GABA Signaling Stimulates ß Cell Regeneration in Diabetic Mice.

GABA and the antimalarial drug artemether, which acts on GABAergic pathways, can drive pancreatic cells with an α-cell phenotype toward a ß-cell-like phenotype. As reported in two papers (Ben-Othman et al. and Li et al.), these drugs can stimulate the production of sufficient numbers of new ß-like cells to reverse severe diabetes in mice.

ANGPTL8/betatrophin does not control pancreatic beta cell expansion.

Recently, it was reported that angiopoietin-like protein 8 (ANGPTL8) was the long-sought "betatrophin" that could control pancreatic beta cell proliferation. However, studies of Angptl8(?/?) mice revealed profound reduction of triglyceride levels, but no abnormalities in glucose homeostasis. We now report that Angptl8(?/?) mice undergo entirely normal beta cell expansion in response to insulin resistance resulting from either a high-fat diet or from the administration of the insulin receptor antagonist S961. Furthermore, overexpression of ANGPTL8 in livers of mice doubles plasma triglyceride levels, but does not alter beta cell expansion nor glucose metabolism. These data indicate that ANGPTL8 does not play a role in controlling beta cell growth, nor can it be given to induce such expansion. The findings that plasma triglyceride levels are reduced by Angptl8 deletion and increased following ANGPTL8 overexpression support the possibility that inhibition of ANGPTL8 represents a therapeutic strategy for hypertriglyceridemia.

PAHSAs reduce cellular senescence and protect pancreatic beta cells from metabolic stress through regulation of Mdm2/p53.

Senescence in pancreatic beta cells plays a major role in beta cell dysfunction, which leads to impaired glucose homeostasis and diabetes. Therefore, prevention of beta cell senescence could reduce the risk of diabetes. Treatment of nonobese diabetic (NOD) mice, a model of type 1 autoimmune diabetes (T1D), with palmitic acid hydroxy stearic acids (PAHSAs), a novel class of endogenous lipids with antidiabetic and antiinflammatory effects, delays the onset and reduces the incidence of T1D from 82% with vehicle treatment to 35% with PAHSAs. Here, we show that a major mechanism by which PAHSAs protect islets of the NOD mice is by directly preventing and reversing the initial steps of metabolic stress-induced senescence. In vitro PAHSAs increased Mdm2 expression, which decreases the stability of p53, a key inducer of senescence-related genes. In addition, PAHSAs enhanced expression of protective genes, such as those regulating DNA repair and glutathione metabolism and promoting autophagy. We demonstrate the translational relevance by showing that PAHSAs prevent and reverse early stages of senescence in metabolically stressed human islets by the same Mdm2 mechanism. Thus, a major mechanism for the dramatic effect of PAHSAs in reducing the incidence of type 1 diabetes in NOD mice is decreasing cellular senescence; PAHSAs may have a similar benefit in humans.

Hyperglycaemia attenuates in vivo reprogramming of pancreatic exocrine cells to beta cells in mice.

AIMS/HYPOTHESIS: Reprogramming of pancreatic exocrine to insulin-producing cells by viral delivery of the genes encoding transcription factors neurogenin-3 (Ngn3), pancreas/duodenum homeobox protein 1 (Pdx1) and MafA is an efficient method for reversing diabetes in murine models. The variables that modulate reprogramming success are currently ill-defined. METHODS: Here, we assess the impact of glycaemia on in vivo reprogramming in a mouse model of streptozotocin-induced beta cell ablation, using subsequent islet transplantation or insulin pellet implantation for creation of groups with differing levels of glycaemia before viral delivery of transcription factors. RESULTS: We observed that hyperglycaemia significantly impaired reprogramming of exocrine to insulin-producing cells in their quantity, differentiation status and function. With hyperglycaemia, the reprogramming of acinar towards beta cells was less complete. Moreover, inflammatory tissue changes within the exocrine pancreas including macrophage accumulation were found, which may represent the tissue's response to clear the pancreas from insufficiently reprogrammed cells. CONCLUSIONS/INTERPRETATION: Our findings shed light on normoglycaemia as a prerequisite for optimal reprogramming success in a diabetes model, which might be important in other tissue engineering approaches and disease models, potentially facilitating their translational applications.

Differentiation of pancreatic endocrine progenitors reversibly blocked by premature induction of MafA.

Specification and maturation of insulin(+) cells accompanies a transition in expression of Maf family of transcription factors. In development, MafA is expressed after specification of insulin(+) cells that are expressing another Maf factor, MafB; after birth, these insulin(+) MafA(+) cells stop MafB expression and gain glucose responsiveness. Current differentiation protocols for deriving insulin-producing ß-cells from stem cells result in ß-cells lacking both MafA expression and glucose-stimulated insulin secretion. So driving expression of MafA, a ß-cell maturation factor in endocrine precursors could potentially generate glucose-responsive MafA(+) ß cells. Using inducible transgenic mice, we characterized the final stages of ß-cell differentiation and maturation with MafA pause/release experiments. We found that forcing MafA transgene expression, out of its normal developmental context, in Ngn3(+) endocrine progenitors blocked endocrine differentiation and prevented the formation of hormone(+) cells. However, this arrest was reversible such that with stopping the transgene expression, the cells resumed their differentiation to hormone(+) cells, including α-cells, indicating that the block likely occurred after progenitors had committed to a specific hormonal fate. Interestingly, this delayed resumption of endocrine differentiation resulted in a greater proportion of immature insulin(+)MafB(+) cells at P5, demonstrating that during maturation the inhibition of MafB in ß-cell transitioning from insulin(+)MafB(+) to insulin(+)MafB(-) stage is regulated by cell-autonomous mechanisms. These results demonstrate the importance of proper context of initiating MafA expression on the endocrine differentiation and suggest that generating mature Insulin(+)MafA(+) ß-cells will require the induction of MafA in a narrow temporal window to achieve normal endocrine differentiation.

Tissue-specific disallowance of housekeeping genes: the other face of cell differentiation.

We report on a hitherto poorly characterized class of genes that are expressed in all tissues, except in one. Often, these genes have been classified as housekeeping genes, based on their nearly ubiquitous expression. However, the specific repression in one tissue defines a special class of "disallowed genes." In this paper, we used the intersection-union test to screen for such genes in a multi-tissue panel of genome-wide mRNA expression data. We propose that disallowed genes need to be repressed in the specific target tissue to ensure correct tissue function. We provide mechanistic data of repression with two metabolic examples, exercise-induced inappropriate insulin release and interference with ketogenesis in liver. Developmentally, this repression is established during tissue maturation in the early postnatal period involving epigenetic changes in histone methylation. In addition, tissue-specific expression of microRNAs can further diminish these repressed mRNAs. Together, we provide a systematic analysis of tissue-specific repression of housekeeping genes, a phenomenon that has not been studied so far on a genome-wide basis and, when perturbed, can lead to human disease.

Renalase inhibition regulates ß cell metabolism to defend against acute and chronic stress.

Renalase (Rnls), annotated as an oxidase enzyme, is a GWAS gene associated with Type 1 Diabetes (T1D) risk. We previously discovered that Rnls inhibition delays diabetes onset in mouse models of T1D in vivo , and protects pancreatic ß cells against autoimmune killing, ER and oxidative stress in vitro . The molecular biochemistry and functions of Rnls are entirely uncharted. Here we find that Rnls inhibition defends against loss of ß cell mass and islet dysfunction in chronically stressed Akita mice in vivo . We used RNA sequencing, untargeted and targeted metabolomics and metabolic function experiments in mouse and human ß cells and discovered a robust and conserved metabolic shift towards glycolysis, amino acid abundance and GSH synthesis to counter protein misfolding stress, in vitro . Our work illustrates a function for Rnls in mammalian cells, and suggests an axis by which manipulating intrinsic properties of ß cells can rewire metabolism to protect against diabetogenic stress.

Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells.

Replenishment of pancreatic beta cells is a key to the cure for diabetes. Beta cells regeneration is achieved predominantly by self-replication especially in rodents, but it was also shown that pancreatic duct cells can transdifferentiate into beta cells. How pancreatic duct cells undergo transdifferentiated and whether we could manipulate the transdifferentiation to replenish beta cell mass is not well understood. Using a genome-wide CRISPR screen, we discovered that loss-of-function of ALDH3B2 is sufficient to transdifferentiate human pancreatic duct cells into functional beta-like cells. The transdifferentiated cells have significant increase in beta cell marker genes expression, secrete insulin in response to glucose, and reduce blood glucose when transplanted into diabetic mice. Our study identifies a novel gene that could potentially be targeted in human pancreatic duct cells to replenish beta cell mass for diabetes therapy.

Making ß cells from adult cells within the pancreas.

Cell therapy is currently considered as a potential therapeutic alternative to traditional treatments of diabetes. Islet and whole pancreas transplantations provided the proof-of-concept of glucose homeostasis restoration after replenishment of the deficiency of ß cells responsible for the disease. Current limitations of these procedures have led to the search for strategies targeting replication of pre-existing ß cells or transdifferentiation of progenitors and adult cells. These investigations revealed an unexpected plasticity towards ß cells of adult cells residing in pancreatic epithelium (eg, acinar, duct, and α cells). Here we discuss recent developments in ß-cell replication and ß-cell transdifferentiation of adult epithelial pancreatic cells, with an emphasis on techniques with a potential for clinical translation.

In vivo imaging of islet transplantation.

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.

Induction of remission in diabetes by lowering blood glucose.

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.

Conflicting Views About Interactions Between Pancreatic α-Cells and ß-Cells.

In type 1 diabetes, the reduced glucagon response to insulin-induced hypoglycemia has been used to argue that ß-cell secretion of insulin is required for the full glucagon counterregulatory response. For years, the concept has been that insulin from the ß-cell core flows downstream to suppress glucagon secretion from the α-cells in the islet mantle. This core-mantle relationship has been supported by perfused pancreas studies that show marked increases in glucagon secretion when insulin was neutralized with antisera. Additional support comes from a growing number of studies focused on vascular anatomy and blood flow. However, in recent years this core-mantle view has generated less interest than the argument that optimal insulin secretion is due to paracrine release of glucagon from α-cells stimulating adjacent ß-cells. This mechanism has been evaluated by knockout of ß-cell receptors and impairment of α-cell function by inhibition of Gi designer receptors exclusively activated by designer drugs. Other studies that support this mechanism have been obtained by pharmacological blocking of glucagon-like peptide 1 receptor in humans. While glucagon has potent effects on ß-cells, there are concerns with the suggested paracrine mechanism, since some of the supporting data are from isolated islets. The study of islets in static incubation or perifusion systems can be informative, but the normal paracrine relationships are disrupted by the isolation process. While this complicates interpretation of data, arguments supporting paracrine interactions between α-cells and ß-cells have growing appeal. We discuss these conflicting views of the relationship between pancreatic α-cells and ß-cells and seek to understand how communication depends on blood flow and/or paracrine mechanisms.

Improving stem cell-derived pancreatic islets using single-cell multi-inferred regulomes.

In this issue of Developmental Cell, Zhu et al. provide integrated data from single-cell/nuclei RNAseq and chromatin accessibility (ATAC) across both a multistaged differentiation protocol-deriving pancreatic islets from hESCs and primary islets. This important resource could be leveraged for optimizing the differentiation and maturation of pancreatic islet cells from hESCs.

Reversing and modulating cellular senescence in beta cells, a new field of opportunities to treat diabetes.

Diabetes constitutes a world-wide pandemic that requires searching for new treatments to halt its progression. Cellular senescence of pancreatic beta cells has been described as a major contributor to development and worsening of diabetes. The concept of reversibility of cellular senescence is critical as is the timing to take actions against this "dormant" senescent state. The reversal of cellular senescence can be considered as rejuvenation of the specific cell if it returns to the original "healthy state" and doesn't behave aberrantly as seen in some cancer cells. In rodents, treatment with senolytics and senomorphics blunted or prevented disease progression, however their use carry drawbacks. Modulators of cellular senescence is a new area of research that seeks to reverse the senescence. More research in each of these modalities should lead to new treatments to stop diabetes development and progression.

Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion.

Pancreatic ß 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 ß 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 ß 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 ß cell glucose toxicity that impairs expression of proteins with critical roles in ß cell function.

Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion.

Pancreatic ß-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 ß-cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion are 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 ß-cell mRNA translation. Prior to 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 of 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 findings uncover a translational regulatory circuit during ß-cell glucose toxicity that impairs expression of proteins with critical roles in ß-cell function.

Activation of pancreatic-duct-derived progenitor cells during pancreas regeneration in adult rats.

The adult pancreas has considerable capacity to regenerate in response to injury. We hypothesized that after partial pancreatectomy (Px) in adult rats, pancreatic-duct cells serve as a source of regeneration by undergoing a reproducible dedifferentiation and redifferentiation. We support this hypothesis by the detection of an early loss of the ductal differentiation marker Hnf6 in the mature ducts, followed by the transient appearance of areas composed of proliferating ductules, called foci of regeneration, which subsequently form new pancreatic lobes. In young foci, ductules express markers of the embryonic pancreatic epithelium - Pdx1, Tcf2 and Sox9 - suggesting that these cells act as progenitors of the regenerating pancreas. The endocrine-lineage-specific transcription factor Neurogenin3, which is found in the developing embryonic pancreas, was transiently detected in the foci. Islets in foci initially resemble embryonic islets in their lack of MafA expression and lower percentage of beta-cells, but with increasing maturation have increasing numbers of MafA(+) insulin(+) cells. Taken together, we provide a mechanism by which adult pancreatic duct cells recapitulate aspects of embryonic pancreas differentiation in response to injury, and contribute to regeneration of the pancreas. This mechanism of regeneration relies mainly on the plasticity of the differentiated cells within the pancreas.

p16INK4a induces an age-dependent decline in islet regenerative potential.

The p16INK4a tumour suppressor accumulates in many tissues as a function of advancing age. p16INK4a is an effector of senescence and a potent inhibitor of the proliferative kinase Cdk4 (ref. 6), which is essential for pancreatic beta-cell proliferation in adult mammals. Here we show that p16INK4a constrains islet proliferation and regeneration in an age-dependent manner. Expression of the p16INK4a transcript is enriched in purified islets compared with the exocrine pancreas, and islet-specific expression of p16INK4a, but not other cyclin-dependent kinase inhibitors, increases markedly with ageing. To determine the physiological significance of p16INK4a accumulation on islet function, we assessed the impact of p16INK4a deficiency and overexpression with increasing age and in the regenerative response after exposure to a specific beta-cell toxin. Transgenic mice that overexpress p16INK4a to a degree seen with ageing demonstrated decreased islet proliferation. Similarly, islet proliferation was unaffected by p16INK4a deficiency in young mice, but was relatively increased in p16(INK4a)-deficient old mice. Survival after toxin-mediated ablation of beta-cells, which requires islet proliferation, declined with advancing age; however, mice lacking p16INK4a demonstrated enhanced islet proliferation and survival after beta-cell ablation. These genetic data support the view that an age-induced increase of p16INK4a expression limits the regenerative capacity of beta-cells with ageing.

Mutations at the BLK locus linked to maturity onset diabetes of the young and beta-cell dysfunction.

Maturity-onset diabetes of the young (MODY) is a subtype of diabetes defined by an autosomal pattern of inheritance and a young age at onset, often before age 25. MODY is genetically heterogeneous, with 8 distinct MODY genes identified to date and more believed to exist. We resequenced 732 kb of genomic sequence at 8p23 in 6 MODY families unlinked to known MODY genes that showed evidence of linkage at that location. Of the 410 sequence differences that we identified, 5 had a frequency <1% in the general population and segregated with diabetes in 3 of the families, including the 2 showing the strongest support for linkage at this location. The 5 mutations were all placed within 100 kb corresponding to the BLK gene. One resulted in an Ala71Thr substitution; the other 4 were noncoding and determined decreased in vitro promoter activity in reporter gene experiments. We found that BLK--a nonreceptor tyrosine-kinase of the src family of proto-oncogenes--is expressed in beta-cells where it enhances insulin synthesis and secretion in response to glucose by up-regulating transcription factors Pdx1 and Nkx6.1. These actions are greatly attenuated by the Ala71Thr mutation. These findings point to BLK as a previously unrecognized modulator of beta-cell function, the deficit of which may lead to the development of diabetes.