Efficient ß-cell regeneration by a combination of neogenesis and replication following ß-cell ablation and reversal of pancreatic duct ligation.

Achieving efficient ß-cell regeneration is a major goal of diabetes research. Previously, we found that a combination of ß-cell ablation and pancreatic duct ligation led to ß-cell regeneration by direct conversion from α-cells. Here, we studied the effect of surgical reversal of the duct ligation, finding that there was a wave of ß-cell replication following reversal. The combination of ß-cell neogenesis prior to reversal of the duct ligation and ß-cell replication following reversal resulted in efficient ß-cell regeneration and eventual recovery of function. This provides an important proof of principle that efficient ß-cell regeneration is possible, even from a starting point of profound ß-cell ablation. This has important implications for efforts to promote ß-cell regeneration.

Induction of ß-cell replication by a synthetic HNF4α antagonist.

Increasing the number of ß cells is critical to a definitive therapy for diabetes. Previously, we discovered potent synthetic small molecule antagonists of the nuclear receptor transcription factor HNF4α. The natural ligands of HNF4α are thought to be fatty acids. Because obesity, in which there are high circulating levels of free fatty acids, is one of the few conditions leading to ß-cell hyperplasia, we tested the hypothesis that a potent HNF4α antagonist might stimulate ß-cell replication. A bioavailable HNF4α antagonist was injected into normal mice and rabbits and ß-cell ablated mice and the effect on ß-cell replication was measured. In normal mice and rabbits, the compound induced ß-cell replication and repressed the expression of multiple cyclin-dependent kinase inhibitors, including p16 that plays a critical role in suppressing ß-cell replication. Interestingly, in ß-cell ablated mice, the compound induced α- and δ-cell, in addition to ß-cell replication, and ß-cell number was substantially increased. Overall, the data presented here are consistent with a model in which the well-known effects of obesity and high fat diet on ß-cell replication occur by inhibition of HNF4α. The availability of a potent synthetic HNF4α antagonist raises the possibility that this effect might be a viable route to promote significant increases in ß-cell replication in diseases with reduced ß-cell mass, including type I and type II diabetes.

Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas.

The nature and even existence of adult pancreatic endocrine stem or progenitor cells is a subject of controversy in the field of beta-cell replacement for diabetes. One place to search for such cells is in the nonendocrine fraction of cells that remain after islet isolation, which consist of a mixture of epithelia and mesenchyme. Culture in G418 resulted in elimination of the mesenchymal cells, leaving a highly purified population of nonendocrine pancreatic epithelial cells (NEPECs). To evaluate their differentiation potential, NEPECs were heritably marked and transplanted under the kidney capsule of immunodeficient mice. When cotransplanted with fetal pancreatic cells, NEPECs were capable of endocrine differentiation. We found no evidence of beta-cell replication or cell fusion that could have explained the appearance of insulin positive cells from a source other than NEPECs. Nonendocrine-to-endocrine differentiation of NEPECs supports the existence of endocrine stem or progenitor cells within the epithelial compartment of the adult human pancreas.

Coordinated regulation by Shp2 tyrosine phosphatase of signaling events controlling insulin biosynthesis in pancreatic beta-cells.

Intracellular signaling by which pancreatic beta-cells synthesize and secrete insulin in control of glucose homeostasis is not fully understood. Here we show that Shp2, a cytoplasmic tyrosine phosphatase possessing 2 SH2 domains, coordinates signaling events required for insulin biosynthesis in beta-cells. Mice with conditional ablation of the Shp2/Ptpn11 gene in the pancreas exhibited defective glucose-stimulated insulin secretion and impaired glucose tolerance. Consistently, siRNA-mediated Shp2-knockdown in rat insulinoma INS-1 832/13 cells resulted in decreased insulin production and secretion despite an increase in cellular ATP. Shp2 modulates the strength of signals flowing through Akt/FoxO1 and Erk pathways, culminating in control of Pdx1 expression and activity on Ins1 and Ins2 promoters, and forced Pdx1 expression rescued insulin production in Shp2-knockdown beta-cells. Therefore, Shp2 acts as a signal coordinator in beta-cells, orchestrating multiple pathways controlling insulin biosynthesis to maintain glucose homeostasis.

Approaches to Inducing ß-Cell Regeneration.

ß-cell number and/or function is reduced in diabetes. Thus, inducing the formation of new ß-cells has been a major goal of diabetes research. However, the pathway(s) by which new ß-cells form when preexisting ß-cells are decreased in number or cease to function has remained obscure. Many pathways have been proposed, but definitive evidence, particularly in humans, has been lacking. Replication of preexisting ß-cells, neogenesis from ducts, redifferentiation from ß-cells that dedifferentiated under metabolic stress, and transdifferentiation from other cell types, particularly within the islet, are the major mechanisms that have been proposed for generating increased numbers of functional ß-cells. Here, I will discuss those approaches critically, with particular attention to transdifferentiation of preexisting α-cells to ß-cells.

A potent HNF4α agonist reveals that HNF4α controls genes important in inflammatory bowel disease and Paneth cells.

HNF4α has been implicated in IBD through a number of genome-wide association studies. Recently, we developed potent HNF4α agonists, including N-trans caffeoyltyramine (NCT). NCT was identified by structural similarity to previously the previously identified but weak HNF4α agonists alverine and benfluorex. Here, we administered NCT to mice fed a high fat diet, with the goal of studying the role of HNF4α in obesity-related diseases. Intestines from NCT-treated mice were examined by RNA-seq to determine the role of HNF4α in that organ. Surprisingly, the major classes of genes altered by HNF4α were involved in IBD and Paneth cell biology. Multiple genes downregulated in IBD were induced by NCT. Paneth cells identified by lysozyme expression were reduced in high fat fed mice. NCT reversed the effect of high fat diet on Paneth cells, with multiple markers being induced, including a number of defensins, which are critical for Paneth cell function and intestinal barrier integrity. NCT upregulated genes that play important role in IBD and that are downregulated in that disease. It reversed the loss of Paneth cell markers that occurred in high fat diet fed mice. These data suggest that HNF4α could be a therapeutic target for IBD and that the agonists that we have identified could be candidate therapeutics.

Long-term oral administration of an HNF4α agonist prevents weight gain and hepatic steatosis by promoting increased mitochondrial mass and function.

We report here that the potent HNF4α agonist N-trans-caffeoyltyramine (NCT) promotes weight loss by inducing an increase in mitochondrial mass and function, including fatty acid oxidation. Previously, we found in a short term trial in obese mice that NCT promoted reversal of hepatic steatosis through a mechanism involving the stimulation of lipophagy by dihydroceramides. NCT led to increased dihydroceramide levels by inhibiting dihydroceramide conversion to ceramides. Here, we were able to administer NCT orally, permitting longer term administration. Mice fed NCT mixed with high fat diet exhibited decreased weight. Examination of RNA-seq data revealed an increase in PPARGC1A, a central regulator of mitochondrial biogenesis. In addition to the decreased hepatic steatosis that we found previously, mice fed a high fat diet containing NCT mice weighed substantially less than control mice fed high fat diet alone. They had increased mitochondrial mass, exhibited increased fatty acid oxidation, and had an increased level of NAD. Markers of liver inflammation such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), which are important in the progression of non-alcoholic fatty liver disease to non-alcoholic steatohepatitis were decreased by NCT. There was no evidence of any toxicity from NCT consumption. These results indicate that HNF4α is an important regulator of mitochondrial mass and function and support that use of HNF4α to treat disorders of fatty acid excess, potentially including obesity, NAFLD, and NASH.

Resolving the conflicts around Par2 opposing roles in regeneration by comparing immune-mediated and toxic-induced injuries.

BACKGROUND: Different factors may lead to hepatitis. Among which are liver inflammation and poisoning. We chose two hepatitis models, typical for these two underlying causes. Thus, we aimed to characterize the role of protease-activated receptor 2 (Par2) in liver regeneration and inflammation to reconcile Par2 conflicting role in many damage models, which sometimes aggravates the induced damage and sometimes alleviates it. METHODS: WT and knockout (Par2KO) mice were injected with concanavalin A (ConA) to induce immune-mediated hepatitis or with carbon tetrachloride (CCl4) to elicit direct hepatic damage. To distinguish the immune component from the liver regenerative response, we conducted bone marrow (BM) replacements of WT and Par2KO mice and repeated the damage models. RESULTS: ConA injection caused limited damage in Par2KO mice livers, while in the WT mice severe damage followed by leukocyte infiltration was evident. Reciprocal BM replacement of WT and Par2KO showed that WT BM-reconstituted Par2KO mice displayed marked liver damage, while in Par2KO BM-reconstituted WT mice, the tissue was generally protected. In the CCl4 direct damage model, hepatocytes regenerated in WT mice, whereas Par2KO mice failed to recover. Reciprocal BM replacement did not show significant differences in hepatic regeneration. In Par2KO mice, hepatitis was more apparent, while WT recovered regardless of the BM origin. CONCLUSIONS: We conclude that Par2 activation in the immune system aggravates hepatitis and that Par2 activation in the damaged tissue promotes liver regeneration. When we incorporate this finding and revisit the literature reports, we reconciled the conflicts surrounding Par2's role in injury, recovery, and inflammation.

Pancreatic ß-cell neogenesis by direct conversion from mature α-cells.

Because type 1 and type 2 diabetes are characterized by loss of ß-cells, ß-cell regeneration has garnered great interest as an approach to diabetes therapy. Here, we developed a new model of ß-cell regeneration, combining pancreatic duct ligation (PDL) with elimination of pre-existing ß-cells with alloxan. In this model, in which virtually all ß-cells observed are neogenic, large numbers of ß-cells were generated within 2 weeks. Strikingly, the neogenic ß-cells arose primarily from α-cells. α-cell proliferation was prominent following PDL plus alloxan, providing a large pool of precursors, but we found that ß-cells could form from α-cells by direct conversion with or without intervening cell division. Thus, classical asymmetric division was not a required feature of the process of α- to ß-cell conversion. Intermediate cells coexpressing α-cell- and ß-cell-specific markers appeared within the first week following PDL plus alloxan, declining gradually in number by 2 weeks as ß-cells with a mature phenotype, as defined by lack of glucagon and expression of MafA, became predominant. In summary, these data revealed a novel function of α-cells as ß-cell progenitors. The high efficiency and rapidity of this process make it attractive for performing the studies required to gain the mechanistic understanding of the process of α- to ß-cell conversion that will be required for eventual clinical translation as a therapy for diabetes.

Liver fat storage is controlled by HNF4α through induction of lipophagy and is reversed by a potent HNF4α agonist.

We report the discovery of strong HNF4α agonists and their use to uncover a previously unknown pathway by which HNF4α controls the level of fat storage in the liver. This involves the induction of lipophagy by dihydroceramides, the synthesis and secretion of which is controlled by genes induced by HNF4α. The HNF4α activators are N-trans caffeoyltyramine (NCT) and N-trans feruloyltyramine (NFT), which are structurally related to the known drugs alverine and benfluorex, which we previously showed to be weak HNF4α activators. In vitro, NCT and NFT induced fat clearance from palmitate-loaded cells. In DIO mice, NCT led to recovery of hepatic HNF4α expression and reduction of steatosis. Mechanistically, increased dihydroceramide production and action downstream of HNF4α occurred through increased expression of HNF4α downstream genes, including SPNS2 and CYP26A1. NCT was completely nontoxic at the highest dose administered and so is a strong candidate for an NAFLD therapeutic.

beta-cell Regeneration: neogenesis, replication or both?

Both type I and type II diabetes are characterized by beta-cell loss and dysfunction. Therefore, a major goal of diabetes therapy is to promote the formation of new beta-cells, either in vitro for transplantation or in vivo, i.e., beta-cell regeneration. The question of whether beta-cell regeneration occurs by replication of preexisting beta-cells or by neogenesis from a precursor within the pancreas is a major focus of interest. Lineage-tracing studies have found evidence only for beta-cell replication, while earlier studies based upon the appearance of insulin-positive cells in areas outside of islets formed the basis for the belief that neogenesis from precursors can occur in adult animals. Recently, we found that nonendocrine pancreatic epithelial cells could be induced to undergo endocrine differentiation under the influence of inductive factors from the human fetal pancreas. One possibility is that, similar to models of hepatocyte regeneration, beta-cells can arise either by neogenesis or replication, depending on the particular stimulus. Clearly, understanding the nature and control of beta-cell regeneration is critical for success in efforts to treat diabetes by beta-cell replacement.

Insulin acts as a repressive factor to inhibit the ability of PAR2 to induce islet cell transdifferentiation.

Recently, we showed that pancreatitis in the context of profound ß-cell deficiency was sufficient to induce islet cell transdifferentiation. In some circumstances, this effect was sufficient to result in recovery from severe diabetes. More recently, we showed that the molecular mechanism by which pancreatitis induced ß-cell neogenesis by transdifferentiation was activation of an atypical GPCR called Protease-Activated Receptor 2 (PAR2). However, the ability of PAR2 to induce transdifferentiation occurred only in the setting of profound ß-cell deficiency, implying the existence of a repressive factor from those cells. Here we show that the repressor from ß-cells is insulin. Treatment of primary islets with a PAR2 agonist (2fLI) in combination with inhibitors of insulin secretion and signaling was sufficient to induce insulin and PAX4 gene expression. Moreover, in primary human islets, this treatment also led to the induction of bihormonal islet cells coexpressing glucagon and insulin, a hallmark of islet cell transdifferentiation. Mechanistically, insulin inhibited the positive effect of a PAR2 agonist on insulin gene expression and also led to an increase in PAX4, which plays an important role in islet cell transdifferentiation. The studies presented here demonstrate that insulin represses transdifferentiation of α- to ß-cells induced by activation of PAR2. This provides a mechanistic explanation for the observation that α- to ß-cell transdifferentiation occurs only in the setting of severe ß-cell ablation. The mechanistic understanding of islet cell transdifferentiation and the ability to modulate that process using available pharmacological reagents represents an important step along the path towards harnessing this novel mechanism of ß-cell neogenesis as a therapy for diabetes.

High-throughput screening and bioinformatic analysis to ascertain compounds that prevent saturated fatty acid-induced ß-cell apoptosis.

Pancreatic ß-cell lipotoxicity is a central feature of the pathogenesis of type 2 diabetes. To study the mechanism by which fatty acids cause ß-cell death and develop novel approaches to prevent it, a high-throughput screen on the ß-cell line INS1 was carried out. The cells were exposed to palmitate to induce cell death and compounds that reversed palmitate-induced cytotoxicity were ascertained. Hits from the screen were analyzed by an increasingly more stringent testing funnel, ending with studies on primary human islets treated with palmitate. MAP4K4 inhibitors, which were not part of the screening libraries but were ascertained by a bioinformatics analysis, and the endocannabinoid anandamide were effective at inhibiting palmitate-induced apoptosis in INS1 cells as well as primary rat and human islets. These targets could serve as the starting point for the development of therapeutics for type 2 diabetes.

Telomere-independent cellular senescence in human fetal cardiomyocytes.

Fetal cardiomyocytes have been proposed as a potential source of cell-based therapy for heart failure. This study examined cellular senescence in cultured human fetal ventricular cardiomyocytes (HFCs). HFCs were isolated and identified by immunocytochemistry and RT-PCR. Cells were found to senesce after 20-25 population doublings, as determined by growth arrest, morphological changes and senescence-associated beta-galactosidase activity. Using the telomeric repeat amplification protocol assay, telomerase activity was undetectable in primary HFCs. Cells were transduced to express the human reverse transcriptase subunit (hTERT) of telomerase. This resulted in greatly increased telomerase activity, but no significant lifespan extension. Analysis of telomere length in primary HFCs revealed that the senescent phenotype was not accompanied by telomere shortening. Telomeres in hTERT-positive cells were elongated in comparison with primary cells, and elongation was retained in senescent cells. Levels of the tumor suppressor protein p16INK4A increased in all senescent cells whether telomerase-positive or -negative. Senescence was accompanied by a decline in transcript levels of the polycomb gene Bmi-1, Ets1 and Ets2 transcription factors, and Id1, Id2 and Id3 helix-loop-helix proteins, suggesting roles for these genes in maintenance of cardiomyocyte proliferative capacity. In addition to offering novel insights into the behavior of human fetal cardiomyocytes in culture, these findings have implications for the development of a cell-based therapy for cardiac injury using primary fetal heart tissue.

PAR2 regulates regeneration, transdifferentiation, and death.

Understanding the mechanisms by which cells sense and respond to injury is central to developing therapies to enhance tissue regeneration. Previously, we showed that pancreatic injury consisting of acinar cell damage+ß-cell ablation led to islet cell transdifferentiation. Here, we report that the molecular mechanism for this requires activating protease-activated receptor-2 (PAR2), a G-protein-coupled receptor. PAR2 modulation was sufficient to induce islet cell transdifferentiation in the absence of ß-cells. Its expression was modulated in an islet cell type-specific manner in murine and human type 1 diabetes (T1D). In addition to transdifferentiation, PAR2 regulated ß-cell apoptosis in pancreatitis. PAR2's role in regeneration is broad, as mice lacking PAR2 had marked phenotypes in response to injury in the liver and in digit regeneration following amputation. These studies provide a pharmacologically relevant target to induce tissue regeneration in a number of diseases, including T1D.

c-Myc controls proliferation versus differentiation in human pancreatic endocrine cells.

Using immortalized human pancreatic endocrine cell lines, we have shown previously that differentiation into hormone-expressing cells requires cell-cell contact acting in synergy with the homeodomain transcription factor pancreatic duodenal homeobox-1 (PDX-1). Although differentiation is associated with a decrease in cell proliferation, the mechanisms behind this relationship are not known. Using TRM-6, a delta cell line, and betalox5, a beta-cell line, we show here that cell-cell contact and subsequent endocrine differentiation lead to a down-regulation of the c-myc protooncogene. Overexpression of c-Myc obtained with an inducible c-Myc-estrogen receptor fusion protein results in an increase in cell proliferation and the ablation of hormone expression. Moreover, we show that although c-Myc is expressed in a subset of cells from the human fetal and adult pancreas, it is absent in differentiated endocrine cells. The mechanism by which c-Myc interferes with hormone expression may be through effects on the homeodomain transcription factor PDX-1, as immunostaining for PDX-1 in cells with activated c-Myc revealed a redistribution of PDX-1 from the nucleus to the cytoplasm. These results suggest that c-Myc plays a central role in a cell-cell contact-mediated switch mechanism by which cell division vs. differentiation in endocrine cells is determined.

Cell-based therapies for diabetes: progress towards a transplantable human beta cell line.

Achieving normoglycemia is the goal of diabetes therapy. Potentially, there are many ways to achieve this goal, including transplantation of cells exhibiting glucose-responsive insulin secretion. However, to be applicable to the large number of people who might benefit from beta cell replacement, an unlimited supply of beta cells must be found. To address this problem, we have been developing cell lines from the human endocrine pancreas. In one case, a cell line, betalox5, has been developed from human islets that can be induced under some circumstances to differentiate into functional beta cells exhibiting appropriate glucose-responsive insulin secretion. Inducing differentiation is complex, requiring the activation of multiple signaling pathways, including those downstream of those involved in cell-cell contact and the glucagon-like peptide-1 receptor. In addition, transfer of the PDX-1 gene is also necessary to render the cells competent for differentiation. However, it is clear that many other genes are involved in maintaining the commitment of betalox5 cells towards the beta cell lineage. Understanding the complement of genes required to establish and maintain a beta cell lineage commitment would be enormously helpful in efforts to develop a cell line that can be used for beta cell replacement therapies. Here, we provide further information on the characteristics of cell lines that we have developed from the human pancreas that are relevant to the development of a beta cell replacement therapy for diabetes.

Islet expression of the DNA repair enzyme 8-oxoguanosine DNA glycosylase (Ogg1) in human type 2 diabetes.

BACKGROUND: It has become increasingly clear that beta-cell failure plays a critical role in the pathogenesis of type 2 diabetes. Free-radical mediated beta-cell damage has been intensively studied in type 1 diabetes, but not in human type 2 diabetes. Therefore, we studied the protein expression of the DNA repair enzyme Ogg1 in pancreases from type 2 diabetics. Ogg1 was studied because it is the major enzyme involved in repairing 7,8-dihydro-8-oxoguanosine DNA adducts, a lesion previously observed in a rat model of type 2 diabetes. Moreover, in a gene expression screen, Ogg1 was over-expressed in islets from a human type 2 diabetic. METHODS: Immunofluorescent staining of Ogg1 was performed on pancreatic specimens from healthy controls and patients with diabetes for 2-23 years. The intensity and islet area stained for Ogg1 was evaluated by semi-quantitative scoring. RESULTS: Both the intensity and the area of islet Ogg1 staining were significantly increased in islets from the type 2 diabetic subjects compared to the healthy controls. A correlation between increased Ogg1 fluorescent staining intensity and duration of diabetes was also found. Most of the staining observed was cytoplasmic, suggesting that mitochondrial Ogg1 accounts primarily for the increased Ogg1 expression. CONCLUSION: We conclude that oxidative stress related DNA damage may be a novel important factor in the pathogenesis of human type 2 diabetes. An increase of Ogg1 in islet cell mitochondria is consistent with a model in which hyperglycemia and consequent increased beta-cell oxidative metabolism lead to DNA damage and the induction of Ogg1 expression.

Maternal embryonic leucine zipper kinase regulates pancreatic ductal, but not ß-cell, regeneration.

The maternal embryonic leucine zipper kinase (MELK) is expressed in stem/progenitor cells in some adult tissues, where it has been implicated in diverse biological processes, including the control of cell proliferation. Here, we described studies on its role in adult pancreatic regeneration in response to injury induced by duct ligation and ß-cell ablation. MELK expression was studied using transgenic mice expressing GFP under the control of the MELK promoter, and the role of MELK was studied using transgenic mice deleted in the MELK kinase domain. Pancreatic damage was initiated using duct ligation and chemical beta-cell ablation. By tracing MELK expression using a MELK promoter-GFP transgene, we determined that expression was extremely low in the normal pancreas. However, following duct ligation and ß-cell ablation, it became highly expressed in pancreatic ductal cells while remaining weakly expressed in α-cells and ß- cells. In a mutant mouse in which the MELK kinase domain was deleted, there was no effect on pancreatic development. There was no apparent effect on islet regeneration, either. However, following duct ligation there was a dramatic increase in the number of small ducts, but no change in the total number of duct cells or duct cell proliferation. In vitro studies indicated that this was likely due to a defect in cell migration. These results implicate MELK in the control of the response of the pancreas to injury, specifically controlling cell migration in normal and transformed pancreatic duct cells.

Identification of alverine and benfluorex as HNF4α activators.

The principal finding of this study is that two drugs, alverine and benfluorex, used in vastly different clinical settings, activated the nuclear receptor transcription factor HNF4α. Both were hits in a high-throughput screen for compounds that reversed the inhibitory effect of the fatty acid palmitate on human insulin promoter activity. Alverine is used in the treatment of irritable bowel syndrome, while benfluorex (Mediator) was used to treat hyperlipidemia and type II diabetes. Benfluorex was withdrawn from the market recently because of serious cardiovascular side effects related to fenfluramine-like activity. Strikingly, alverine and benfluorex have a previously unrecognized structural similarity, consistent with a common mechanism of action. Gene expression and biochemical studies revealed that they both activate HNF4α. This novel mechanism of action should lead to a reinterpretation of previous studies with these drugs and suggests a path toward the development of therapies for diseases such as inflammatory bowel and diabetes that may respond to HNF4α activators.