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After birth the endocrine pancreas continues its development, a complex process that involves both the maturation of islet cells and a marked expansion of their numbers. New beta cells are formed both by duplication of pre-existing cells and by new differentiation (neogenesis) across the first postnatal weeks, with the result of beta cells of different stages of maturation even after weaning. Improving our understanding of this period of beta cell expansion could provide valuable therapeutic insights.

Pancreatic duodenal homeobox-1 (Pdx1), a transcription factor required for pancreatic development and maintenance of β-cell function, was assessed for a possible role in postnatal β-cell formation from progenitors in the pancreatic ducts by selectively deleting Pdx1 from the ducts. Carbonic anhydrase II (CAII)(Cre);Pdx1(Fl) mice were euglycemic for the first 2 postnatal weeks but showed moderate hyperglycemia from 3 to 7 weeks of age. By 10 weeks, they had near-normal morning fed glucose levels but showed severely impaired glucose tolerance and insulin secretion. Yet the loss of Pdx1 did not result in decreased islet and β-cell mass at 4 and 10 weeks of age. Within the same pancreas, there was a mixed population of islets, with PDX1 and MAFA protein expression normal in some cells and severely diminished in others. Even at 10 weeks, islets expressed immaturity markers. Thus, we conclude that Pdx1 is not necessary for the postnatal formation of β-cells but is essential for their full maturation to glucose-responsive β-cells.

This commentary discusses the concept of β-cell dedifferentiation in diabetes, which is important but not well defined. A broad interpretation is that a state of differentiation has been lost, which means changes in gene expression as well as in structural and functional elements. Thus, a fully mature healthy β cell will have its unique differentiation characteristics, but maturing cells and old β cells will have different patterns of gene expression and might therefore be considered as dedifferentiated. The meaning of dedifferentiation is now being debated because β cells in the diabetic state lose components of their differentiated state, which results in severe dysfunction of insulin secretion. The major cause of this change is thought to be glucose toxicity (glucotoxicity) and that lowering glucose levels with treatment results in some restoration of function. An issue to be discussed is whether dedifferentiated β cells return to a multipotent precursor cell phenotype or whether they follow a different pathway of dedifferentiation.

Neonatal β cells do not secrete glucose-responsive insulin and are considered immature. We previously showed the transcription factor MAFA is key for the functional maturation of β cells, but the physiological regulators of this process are unknown. Here we show that postnatal rat β cells express thyroid hormone (TH) receptor isoforms and deiodinases in an age-dependent pattern as glucose responsiveness develops. In vivo neonatal triiodothyronine supplementation and TH inhibition, respectively, accelerated and delayed metabolic development. In vitro exposure of immature islets to triiodothyronine enhanced the expression of Mafa, the secretion of glucose-responsive insulin, and the proportion of responsive cells, all of which are effects that were abolished in the presence of dominant-negative Mafa. Using chromatin immunoprecipitation and electrophoretic mobility shift assay, we show that TH has a direct receptor-ligand interaction with the Mafa promoter and, using a luciferase reporter, that this interaction was functional. Thus, TH can be considered a physiological regulator of functional maturation of β cells via its induction of Mafa.

There is growing information about the heterogeneity of pancreatic β-cells and how it relates to insulin secretion. This study used the approach of flow cytometry to sort and analyze β-cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter. Three populations of β-cells with differing GFP brightness could be identified, which were classified as GFP-low, GFP-medium, and GFP-bright. The GFP-medium population comprised about 70% of the total. The GFP-low population had less insulin secretion as determined by the reverse hemolytic plaque assay and reduced insulin gene expression. Additionally, all three subpopulations of β-cells were found in mice of varying ages (embryonic d 15.5 and postnatal wk 1-9). The three populations from the youngest had larger cells (forward scatter) and less granularity (side scatter) than those from the adults. This approach opens up new ways to advance knowledge about β-cell heterogeneity.

For decades, investigators have made numerous attempts to generate human pancreatic β cell lines that could be used to advance β cell biology, facilitate drug discovery, and provide a pathway to β cell replacement therapy for the treatment of diabetes. In this issue of the JCI, Ravassard and colleagues report that this has finally been achieved successfully with a multistep process that led to the generation of cells, which they termed EndoC-βH1 cells, that secreted insulin in response to glucose challenge.

Objective: To evaluate the extent of pancreatic β-cell function in a large number of insulin-dependent diabetic patients with a disease duration of 50 years or longer (Medalists)., Research Design and Methods: Characterization of clinical and biochemical parameters and β-cell function of 411 Medalists with correlation with postmortem morphologic findings of 9 Medalists., Results: The Medalist cohort, with a mean ± SD disease duration and age of 56.2 ± 5.8 and 67.2 ± 7.5 years, respectively, has a clinical phenotype similar to type 1 diabetes (type 1 diabetes): mean ± SD onset at 11.0 ± 6.4 years, BMI at 26.0 ± 5.1 kg/m(2), insulin dose of 0.46 ± 0.2 u/kg, ∼94% positive for DR3 and/or DR4, and 29.5% positive for either IA2 or glutamic acid decarboxylase (GAD) autoantibodies. Random serum C-peptide levels showed that more than 67.4% of the participants had levels in the minimal (0.03-0.2 nmol/l) or sustained range (≥ 0.2 nmol/l). Parameters associated with higher random C-peptide were lower hemoglobin A1C, older age of onset, higher frequency of HLA DR3 genotype, and responsiveness to a mixed-meal tolerance test (MMTT). Over half of the Medalists with fasting C-peptide > 0.17 nmol/l responded in MMTT by a twofold or greater rise over the course of the test compared to fasting. Postmortem examination of pancreases from nine Medalists showed that all had insulin+ β-cells with some positive for TUNEL staining, indicating apoptosis., Conclusions: Demonstration of persistence and function of insulin-producing pancreatic cells suggests the possibility of a steady state of turnover in which stimuli to enhance endogenous β cells could be a viable therapeutic approach in a significant number of patients with type 1 diabetes, even for those with chronic duration.

We have previously found that cyclin A expression is markedly reduced in pancreatic beta-cells by cell-specific overexpression of repressor inducible cyclic AMP early repressor (ICER Igamma) in transgenic mice. Here we further examined regulatory effects of ICER Igamma on cyclin A gene expression using Min6 cells, an insulin-producing cell line. The cyclin A promoter luciferase assay showed that ICER Igamma directly repressed cyclin A gene transcription. In addition, upon ICER Igamma overexpression, cyclin A mRNA levels markedly decreased, thereby confirming an inhibitory effect of ICER Igamma on cyclin A expression. Suppression of cyclin A results in inhibition of BrdU incorporation. Under normal culture conditions endogenous cyclin A is abundant in these cells, whereas ICER is hardly detectable. However, serum starvation of Min6 cells induces ICER Igamma expression with a concomitant very low expression level of cyclin A. Cyclin A protein is not expressed unless the cells are in active DNA replication. These results indicate a potentially important anti-proliferative effect of ICER Igamma in pancreatic beta cells. Since ICER Igamma is greatly increased in diabetes as well as in FFA- or high glucose-treated islets, this effect may in part exacerbate diabetes by limiting beta-cell proliferation.

beta cell dysfunction is an important component of type 2 diabetes, but the molecular basis for this defect is poorly understood. The transcriptional coactivator PGC-1alpha mRNA and protein levels are significantly elevated in islets from multiple animal models of diabetes; adenovirus-mediated expression of PGC-1alpha to levels similar to those present in diabetic rodents produces a marked inhibition of glucose-stimulated insulin secretion from islets in culture and in live mice. This inhibition coincides with changes in metabolic gene expression associated with impaired beta cell function, including the induction of glucose-6-phosphatase and suppression of GLUT2, glucokinase, and glycerol-3-phosphate dehydrogenase. These changes result in blunting of the glucose-induced rise in cellular ATP levels and membrane electrical activity responsible for Ca(2+) influx and insulin exocytosis. These results strongly suggest that PGC-1alpha plays a key functional role in the beta cell and is involved in the pathogenesis of the diabetic phenotype.

Oxidative stress, which is found in pancreatic beta-cells in the diabetic state, suppresses insulin gene transcription and secretion, but the signaling pathways involved in the beta-cell dysfunction induced by oxidative stress remain unknown. In this study, subjecting rat islets to oxidative stress activates JNK, p38 MAPK, and protein kinase C, preceding the decrease of insulin gene expression. Adenovirus-mediated overexpression of dominant-negative type (DN) JNK, but not the p38 MAPK inhibitor SB203580 nor the protein kinase C inhibitor GF109203X, protected insulin gene expression and secretion from oxidative stress. Moreover, wild type JNK overexpression suppressed both insulin gene expression and secretion. These results were correlated with changes in the binding of the important transcription factor PDX-1 to the insulin promoter; adenoviral overexpression of DN-JNK preserved PDX-1 DNA binding activity in the face of oxidative stress, whereas wild type JNK overexpression decreased PDX-1 DNA binding activity. Furthermore, to examine whether suppression of the JNK pathway can protect beta-cells from the toxic effects of hyperglycemia, rat islets were infected with DN-JNK expressing adenovirus or control adenovirus and transplanted under renal capsules of streptozotocin-induced diabetic nude mice. In mice receiving DN-JNK overexpressing islets, insulin gene expression in islet grafts was preserved, and hyperglycemia was ameliorated compared with control mice. In conclusion, activation of JNK is involved in the reduction of insulin gene expression by oxidative stress, and suppression of the JNK pathway protects beta-cells from oxidative stress.

To test the hypothesis that c-Myc plays an important role in beta-cell growth and differentiation, we generated transgenic mice overexpressing c-Myc in beta-cells under control of the rat insulin II promoter. F(1) transgenic mice from two founders developed neonatal diabetes (associated with reduced plasma insulin levels) and died of hyperglycemia 3 days after birth. In pancreata of transgenic mice, marked hyperplasia of cells with an altered phenotype and amorphous islet organization was displayed: islet volume was increased threefold versus wild-type littermates. Apoptotic nuclei were increased fourfold in transgenic versus wild-type mice, suggesting an increased turnover of beta-cells. Very few cells immunostained for insulin; pancreatic insulin mRNA and content were markedly reduced. GLUT2 mRNA was decreased, but other beta-cell-associated genes (IAPP [islet amyloid pancreatic polypeptide], PDX-1 [pancreatic and duodenal homeobox-1], and BETA2/NeuroD) were expressed at near-normal levels. Immunostaining for both GLUT2 and Nkx6.1 was mainly cytoplasmic. The defect in beta-cell phenotype in transgenic embryos (embryonic days 17-18) and neonates (days 1-2) was similar and, therefore, was not secondary to overt hyperglycemia. When pancreata were transplanted under the kidney capsules of athymic mice to analyze the long-term effects of c-Myc activation, beta-cell depletion was found, suggesting that, ultimately, apoptosis predominates over proliferation. In conclusion, these studies demonstrate that activation of c-Myc in beta-cells leads to 1) increased proliferation and apoptosis, 2) initial hyperplasia with amorphous islet organization, and 3) selective downregulation of insulin gene expression and the development of overt diabetes.

Insulin biosynthesis and secretion are critical for pancreatic beta-cell function, but both are impaired under diabetic conditions. We have found that hyperglycemia induces the expression of the basic helix-loop-helix transcription factor c-Myc in islets in several different diabetic models. To examine the possible implication of c-Myc in beta-cell dysfunction, c-Myc was overexpressed in isolated rat islets using adenovirus. Adenovirus-mediated c-Myc overexpression suppressed both insulin gene transcription and glucose-stimulated insulin secretion. Insulin protein content, determined by immunostaining, was markedly decreased in c-Myc-overexpressing cells. In gel-shift assays c-Myc bound to the E-box in the insulin gene promoter region. Furthermore, in betaTC1, MIN6, and HIT-T15 cells and primary rat islets, wild type insulin gene promoter activity was dramatically decreased by c-Myc overexpression, whereas the activity of an E-box mutated insulin promoter was not affected. In HeLa and HepG2 cells c-Myc exerted a suppressive effect on the insulin promoter activity only in the presence of NeuroD/BETA2 but not PDX-1. Both c-Myc and NeuroD can bind the E-box element in the insulin promoter, but unlike NeuroD, the c-Myc transactivation domain lacked the ability to activate insulin gene expression. Additionally p300, a co-activator of NeuroD, did not function as a co-activator of c-Myc. In conclusion, increased expression of c-Myc in beta-cells suppresses the insulin gene transcription by inhibiting NeuroD-mediated transcriptional activation. This mechanism may explain some of the beta-cell dysfunction found in diabetes.

Pancreatic [beta] cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair [beta] cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion is similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome- wide impact of sustained high glucose on [beta] cell mRNA translation. Before induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during [beta] cell glucose toxicity that impairs expression of proteins with critical roles in [beta] cell function., Introduction Diabetes results from failure of pancreatic [beta] cells to secrete sufficient insulin to regulate glucose homeostasis. Progressive decline in [beta] cell function occurs in the setting of hyperglycemia during [...]

BACKGROUND. In the Joslin Medalist Study (Medalists), we determined whether significant associations exist between [beta] cell function and pathology and clinical characteristics. METHODS. Individuals with type 1 diabetes (T1D) for 50 or more years underwent evaluation including HLA analysis, basal and longitudinal autoantibody (AAb) status, and [beta] cell function by a mixed-meal tolerance test (MMTT) and a hyperglycemia/ arginine clamp procedure. Postmortem analysis of pancreases from 68 Medalists was performed. Monogenic diabetes genes were screened for the entire cohort. RESULTS. Of the 1019 Medalists, 32.4% retained detectable C-peptide levels (>0.05 ng/mL, median: 0.21 ng/mL). In those who underwent a MMTT (n = 516), 5.8% responded with a doubling of baseline C-peptide levels. Longitudinally (n = 181, median: 4 years), C-peptide levels increased in 12.2% (n = 22) and decreased in 37% (n = 67) of the Medalists. Among those with repeated MMTTs, 5.4% (3 of 56) and 16.1% (9 of 56) had waxing and waning responses, respectively. Thirty Medalists with baseline C-peptide levels of 0.1 ng/mL or higher underwent the clamp procedure, with [HLA.sup.-]/[AAb.sup.-] and [HLA.sup.+]/[AAb.sup.-] Medalists being most responsive. Postmortem examination of pancreases from 68 Medalists showed that all had scattered insulin-positive cells; 59 additionally had few insulin-positive cells within a few islets; and 14 additionally had lobes with multiple islets with numerous insulin-positive cells. Genetic analysis revealed that 280 Medalists (27.5%) had monogenic diabetes variants; in 80 (7.9%) of these Medalists, the variants were classified as 'likely pathogenic' (rare exome variant ensemble learner [REVEL] >0.75). CONCLUSION. All Medalists retained insulin-positive [beta] cells, with many responding to metabolic stimuli even after 50 years of T1D. The Medalists were heterogeneous with respect to [beta] cell function, and many with [HLA.sup.+] diabetes risk alleles also had monogenic diabetes variants, indicating the importance of genetic testing for clinically diagnosed T1D. FUNDING. Funding for this work was provided by the Dianne Nunnally Hoppes Fund; the Beatson Pledge Fund; the NIH, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK); and the American Diabetes Association (ADA)., Introduction Pancreatic [beta] cell depletion attributed to autoimmunity is the hallmark of type 1 diabetes (T1D). However, recent studies have shown that residual [beta] cells may exist despite very long-duration [...]

As diabetes continues to grow as major health problem, there has been great progress in understanding the important role of pancreatic beta-cells in its pathogenesis. Diabetes develops when the normal interplay between insulin secretion and the insulin sensitivity of target tissues is disrupted. With type 2 diabetes (T2D), glucose levels start to rise when beta-cells are unable to meet the demands of insulin resistance. For type 1 diabetes (T1D) glucose levels rise as beta-cells are killed off by autoimmunity. In both cases the increased glucose levels have a toxic effect on beta-cells. This process, called glucose toxicity, has a major inhibitory effect on insulin secretion. This beta-cell dysfunction can be reversed by therapies that reduce glucose levels. Thus, it is becoming increasingly apparent that an opportunity exists to produce a complete or partial remission for T2D, both of which will provide health benefit. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.06.029 Byline: Wataru Nishimura, Susan Bonner-Weir, Arun Sharma Keywords: MafA; Pancreas; [beta]-cell; Insulin gene transcription factor; Endocrine differentiation; Pancreatic development Abstract: The transcription factor MafA regulates glucose-responsive expression of insulin. MafA-deficient mice have a normal proportion of insulin.sup.+ cells at birth but develop diabetes gradually with age, suggesting that MafA is required for maturation and not specification of pancreatic [beta]-cells. However, several studies show that ectopic expression of MafA may have a role in specification as it induces insulin.sup.+ cells in chicken gut epithelium, reprograms adult murine acinar cells into insulin.sup.+ cells in combination with Ngn3 and Pdx1, and triggers the lens differentiation. Hence, we examined whether MafA can induce specification of [beta]-cells during pancreatic development. When the MafA transgene is expressed in Pdx1.sup.+ pancreatic progenitors, both pancreatic mass and proliferation of progenitors are reduced, at least partially due to induction of cyclin kinase inhibitors p27 and p57. Expression of MafA in Pdx1.sup.+ cells until E12.5 was sufficient to cause these effects and to disproportionately inhibit the formation of endocrine cells in the remnant pancreas. Thus, in mice, MafA expression in Pdx1.sup.+ pancreatic progenitors is not sufficient to specify insulin.sup.+ cells but in fact deters pancreatic development and the differentiation of endocrine cells. These findings imply that MafA should be used to enhance maturation, rather than specification, of [beta]-cells from stem/progenitor cells. Author Affiliation: Section of Islet Transplantation & Cell Biology, Joslin Diabetes Center, Boston, Massachusetts, USA Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA Article History: Received 23 January 2009; Revised 11 June 2009; Accepted 22 June 2009 Article Note: (footnote) [star] Effect of early expression of MafA in pancreatic progenitors.

During progression to both types 1 and 2 diabetes (T1D, T2D), there is a striking loss of glucose-induced first-phase insulin release (FPIR), which is known to predict the onset of T1D. The contribution of reduced β cell mass to the onset of hyperglycemia remains unclear. In this issue of the JCI, Mezza et al. report on their study of patients with pancreatic neoplasms before and after partial pancreatectomy to evaluate the impact of reduced β cell mass on the development of diabetes. The authors found that reduced FPIR predicted diabetes when 50% of the pancreas was removed. These findings suggest that low or absent FPIR indicates that β cell mass can no longer compensate for increased insulin needs. Notably, clinicians may use reduction of FPIR as a warning that progression to T2D is underway. [ABSTRACT FROM AUTHOR]

Conversion of one terminally differentiated cell type into another (or transdifferentiation) usually requires the forced expression of key transcription factors. We examined the plasticity of human insulin-producing β-cells in a model of islet cell aggregate formation. Here, we show that primary human β-cells can undergo a conversion into glucagon-producing α-cells without introduction of any genetic modification. The process occurs within days as revealed by lentivirus-mediated β-cell lineage tracing. Converted cells are indistinguishable from native α-cells based on ultrastructural morphology and maintain their α-cell phenotype after transplantation in vivo. Transition of β-cells into α-cells occurs after β-cell degranulation and is characterized by the presence of β-cell-specific transcription factors Pdx1 and Nkx6.1 in glucagon+ cells. Finally, we show that lentivirus-mediated knockdown of Arx, a determinant of the α-cell lineage, inhibits the conversion. Our findings reveal an unknown plasticity of human adult endocrine cells that can be modulated. This endocrine cell plasticity could have implications for islet development, (patho) physiology, and regeneration. [ABSTRACT FROM AUTHOR]

Background: Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D) should provide insights into their abnormal insulin secretion and turnover. Methodology/Principal Findings: Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM). RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip) and Gene Set Enrichment Analysis (GSEA) software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG) family and metalloproteinase 7 (MMP7). Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2) were upregulated while JAZF1 and SLC30A8 were downregulated. Conclusions/Significance: This study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D. [ABSTRACT FROM AUTHOR]

β cells in the hyperglycemic environment of diabetes have marked changes in phenotype and function that are largely reversible if glucose levels can be returned to normal. A leading hypothesis is that these changes are caused by the elevated glucose levels leading to the concept of glucose toxicity. Support for the glucose toxicity hypothesis is largely circumstantial, but little progress has been made in defining the responsible mechanisms. Then questions emerge that are difficult to answer. In the very earliest stages of diabetes development, there is a dramatic loss of glucose-induced first-phase insulin release (FPIR) with only trivial elevations of blood glucose levels. A related question is how impaired insulin action on target tissues such as liver, muscle and fat can cause increased insulin secretion. The existence of a sophisticated feedback mechanism between insulin secretion and insulin action on peripheral tissues driven by glucose has been postulated, but it has been difficult to measure increases in blood glucose levels that might have been expected. These complexities force us to challenge the simplicity of the glucose toxicity hypothesis and feedback mechanisms. It may turn out that glucose is somehow driving all of these changes, but we must develop new questions and experimental approaches to test the hypothesis. • With only slight increases in plasma glucose levels, glucose-induced first phase insulin release (FPIR) is abolished. • This loss of FPIR is associated with increased risk of progressing to diabetes. • The most attractive way to explain these changes is that they are driven by glucose. • However, while the circumstantial evidence supporting the relationship is strong, the responsible mechanisms have not been found. • The complexities of addressing this challenge are discussed and suggestions for experiments are proposed. [ABSTRACT FROM AUTHOR]

Islets are composed mostly of β-cells, and therefore stem cell research has concentrated on generating purified β-cells, neglecting the other endocrine cell types in the islet. We investigated the presence of endocrine non-β-cells after islet transplantation. In addition, we studied whether the transplantation of pure β-cells, in volumes similar to that used in islet transplantation, would suffice to reverse hyperglycemia in diabetic mice. Rat islets were dispersed and β-cells were purified by fluorescence-activated cell sorting according to their endogenous fluorescence. After reaggregation, 600 islet equivalents of the purified β-cell aggregates were implanted into diabetic SCID mice. In mice implanted with β-cell-enriched aggregates, the hyperglycemia was reversed and good graft function over a 12-week period was observed with regard to glucose and insulin levels, glucose tolerance tests, and graft insulin content. The endocrine cell composition of the β-cell-enriched aggregates remained constant; before and 12 weeks after transplantation, the β-cell-enriched aggregates comprised 95% β-cells and 5% endocrine non-β-cells. However, islet grafts, despite originally having comprised 75% β-cells and 25% endocrine non-β-cells, comprised just 5% endocrine non-β-cells after transplantation, indicating a loss of these cells. β-Cell-enriched aggregates can effectively reverse hyperglycemia in mice, and transplanted intact islets are depleted in non-β-cells. It is therefore likely that islet non-β-cells are not essential for successful islet transplantation. Diabetes 56:2312-2318, 2007 [ABSTRACT FROM AUTHOR]

To test whether pancreatic duct cells are in vitro progenitors, they were purified from dispersed islet-depleted human pancreatic tissue using CA19-9 antibody. The purified fraction was almost entirely CK19[sup +] with no insulin[sup +] cells, whereas the unpurified cells (crude duct) were 56% CK19[sup +] and 0.4% insulin[sup +] of total cells (0.7% of CK19[sup +] cells). These cells were expanded as monolayers, aggregated under serum-free conditions, and transplanted into normoglycemic NOD/SCID mice. In crude duct grafts, insulin[sup +] cells increased to 6.1% of CK19[sup +] cells. Purified duct cells had slow expansion and poor aggregation, as well as engraftment. The addition of 0.1% cultured stromal cells improved these parameters. These stromal cells contained no CK19[sup +] cells and no insulin by either quantitative RT-PCR or immunohistochemistry; stromal cell aggregates and grafts contained no insulin[sup +] cells. Aggregation of purified duct plus stromal preparations induced insulin[sup +] cells (0.1% of CK19[sup +] cells), with further increase to 1.1% in grafts. Insulin mRNA mirrored these changes. In these grafts, all insulin[sup +] cells were in duct-like structures, while in crude duct grafts, 85% were. Some insulin[sup +] cells coexpressed duct markers (CK19 and CA19-9) and heat shock protein (HSP)27, a marker of nonislet cells, suggesting the transition from duct. Thus, purified duct cells from adult human pancreas can differentiate to insulin-producing cells. Diabetes 56:1802-1809, 2007 [ABSTRACT FROM AUTHOR]

Stimulation of insulin secretion by the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) has been found to be diminished in type 2 diabetes. We hypothesized that this impairment is due to a defect at the receptor level induced by the diabetic state, particularly hyperglycemia. Gene expression of incretin receptors, GLP-1R and GIPR, were significantly decreased in islets of 90% pancreatectomized (Px) hyperglycemic rats, with recovery when glucose levels were normalized by phlorizin. Perifused islets isolated from hyperglycemic Px rats showed reduced insulin responses to GLP-1 and GIP. To examine the acute effect of hyperglycemia on incretin receptor expression, a hyperglycemic clamp study was performed for 96 h with reduction of GLP-1 receptor expression but increase in GIP receptor expression. Similar findings were found when islets were cultured at high glucose concentrations for 48 h. The reduction of GLP-1 receptor expression by high glucose was prevented by dominant-negative protein kinase C (PKC)α overexpression, whereas GLP-1 receptor expression was reduced with wild-type PKCα overexpression. Taken together, GLP-1 and GIP receptor expression is decreased with chronic hyperglycemia, and this decrease likely contributes to the impaired incretin effects found in diabetes. Diabetes 56:1551-1558, 2007 [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome pathway is crucial for protein turnover. Part of the pathway involves deubiquitination, which is carried out by cystein proteases known as ubiquitin COOH-terminal hydrolases. The isoform Uch-L1 was found to be present in large amounts in rat islets by immunostaining, Western blot analysis, and RT-PCR. Culturing islets in high glucose concentrations (16.7 mmol/l) for 24 h led to decreased gene expression. Exposure to chronic hyperglycemia following 90% partial pancreatectomy also led to reduced Uch-L1 expression. Expression of other members of the ubiquitin-proteasome pathway studied after culturing islets at high glucose concentrations revealed little change except for modest declines in parkin, human ubiquitin-conjugating enzyme 5 (UbcH5), and β-TRCP (transducin repeat-containing protein). With the pancreatectomy model, expression of polyubiquitin-B and c-Cbl were increased and E6-associated protein was reduced. Further insight about the proteasome pathway was obtained with the proteasome inhibitor lactacystin, which in short-term 2-h experiments enhanced glucose-induced insulin secretion. An important role for the ubiquitin-proteasome pathways in β-cells is suggested by the findings that changes in glucose concentration influence expression of genes in the pathway and that blockade of the proteasome degradation machinery enhances glucose-stimulated insulin secretion. Diabetes 55:1223-1231, 2006 [ABSTRACT FROM AUTHOR]

Type 1 diabetes mellitus is characterized by the selective destruction of insulin-producing beta cells, which leads to a deficiency in insulin secretion and, as a result, to hyperglycemia. At present, transplantation of pancreatic islets is an emerging and promising clinical modality, which can render individuals with type 1 diabetes insulin independent without increasing the incidence of hypoglycemic events. To monitor transplantation efficiency and graft survival, reliable noninvasive imaging methods are needed. If such methods were introduced into the clinic, essential information could be obtained repeatedly and noninvasively. Here we report on the in vivo detection of transplanted human pancreatic islets using magnetic resonance imaging (MRI) that allowed noninvasive monitoring of islet grafts in diabetic mice in real time. We anticipate that the information obtained in this study would ultimately result in the ability to detect and monitor islet engraftment in humans, which would greatly aid the clinical management of this disease. [ABSTRACT FROM AUTHOR]

BETA2/NeuroD, a basic helix-loop-helix transcription factor, is a key regulator of pancreatic islet morphogenesis and insulin gene transcription. Here we report for the first time that the BETA2/NeuroD protein can permeate several cells, including pancreatic islets, due to an arginine- and lysine-rich protein transduction domain sequence in its structure. The BETA2/NeuroD protein was transduced in a dose-dependent manner up to 1 micromol/l. Transduced BETA2/NeuroD functions similarly to endogenous BETA2/NeuroD: it binds to the insulin promoter and activates its expression. We also investigated the mechanism of BETA2/NeuroD protein transduction. The BETA2/NeuroD protein penetrated cells by macropinocytosis and was released from endosomes homogeneously in cytoplasm and nuclei. These data suggest that BETA2/NeuroD protein transduction could be a safe and valuable strategy for enhancing insulin gene transcription without requiring gene transfer technology. [ABSTRACT FROM AUTHOR]

This article proposes five stages in the progression of diabetes, each of which is characterized by different changes in β-cell mass, phenotype, and function. Stage 1 is compensation: insulin secretion increases to maintain normoglycemia in the face of insulin resistance and/or decreasing β-cell mass. This stage is characterized by maintenance of differentiated function with intact acute glucose-stimulated insulin secretion (GSIS). Stage 2 occurs when glucose levels start to rise, reaching ∼5.0-6.5 mmol/l; this is a stable state of β-cell adaptation with loss of β-cell mass and disruption of function as evidenced by diminished GSIS and β-cell dedifferentiation. Stage 3 is a transient unstable period of early decompensation in which glucose levels rise relatively rapidly to the frank diabetes of stage 4, which is characterized as stable decompensation with more severe β-cell dedifferentiation. Finally, stage 5 is characterized by severe decompensation representing a profound reduction in β-cell mass with progression to ketosis. Movement across stages 1-4 can be in either direction. For example, individuals with treated type 2 diabetes can move from stage 4 to stage 1 or stage 2. For type I diabetes, as remission develops, progression from stage 4 to stage 2 is typically found. Delineation of these stages provides insight into the pathophysiology of both progression and remission of diabetes. Diabetes 53 (Suppl. 3):S16-S210, 2004 [ABSTRACT FROM AUTHOR]

Omer A, Keegan M, Czismadia E, De Vos P, Van Rooijen N, Bonner-Weir S and Weir GC. Macrophage depletion improves survival of porcine neonatal pancreatic cell clusters contained in alginate macrocapsules transplanted into rats. Xenotransplantation 2003; 10: 240–251. © Blackwell Munksgaard 2003 Background: Macrophages can accumulate on the surface of empty and islet-containing alginate capsules, leading to loss of functional tissue. In this study, the effect of peritoneal macrophage depletion on the biocompatibility of alginate macrocapsules and function of macroencapsulated porcine neonatal pancreatic cell clusters (NPCCs) was investigated. Methods: Clodronate liposomes were injected into the peritoneal cavities of normoglycemic Lewis rats 5 and 2 days before the transplantation. Empty or NPCC-containing Ca-alginate poly L-lysine (PLL)-coated macrocapsules were transplanted into the peritoneal cavities of rats injected with either clodronate liposomes or saline. On days 7, 14 and 21, samples were evaluated by immunohistochemistry for cellular immune responses on the surface of the macrocapsules and for macrophage populations in omental tissue. To assess the function of macroencapsulated NPCCs, insulin secretory responses to glucose and theophylline were measured after capsule retrieval. Results: In saline-injected control groups, all of the empty and NPCC-containing macrocapsules were overgrown with macrophages, this being especially severe on NPCC-containing macrocapsules. In the clodronate liposomes-injected group, the majority of the empty macrocapsules were free of macrophage accumulation and the NPCC-containing macrocapsules were less overgrown than in control animals. Higher insulin responses to glucose and theophylline were observed in NPCCs retrieved from rats injected with clodronate liposomes. Conclusion: We conclude that depletion of peritoneal macrophages with clodronate liposomes improve the survival of macroencapsulated NPCCs. [ABSTRACT FROM AUTHOR]

Type 2 diabetes is characterized by impaired insulin secretion. Some but not all studies suggest that a decrease in beta-cell mass contributes to this. We examined pancreatic tissue from 124 autopsies: 91 obese cases (BMI >27 kg/m(2); 41 with type 2 diabetes, 15 with impaired fasting glucose [IFG], and 35 nondiabetic subjects) and 33 lean cases (BMI <25 kg/m(2); 16 type 2 diabetic and 17 nondiabetic subjects). We measured relative beta-cell volume, frequency of beta-cell apoptosis and replication, and new islet formation from exocrine ducts (neogenesis). Relative beta-cell volume was increased in obese versus lean nondiabetic cases (P = 0.05) through the mechanism of increased neogenesis (P < 0.05). Obese humans with IFG and type 2 diabetes had a 40% (P < 0.05) and 63% (P < 0.01) deficit and lean cases of type 2 diabetes had a 41% deficit (P < 0.05) in relative beta-cell volume compared with nondiabetic obese and lean cases, respectively. The frequency of beta-cell replication was very low in all cases and no different among groups. Neogenesis, while increased with obesity, was comparable in obese type 2 diabetic, IFG, or nondiabetic subjects and in lean type 2 diabetic or nondiabetic subjects. However, the frequency of beta-cell apoptosis was increased 10-fold in lean and 3-fold in obese cases of type 2 diabetes compared with their respective nondiabetic control group (P < 0.05). We conclude that beta-cell mass is decreased in type 2 diabetes and that the mechanism underlying this is increased beta-cell apoptosis. Since the major defect leading to a decrease in beta-cell mass in type 2 diabetes is increased apoptosis, while new islet formation and beta-cell replication are normal, therapeutic approaches designed to arrest apoptosis could be a significant new development in the management of type 2 diabetes, because this approach might actually reverse the disease to a degree rather than just palliate glycemia. [ABSTRACT FROM AUTHOR]

To test the hypothesis that c-Myc plays an important role in β-cell growth and differentiation, we generated transgenic mice overexpressing c-Myc in β-cells under control of the rat insulin II promoter. F[sub 1] transgenic mice from two founders developed neonatal diabetes (associated with reduced plasma insulin levels) and died of hyperglycemia 3 days after birth. In pancreata of transgenic mice, marked hyperplasia of cells with an altered phenotype and amorphous islet organization was displayed: islet volume was increased threefold versus wild-type littermates. Apoptotic nuclei were increased fourfold in transgenic versus wild-type mice, suggesting an increased turnover of β-cells. Very few cells immunostained for insulin; pancreatic insulin mRNA and content were markedly reduced. GLUT2 mRNA was decreased, but other β-cell-associated genes (IAPP [islet amyloid pancreatic polypeptide], PDX-1 [pancreatic and duodenal homeobox-1], and BETA2/NeuroD) were expressed at near-normal levels. Immunostaining for both GLUT2 and Nkx6.1 was mainly cytoplasmic. The defect in β-cell phenotype in transgenic embryos (embryonic days 17-18) and neonates (days 1-2) was similar and, therefore, was not secondary to overt hyperglycemia. When pancreata were transplanted under the kidney capsules of athymic mice to analyze the long-term effects of c-Myc activation, β-cell depletion was found, suggesting that, ultimately, apoptosis predominates over proliferation. In conclusion, these studies demonstrate that activation of c-Myc in β-cells leads to 1) increased proliferation and apoptosis, 2) initial hyperplasia with amorphous islet organization, and 3) selective downregulation of insulin gene expression and the development of overt diabetes. [ABSTRACT FROM AUTHOR]

Pancreatic beta-cell mass (BCM) is a major determinant of the quantity of insulin that can be secreted. BCM is markedly reduced in type 1 diabetes because of selective autoimmune destruction of beta-cells. Accurate assessment of BCM in human diabetes is limited to autopsy studies, which usually suffer from inadequate clinical information; thus, the development of noninvasive means of BCM measurement could be important in intervention therapy. The goal of this study was to develop such noninvasive methods for measuring BCM featuring target-specific imaging probes and to investigate whether this technique is feasible, accurate, and predictive of BCM in normal and diabetic states. Using a beta-cell-specific monoclonal antibody IC2, modified with a radioisotope chelator for nuclear imaging, we showed that highly specific binding and accumulation to beta-cells occurs after intravenous administration of the probe, with virtually no binding to exocrine pancreas or stromal tissues. Furthermore, we observed a direct correlation between accumulation of the probe with BCM in diabetic and normal animals. Nuclear imaging of the animals that received an injection of the radioactive probe showed major difference in signal intensity between normal and diabetic pancreases. The results from this study set the route for further development of imaging probes for measuring BCM that would aid in diagnosis and treatment of diabetic patients in the clinic. [ABSTRACT FROM AUTHOR]

Presents information on a study which determined whether beta-cells can adapt to the increase in insulin demand during glucose or lipid infusion and the molecular mechanisms behind the adaptation. Background on adaptive beta-cells; Materials and methodology used; Results and discussion.

Diabetes is caused by a failure of the pancreas to produce insulin in amounts sufficient to meet the body's needs. A hallmark of diabetes is an absolute (type 1) or relative (type 2) reduction in the mass of pancreatic beta-cells that produce insulin. Mature beta-cells have a lifespan of approximately 48-56 days (rat) and are replaced by the replication of preexisting beta-cells and by the differentiation and proliferation of new beta-cells (neogenesis) derived from the pancreatic ducts. Here, we show that the insulinotropic hormone glucagon-like peptide (GLP)-1, which is produced by the intestine, enhances the pancreatic expression of the homeodomain transcription factor IDX-1 that is critical for pancreas development and the transcriptional regulation of the insulin gene. Concomitantly, GLP-1 administered to diabetic mice stimulates insulin secretion and effectively lowers their blood sugar levels. GLP-1 also enhances beta-cell neogenesis and islet size. Thus, in addition to stimulating insulin secretion, GLP-1 stimulates the expression of the transcription factor IDX-1 while stimulating beta-cell neogenesis and may thereby be an effective treatment for diabetes. [ABSTRACT FROM AUTHOR]

The article presents information on the efficiency of weight loss or bariatric surgery in treating type 2 diabetes. It is mentioned that a duodenal-jejunal bypass (DJB) was carried out on rats induced with diabetes and it was found that it lowered blood glucose concentrations as delivery of nutrients to the jejunum increased without increase in insulin secretion.

The article comments on a study published in this issue which explored the association between insulin secretion and functional β-cell mass. It notes that the study can serve as example of how animal research can discover new mechanisms in humans. It points out the important role of pancreatic insulin secretion in keeping glucose levels in the normal range. Particular focus is given to ways in which regeneration might occur in β-cells.

A letter to the editor is presented in response to the article "Residual Insulin Production and Pancreatic β-Cell Turnover After 50 Years of Diabetes: Joslin Medalist Study," by Hillary A. Keenan, Susan Bonner-Weir and George L. King in a 2010 issue of the journal, as well as a response by the authors to the letter.

MafA/RIPE3b1 is an important glucose-responsive insulin gene transcription factor; its expression is differentially regulated by acute and chronic hyperglycemic conditions. Our understanding of the molecular mechanisms regulating MafA expression is still limited. Using RT-PCR and Western blot analyses; we demonstrate that the induction of MafA expression in response to acute increases in the glucose level requires both new RNA and protein synthesis. Furthermore, chronic hyperglycemia, as observed in diabetic rats, inhibited expression of both MafA mRNA and protein in pancreatic islets. Normalizing hyperglycemia with Phlorizin treatment reversed this reduction in MafA expression. These observations demonstrate a direct correlation between loss of MafA and β-cell dysfunction. We show that mammalian MafA, like its avian counterpart, is a phospho-protein and that phosphorylation affects its stability and migration. Increased phosphorylation of MafA reduces its stability. Consistent with these observations, specific inhibition of ERK or p38 MAPK increased MafA protein stability. Chronic hyperglycemia can trigger oxidative stress; treatment of MIN6 cells with tea-Butyl hydroperoxide, an inducer of oxidative stress, reduces MafA stability by increasing p38 MAPK activity. Interestingly, even under conditions of reduced oxidative stress such as low glucose concentrations, inhibiting p38 MAPK pathway enhanced the MafA protein stability. These results suggest that p38 MAPK pathway represents a common mechanism for regulating MafA levels under basal, acute and chronic hyperglycemic conditions. Results demonstrate that p38 MAPK mediated phosphorylation of MafA is important for its activity, this would suggest that the active MafA will be rapidly degraded in a cell, possibly to prevent any detrimental action of its uncontrolled activity. Thus identifying mechanisms that will permit regulation of p38 MAPK mediated activation of MafA without increasing its degradation may provide novel approaches to improve β-cell dysfunction. [ABSTRACT FROM AUTHOR]

To investigate islet hormone gene expression in single beta cells of adult and neonatal mice, we performed nested PCR on FACS-sorted GFP+ beta cells from mouse insulin 1 promoter (MIP)-GFP mice. Islets isolated from MIP-GFP mice (16-24 weeks old) or whole pancreatic tissue from MIP-GFP neonates (1.5 day-old) were digested with Liberase, dispersed by trypsin and then double sorted for GFP to obtain pure high expression GFP+ beta cells. Sorted cells were directly plated singly into wells containing cell lysis mixture, RT buffer, and gene specific reverse primers (insulin1, insulin2, glucagon, somatostatin, pancreatic polypeptide (PP), pdx-1, neuroD and HPRT). After RT, forward primers were added for multiplex PCR. After 1st PCR, 2nd round nested PCR was performed using single cell sample primers set within the 1st primers. By single cell nested PCR analysis, every GFP+ cell from adult or neonate expressed insulin 1, insulin 2, pdx-1 and neuroD. In adult mice, 60-70% of GFP+cells were positive for only insulin, while 30-40% of GFP+cells expressed two or more islet hormones. 3% of GFP+ cells expressed all 4 islets hormones. Single neonatal GFP+ cells also expressed multiple islet hormones, hut, notably, 80% of GFP+cells expressed PP in addition to insulin (versus about 30% in adult). Of these, 64% expressed just PP and insulin, and no other islet hormones (versus 10% in the adult). Only 1-2% of neonatal GFP +cells expressed all 4 islets hormones. For both adult and neonatal cells, expression of these other islet hormones was at a low level, and protein was not found with immunostaining. In conclusion, single beta cells express not only insulin but low levels of mRNA for other islet hormones. The pattern of expression in adult and neonatal beta cells differs; neonatal cells express PP much more frequently. These findings support the hypothesis that expression of small amounts of PP mRNA is a marker of young beta cells. [ABSTRACT FROM AUTHOR]

During pancreatic development insulin+ cells transition from a MafA- to MafA+ state. Our previous results suggested that the expression of transcription factor MafB in insulin+ cells precedes this maturation step. To directly examine the role of MafB in this process, we analyzed embryonic pancreas from mice carrying a kreisler mutation in the mafBkr/kr gene. We show that this kr allele, for which serine is replaced by asparagine in the conserved, DNA-binding domain found in all bZIP family members, reduced the ability of MafB to activate both insulin and glucagon gene expression and that mice with this mutation had a significant reduction in the insulin and glucagon expressing cells. Mutation also resulted in the reduction of PDX-1+ and MafA+ cells with only a minor reduction in MafB+ cells. Interestingly, the proportion of MafB+ or PDX-1+ cells that also expressed insulin was reduced. However mutation in MafB did not affect the expression of Nkx2.2, Nkx6.1, Ngn3 and Pax6 suggesting that MafB does not affect the endocrine specification but rather plays a role in the maturation of insulin+ cells. Examination of Pax6 sey/sey mutant mice demonstrated a reduction in the insulin, glucagon, MatB, PDX-1 and MafA expressing cells. Since Pax6 expression was not altered in kreisler mice, our results suggest a role of MafB in the reduction of insulin and glucagon expressing cells in sey/sey mice. Furthermore, as in kreisler mice, the proportion of MafB+ or PDX-1+ cells expressing insulin was reduced in sey/sey mice, suggesting roles for both Pax6 and MafB in regulating the efficiency of terminal differentiation/maturation of insulin+ cells. Interestingly, unlike MafB and PDX-1 that were also expressed in some insulin- cells, all MafA+ cells in these mutant mice expressed insulin. Thus, our results indicate that MafA is a marker of terminally differentiated β-cell, and that Pax6 acts upstream of MafB, which in turn contributes to the expression of PDX-1 and MafA in differentiating insulin+ cells. [ABSTRACT FROM AUTHOR]

Extended Abstract β-Cell replacement therapy via islet transplantation has received renewed interest due to the recent improved success. In order to make such a therapy available to more than a few of the thousands of patients with diabetes, new sources of insulin-producing cells must be readily available. The most promising sources are stem cells, with efforts of deriving new β-cells from both embryonic and adult stem cells. Several groups have reported generating insulin-producing cells from mouse embryonic stem cells. The strategies in the first two acclaimed reports were very different. One strategy, used by Soria’s group, is gene trapping in which an introduced antibiotic resistance under the control of the insulin promoter allowed the selection of insulin-expressing cells that had spontaneously differentiated within embryoid bodies. Another strategy, used by McKay’s group, manipulated culture conditions in a multistep protocol used for generating neural cells but with changed final conditions. Since these reports, there have been modifications of the protocols in efforts to improve the yields and maturity of the resulting cells. While it is unclear if the insulin-producing cells in any of these studies are truly mature β-cells, these studies show the clear potential of embryonic stem cells and support optimism that similar results will be possible with human embryonic stem cells. We know that new β-cells are generated throughout adult life, but the identity of adult pancreatic stem cells has been elusive. The potential for expansion and differentiation of pluripotent adult stem cells, whether from bone marrow or as non-pancreas tissue resident SP cells, is being explored but has not yet yielded insulin-producing tissue. In contrast, insulin-producing cells have been generated in vitro from adult pancreatic tissues. We have been examining the hypothesis that the functional source for new β-cells in the adult pancreas are mature duct epithelial cells that have regressed or lost their mature phenotype after replication. Others have isolated putative stem cells from islets and ducts. For adult cells the issue of expansion as well as of differentiation is a question. The field of generating new β-cells from stem cells, either embryonic or adult, is still in its infancy. Each new report has been met with a mixture of excitement and skepticism. With continued efforts and rigorous assessments, hopefully the potential of generating enough new β-cells from stem cells will be realized. Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Mentions two cases that describe the instability of lispro insulin, which is not specific to a particular infusion catheter or type of pump. The erratic and unpredictable glucose fluctuations with lispro insulin that have resolved when the treatment was changed to buffered regular insulin and aspart insulin; Confirmation of insulin precipitation.