrast, other-than-ß endocrine islet cells did not exhibit the same glucose-dependent proliferative responses. Rather, disease-associated alterations of BrdU-incorporation rates of δ-cells (minor decrease), and non-endocrine islet cells (slight increase) were not affected by blood glucose levels, or were inversely related to glycemia control after diabetes onset (α-cells).ConclusionWe conclude that murine β-cells' ability to proliferate in response to metabolic need (i.e. rising blood glucose concentrations) is remarkably well preserved during severe, chronic β-cell autoimmunity. These data suggest that timely control of the destructive immune response after disease manifestation could allow spontaneous regeneration of sufficient β-cell mass to restore normal glucose homeostasis.

Taking a closer look at the pancreas

Our current understanding of how pancreatic beta cell mass fluctuates during the course of diabetes development is largely based on indirect evidence. Various approaches based on in vivo metabolic testing have been described, each reflecting specific aspects of beta cell function [1]. These measurements are correlated with actual beta cell mass only to a certain extent, and were developed to allow estimates of mass following surgery, therapy or transplantation. Direct visualisation of beta cell mass by means of novel imaging technologies is anticipated to provide more detailed insight into the true kinetics of key processes such as physiological beta cell turnover, islet graft loss and the pathophysiology of type 1 and type 2 diabetes. Likewise, a substantial part of what we know about the autoimmune component of type 1 diabetes has historically been derived from ex vivo, cross-sectional studies in rodents. Improved intravital microscopy techniques could also shed new light on the in situ behaviour of immune cells—after all, seeing is believing. In this issue of Diabetologia, Holmberg and Ahlgren review recent progress in the area of optical imaging technologies that is specifically applicable to monitoring the pancreas [2]. Although most investigators may be familiar with conventional non-optical imaging modalities such as magnetic resonance imaging (MRI) and positron emission tomography (PET), and the ongoing endeavour to optimise these technologies for non-invasive monitoring of beta cell mass in patients, there are a number of lesser known optical strategies that are likely to contribute essential information in the near future. In an effort to improve our understanding of emerging technologies such as optical projection tomography (OPT), bioluminescence imaging, confocal microscopy and optical coherence tomography, the authors outline the characteristics, benefits and limitations of each technique. Perhaps one of the most elegant examples of this new generation of imaging modalities is optical projection tomography, an approach that has recently been advanced by the authors to allow for detailed whole organ assessment and accurate quantification of beta cell mass ex vivo. The three-dimensional OPT image provided in their current review showcases the remarkable quantitative resolution that is characteristic of OPT, enabling us to observe individual islets and their position relative to each other. The authors conclude that, although OPTand the other technologies discussed are at present merely suitable for pre-clinical research, they could prove clinically relevant in the long run. So which questions need to be addressed by optical imaging? (see text box: Questions to be addressed by optical imaging). We and others have recently proposed a cyclical model for type 1 diabetes, describing the disease as a relapsing–remitting condition, based on our understanding of its key cellular constituents [3]. According to this hypothesis, the kinetics of beta cell degeneration are directed by the degree of epitope spreading, the rate of beta cell proliferation in response to autoimmune attack and the immune modulatory action of regulatory T cells (Tregs). As a result of the dominance of the autoreactive effector T cell responses, beta cell mass will gradually decrease in atrisk individuals, ultimately leading to disease manifestation once a critical threshold has been passed. Support for the existence of this delicate balance was obtained in the NOD mouse, still the most widely studied animal model of type 1 diabetes. Sensitive longitudinal detection of alterations in beta cell mass during diabetes development in these animals may put this theory to the test and may Diabetologia (2008) 51:2145–2147 DOI 10.1007/s00125-008-1181-y

Expanding human beta cells

To the Editor: An understanding of how beta cell number is regulated during normal life and regeneration is crucial not only from a cognitive point of view, but also forms a basis for the development of new strategies to treat or cure diabetic patients. Beta cell homeostasis is the balance between beta cell formation and death. Beta cells can be formed by neogenesis from non-beta cells (differentiation from progenitors, transdifferentiation from exocrine cells) or by the proliferation of beta cells already present in the pancreas. For a long time, it was thought that the rate of beta cell proliferation was extremely low and played a minor role in beta cell expansion occurring during adult life. This hypothesis was convincingly challenged by lineage-based approaches demonstrating that, during physiological adult life, newly formed beta cells are derived from pre-existing beta cells [1]. This important finding led to a large number of teams entering the field of beta cell proliferation, which produced major new information and a better understanding of this important topic [2]. It is thus now proposed that strategies that would enhance beta cell proliferation can be used to develop human endocrine cells in large quantities to be used for diabetes therapy in humans. However, it should be kept in mind that the vast majority of the recent data on beta cell proliferation are derived from rodent models. Thus, in my view, a major question is: is the recent information demonstrating the major role of beta cell proliferation in the rodent true for human beta cells? Although human and rodent beta cells share a large number of similarities, they are different in several respects. For example, it has long been known that beta cell function and sensitivity to destruction are not perfectly identical in rodents and humans [3, 4]. Moreover, islet cytoarchitecture—thought to have important functional implications— is also different [5]. Finally, there is debate over the proliferative potential of human beta cells. Some groups claim that human beta cells proliferate efficiently [6], while others argue that the proliferation of human adult beta cells is either extremely low or null [7]. In an article published in Diabetologia, Halban and colleagues compared beta cell proliferation in rodents and humans [8]. For this purpose, the authors sorted beta cells from adult rats and from 16 human donors, cultured the cells under different conditions, and analysed the effect of different exogenous growth factors on beta cell proliferation. For quantification of beta cell proliferation the cells were incubated with BrdU and then immunostained for BrdU and insulin or pancreatic and duodenal homeobox protein 1 (PDX1). Samples of cells were also stained with Ki-67, another marker used to label proliferating cells. A major finding was that rat beta cells proliferated under some of the different conditions tested, whereas human beta cells did not. This suggests that rat and human beta cells have different rates of proliferation. At this point, the question is: what is the exact rate of beta cell proliferation in humans, if above zero? One possible explanation for the findings of Halban and colleagues [8] is that human beta cells proliferate, but at a lower rate than rodent beta cells. However, given that not a Diabetologia (2008) 51:692–693 DOI 10.1007/s00125-007-0909-4

Rapamycin Impairs In Vivo Proliferation of Islet Beta-Cells

Progressive graft dysfunction is commonly observed in recipients of islet allografts treated with high doses of rapamycin. This study aimed at evaluating the effect of rapamycin on pancreatic islet cell proliferation in vivo. The murine pregnancy model was utilized, since a high rate of beta-cell proliferation occurs in a well-defined time frame. Rapamycin (0.2 mg/kg/day) was given to C57BL/6 mice for 5-7 days starting on day 7.5 of pregnancy. Cell proliferation was evaluated by detection of bromodeoxyuridine incorporation by immunohistochemistry. Pregnancy led to increased beta-cell proliferation and islet yield with skewing in islet size distribution as well as higher pancreatic insulin content, when compared to that of nonpregnant females. These effects of pregnancy on beta-cell proliferation and mass were significantly blunted by rapamycin treatment. Minimal effect of rapamycin was observed on islet function both in vivo and in vitro. Rapamycin treatment of islets in vitro resulted in reduced p70s6k phosphorylation, which was paralleled by increased ERK1/2 phosphorylation. Rapamycin treatment reduces the rate of beta-cell proliferation in vivo. This phenomenon may contribute to impair beta-cell renewal in transplanted patients and to the progressive dysfunction observed in islet graft recipients.

Role of oxidative stress in pancreatic beta-cell dysfunction.

Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic beta-cell dysfunction found in diabetes. Possibly caused by low levels of antioxidant enzyme expressions, pancreatic beta-cells are vulnerable to oxidative stress. When beta-cell-derived HIT-T15 cells or isolated rat islets were exposed to oxidative stress, insulin gene expression was markedly decreased. To investigate the significance of oxidative stress in the progression of pancreatic beta-cell dysfunction in type 2 diabetes, we evaluated the effects of antioxidants in diabetic C57BL/KsJ-db/db mice. According to an intraperitoneal glucose tolerance test, the treatment with antioxidants retained glucose-stimulated insulin secretion and moderately decreased blood glucose levels. Histological analyses of the pancreata revealed that the beta-cell mass was significantly larger in the mice treated with the antioxidants, and the antioxidant treatment suppressed apoptosis in beta-cells without changing the rate of beta-cell proliferation. The antioxidant treatment also preserved the amounts of insulin content and insulin mRNA, making the extent of insulin degranulation less evident. As possible mechanism underlying the phenomena, expression of pancreatic and duodenal homeobox factor-1 (also known as IDX-1/STF-1/IPF1), an important transcription factor for the insulin gene, was more clearly visible in the nuclei of islet cells after the antioxidant treatment. Under diabetic conditions, JNK is activated by oxidative stress and involved in the suppression of insulin gene expression. This JNK effect appears to be mediated in part by nucleocytoplasmic translocation of PDX-1, which is also downstream of JNK activation. Taken together, oxidative stress and consequent activation of the JNK pathway are involved in progression of beta-cell dysfunction found in diabetes. Antioxidants may serve as a novel mechanism-based therapy for type 2 diabetes.

Specific and combined effects of insulin and glucose on functional pancreatic beta-cell mass in vivo in adult rats.

We investigated the specific and associated effects of insulin and glucose on beta-cell growth and function in adult rats. By combining simultaneous infusion either of glucose and/or insulin or glucose and diazoxide, three groups of rats were constituted: hyperglycemic-hyperinsulinemic rats (high glucose-high insulin), hyperglycemic-euinsulinemic rats (high glucose), and euglycemic-hyperinsulinemic rats (high insulin). All the infusions lasted 48 h. Control rats were infused with 0.9% NaCl (saline controls). In all groups, beta-cell mass was significantly increased, compared with controls (by 70% in high glucose-high insulin rats, 65% in high glucose rats, and 50% in high insulin rats). The stimulation of neogenesis was suggested by the high number of islets budding from pancreatic ducts in high glucose-high insulin and high glucose rats and by the presence of numerous clusters of few beta-cells within the exocrine pancreas in high insulin rats. beta-Cell hypertrophy was observed only in high glucose-high insulin rats. The rate of beta-cell proliferation was similar to that of controls in high glucose-high insulin rats after a 48-h glucose infusion, dropped dramatically in high insulin rats, and dropped to a lesser extent in high glucose rats. In high glucose-high insulin and high glucose rats, beta-cell mass increase was related to a higher beta-cell responsiveness to glucose in vitro as measured by islet perifusion studies, whereas in high insulin rats, no significant enhancement of glucose induced insulin secretion could be noticed. The data show that glucose and insulin may have specific stimulating effects on beta-cell growth and function in vivo in adult rats independently of the influence they exert each other on their respective plasma concentration.

Decreased beta-cell proliferation impairs the adaptation to pregnancy in rats malnourished during perinatal life.

We investigated the cellular mechanisms responsible for the inability of 8-month-old previously malnourished (PM) females to adapt their beta-cell mass during pregnancy. The evolution during pregnancy of beta-cell fraction, size and proliferation was studied. At day 21 of pregnancy beta-cell fraction increased less in PM than in control females, compared with their non-pregnant values. A slight beta-cell hypertrophy was observed during pregnancy in both groups. In control females, beta-cell 5-bromo-2'-deoxyuridine (BrdU) labelling index (LI) increased from 0.07+/-0.04% before pregnancy to 1.13+/-0.20% at day 12 and decreased thereafter to reach again basal levels at day 21. In PM females, beta-cell proliferation rate was decreased at day 12 (0.74+/-0.15%, P<0.05) but similar to controls at all other stages studied. Separate analysis of the head and tail parts of the pancreas in control animals revealed that the beta-cell fraction during pregnancy increased more in the head than in the tail; similarly, BrdU LI increased 20-fold in the head and 10-fold in the tail, compared with non-pregnant values. In PM females, no adaptation of beta-cell fraction could be observed in the head, where BrdU LI was decreased by half at day 12 of pregnancy. In PM females the lactogenic activity was twice that of controls at day 12 whereas all beta-cells expressed the prolactin receptor. In conclusion, perinatal malnutrition impairs subsequent adaptation to pregnancy by decreasing beta-cell proliferation in the head of the pancreas at a critical time during pregnancy.

Beta-cell glucotoxicity in the Psammomys obesus model of type 2 diabetes.

Deficient insulin secretion and relative hyperproinsulinemia are characteristic features of type 2 diabetes. The gerbil Psammomys obesus appears to be an ideal natural model of the human disease because it shows increased tendency to develop diet-induced diabetes, which is associated with moderate obesity. The disease is characterized by initial hyperinsulinemia, progressing to hypoinsulinemia associated with depleted pancreatic insulin stores and an increased proportion of insulin precursor molecules in the blood and islets. Although the proinsulin translational efficacy was found to be increased in hyperglycemic animals, insulin mRNA levels were not augmented and exhibited a gradual decrease with disease progression. The development of hyperglycemia was associated with a transient increase in beta-cell proliferative activity, as opposed to a prolonged increase in the rate of beta-cell death, culminating in disruption of islet architecture. The hypothesis that glucotoxicity is responsible in part for these in vivo changes was investigated in vitro in primary islet cultures. Islets from diabetes-prone P. obesus cultured at high glucose concentrations displayed changes in beta-cell function that mimic those observed in diabetic animals. These changes include deficient insulin secretion, depleted insulin content, an increased proportion of insulin precursor molecules, a progressive increase of DNA fragmentation, and a transient proliferative response. Furthermore, insulin mRNA was not increased by short-term exposure of P. obesus islets to elevated glucose in vitro. It is proposed that beta-cell glucotoxicity in P. obesus results from the inability of proinsulin biosynthesis to keep pace with chronic insulin hypersecretion. The resulting depletion of the insulin stores may be related to deficient glucose-regulated insulin gene transcription, possibly due to defective PDX-1 (pancreatic duodenal homeobox factor-1) expression in the adult P. obesus. An additional glucotoxic effect involves the loss of beta-cell mass in hyperglycemic P. obesus as a result of progressive beta-cell death without an adequate increase in the rate of beta-cell proliferation.