Biophysical insights into glucose-dependent transcriptional regulation by PDX1.

The pancreatic and duodenal homeobox 1 (PDX1) is a central regulator of glucose-dependent transcription of insulin in pancreatic ß cells. PDX1 transcription factor activity is integral to the development and sustained health of the pancreas; accordingly, deciphering the complex network of cellular cues that lead to PDX1 activation or inactivation is an important step toward understanding the etiopathologies of pancreatic diseases and the development of novel therapeutics. Despite nearly 3 decades of research into PDX1 control of Insulin expression, the molecular mechanisms that dictate the function of PDX1 in response to glucose are still elusive. The transcriptional activation functions of PDX1 are regulated, in part, by its two intrinsically disordered regions, which pose a barrier to its structural and biophysical characterization. Indeed, many studies of PDX1 interactions, clinical mutations, and posttranslational modifications lack molecular level detail. Emerging methods for the quantitative study of intrinsically disordered regions and refined models for transactivation now enable us to validate and interrogate the biochemical and biophysical features of PDX1 that dictate its function. The goal of this review is to summarize existing PDX1 studies and, further, to generate a comprehensive resource for future studies of transcriptional control via PDX1.

Unknown presentation of a rare genetic disorder: Monogenic diabetes in young type 4 presenting with hepatic cysts and procoagulant state.

Maturity onset diabetes in young (MODY) is the most common form of monogenic diabetes, which characteristically presents in adolescents and young adults. Till date, pathogenic variations involving 14 different genes have been causally implicated with the development of MODY. Maturity onset diabetes in young type 4 (MODY-4) is a very rare form of MODY. We present here case of 28-year-old nonobese male patient with distinct family history of diabetes spanning two generations, incidentally, detected to have a rare form of diabetes on genetic analysis when he presented with recurrent thromboembolic manifestations: deep vein thrombosis and pulmonary thromboembolism. Our case highlights a previously unknown disease association of a rare genetic disorder. Increasing awareness about this genetic disorder and early identification of such cases will enhance our understanding of hitherto unknown disease associations and the pathophysiological role of genetic mutations. This may contribute to the improved treatment and prevention of debilitating diseases such as diabetes.

FoxO1 inhibition promotes differentiation of human embryonic stem cells into insulin producing cells.

Insulin-producing cells (IPCs) derived from human embryonic stem cells (hESCs) hold great potential for cell transplantation therapy in diabetes. Tremendous progress has been made in inducing differentiation of hESCs into IPCs in vitro, of which definitive endoderm (DE) protocol mimicking foetal pancreatic development has been widely used. However, immaturity of the obtained IPCs limits their further applications in treating diabetes. Forkhead box O1 (FoxO1) is involved in the differentiation and functional maintenance of murine pancreatic ß cells, but its role in human ß cell differentiation is under elucidation. Here, we showed that although FoxO1 expression level remained consistent, cytoplasmic phosphorylated FoxO1 protein level increased during IPC differentiation of hESCs induced by DE protocol. Lentiviral silencing of FoxO1 in pancreatic progenitors upregulated the levels of pancreatic islet differentiation-related genes and improved glucose-stimulated insulin secretion response in their progeny IPCs, whereas overexpression of FoxO1 showed the opposite effects. Notably, treatment with the FoxO1 inhibitor AS1842856 displayed similar effects with FoxO1 knockdown in pancreatic progenitors. These effects were closely associated with the mutually exclusive nucleocytoplasmic shuttling of FoxO1 and Pdx1 in the AS1842856-treated pancreatic progenitors. Our data demonstrated a promising effect of FoxO1 inhibition by the small molecule on gene expression profile during the differentiation, and in turn, on determining IPC maturation via modulating subcellular location of FoxO1 and Pdx1. Therefore, we identify a novel role of FoxO1 inhibition in promoting IPC differentiation of hESCs, which may provide clues for induction of mature ß cells from hESCs and clinical applications in regenerative medicine.

Cooperative function of Pdx1 and Oc1 in multipotent pancreatic progenitors impacts postnatal islet maturation and adaptability.

The transcription factors pancreatic and duodenal homeobox 1 (Pdx1) and onecut1 (Oc1) are coexpressed in multipotent pancreatic progenitors (MPCs), but their expression patterns diverge in hormone-expressing cells, with Oc1 expression being extinguished in the endocrine lineage and Pdx1 being maintained at high levels in ß-cells. We previously demonstrated that cooperative function of these two factors in MPCs is necessary for proper specification and differentiation of pancreatic endocrine cells. In those studies, we observed a persistent decrease in expression of the ß-cell maturity factor MafA. We therefore hypothesized that Pdx1 and Oc1 cooperativity in MPCs impacts postnatal ß-cell maturation and function. Here our model of Pdx1-Oc1 double heterozygosity was used to investigate the impact of haploinsufficiency for both of these factors on postnatal ß-cell maturation, function, and adaptability. Examining mice at postnatal day (P) 14, we observed alterations in pancreatic insulin content in both Pdx1 heterozygotes and double heterozygotes. Gene expression analysis at this age revealed significantly decreased expression of many genes important for glucose-stimulated insulin secretion (e.g., Glut2, Pcsk1/2, Abcc8) exclusively in double heterozygotes. Analysis of P14 islets revealed an increase in the number of mixed islets in double heterozygotes. We predicted that double-heterozygous ß-cells would have an impaired ability to respond to stress. Indeed, we observed that ß-cell proliferation fails to increase in double heterozygotes in response to either high-fat diet or placental lactogen. We thus report here the importance of cooperation between regulatory factors early in development for postnatal islet maturation and adaptability.

Pancreatic and duodenal homeobox protein 1 (Pdx-1) maintains endoplasmic reticulum calcium levels through transcriptional regulation of sarco-endoplasmic reticulum calcium ATPase 2b (SERCA2b) in the islet ß cell.

Although the pancreatic duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in ß cell development and secretory function, recent data also implicate Pdx-1 in the maintenance of endoplasmic reticulum (ER) health. The sarco-endoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) gradient between the cytosol and ER lumen. In models of diabetes, our data demonstrated loss of ß cell Pdx-1 that occurs in parallel with altered SERCA2b expression, whereas in silico analysis of the SERCA2b promoter revealed multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b levels and activity with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. The results revealed reduced SERCA2b expression and decreased ER Ca(2+), which was measured using fluorescence lifetime imaging microscopy. Cotransfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity 3- to 4-fold relative to an empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1(+/-) mice were fed a high-fat diet. Isolated islets demonstrated an increased spliced-to-total Xbp1 ratio, whereas SERCA2b overexpression reduced the Xbp1 ratio to that of wild-type controls. Together, these results identify SERCA2b as a novel transcriptional target of Pdx-1 and define a role for altered ER Ca(2+) regulation in Pdx-1-deficient states.

A high-fat diet activates oncogenic Kras and COX2 to induce development of pancreatic ductal adenocarcinoma in mice.

BACKGROUND & AIMS: Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), but it is not clear how obesity contributes to pancreatic carcinogenesis. The oncogenic form of KRAS is expressed during early stages of PDAC development and is detected in almost all of these tumors. However, there is evidence that mutant KRAS requires an additional stimulus to activate its full oncogenic activity and that this stimulus involves the inflammatory response. We investigated whether the inflammation induced by a high-fat diet, and the accompanying up-regulation of cyclooxygenase-2 (COX2), increases Kras activity during pancreatic carcinogenesis in mice. METHODS: We studied mice with acinar cell-specific expression of KrasG12D (LSL-Kras/Ela-CreERT mice) alone or crossed with COX2 conditional knockout mice (COXKO/LSL-Kras/Ela-CreERT). We also studied LSL-Kras/PDX1-Cre mice. All mice were fed isocaloric diets with different amounts of fat, and a COX2 inhibitor was administered to some LSL-Kras/Ela-CreERT mice. Pancreata were collected from mice and analyzed for Kras activity, levels of phosphorylated extracellular-regulated kinase, inflammation, fibrosis, pancreatic intraepithelial neoplasia (PanIN), and PDACs. RESULTS: Pancreatic tissues from LSL-Kras/Ela-CreERT mice fed high-fat diets (HFDs) had increased Kras activity, fibrotic stroma, and numbers of PanINs and PDACs than LSL-Kras/Ela-CreERT mice fed control diets; the mice fed the HFDs also had shorter survival times than mice fed control diets. Administration of a COX2 inhibitor to LSL-Kras/Ela-CreERT mice prevented these effects of HFDs. We also observed a significant reduction in survival times of mice fed HFDs. COXKO/LSL-Kras/Ela-CreERT mice fed HFDs had no evidence for increased numbers of PanIN lesions, inflammation, or fibrosis, as opposed to the increases observed in LSL-Kras/Ela-CreERT mice fed HFDs. CONCLUSIONS: In mice, an HFD can activate oncogenic Kras via COX2, leading to pancreatic inflammation and fibrosis and development of PanINs and PDAC. This mechanism might be involved in the association between risk for PDAC and HFDs.

Reprogramming of enteroendocrine K cells to pancreatic ß-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.

Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic ß-cells in a process called cell reprogramming. Enteroendocrine cells and ß-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in ß-cells. Thus, K cells could be reprogrammed to ß-cells under certain conditions. However, there is no clear evidence on whether these cells convert to ß-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1(+)-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1(+)-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1(+)-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to ß-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.

Neuroendocrine carcinoma arising in a hepatitis C virus-infected liver: mechanism of the tumor development may be similar to that of development of pancreatic neuroendocrine cells.

We experienced a case of neuroendocrine carcinoma (NC). The tumor developed in the cirrhotic liver of a 62-year-old Japanese man who had been infected with hepatitis C virus. The tumor cells showed high N/C ratio, formed many rosettes, and expressed CD56, synaptophysin, HepPar1 and pancreatic and duodenal homeobox 1. MIB1 expression was 65%. Because both liver and pancreas are derived from a common endodermal layer during fetal development, we speculated that the tumor may have formed via the interaction of neurogenin 3, insulinoma-associated 1 gene and NeuroD/beta2, which are involved in the stage at which some pancreatic cells commit to becoming endocrine cells. Molecular analysis revealed that the NC had higher relative expression levels of mRNA of the three molecules than did the nontumorous liver. The results indicate that the NC in this patient may have formed via the same mechanism that acts in the development of pancreatic neuroendocrine cells.

Specific reprogramming of alpha cells to insulin-producing cells by short glucagon promoter-driven Pdx1 and MafA.

Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we used an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 (AAV8) can be used to successfully deliver Pdx1 and MafA to pancreatic alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells were also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.

Mechanistic Investigation of GHS-R Mediated Glucose-Stimulated Insulin Secretion in Pancreatic Islets.

Ghrelin receptor, a growth hormone secretagogue receptor (GHS-R), is expressed in the pancreas. Emerging evidence indicates that GHS-R is involved in the regulation of glucose-stimulated insulin secretion (GSIS), but the mechanism by which GHS-R regulates GSIS in the pancreas is unclear. In this study, we investigated the role of GHS-R on GSIS in detail using global Ghsr-/- mice (in vivo) and Ghsr-ablated pancreatic islets (ex vivo). GSIS was attenuated in both Ghsr-/- mice and Ghsr-ablated islets, while the islet morphology was similar between WT and Ghsr-/- mice. To elucidate the mechanism underpinning Ghsr-mediated GSIS, we investigated the key steps of the GSIS signaling cascade. The gene expression of glucose transporter 2 (Glut2) and the glucose-metabolic intermediate-glucose-6-phosphate (G6P) were reduced in Ghsr-ablated islets, supporting decreased glucose uptake. There was no difference in mitochondrial DNA content in the islets of WT and Ghsr-/- mice, but the ATP/ADP ratio in Ghsr-/- islets was significantly lower than that of WT islets. Moreover, the expression of pancreatic and duodenal homeobox 1 (Pdx1), as well as insulin signaling genes of insulin receptor (IR) and insulin receptor substrates 1 and 2 (IRS1/IRS2), was downregulated in Ghsr-/- islets. Akt is the key mediator of the insulin signaling cascade. Concurrently, Akt phosphorylation was reduced in the pancreas of Ghsr-/- mice under both insulin-stimulated and homeostatic conditions. These findings demonstrate that GHS-R ablation affects key components of the insulin signaling pathway in the pancreas, suggesting the existence of a cross-talk between GHS-R and the insulin signaling pathway in pancreatic islets, and GHS-R likely regulates GSIS via the Akt-Pdx1-GLUT2 pathway.

Annexin A10 is involved in the induction of pancreatic duodenal homeobox­1 in gastric cancer tissue, cells and organoids.

Gastric cancer (GC) is the third most common cause of cancer­related death in the world. Annexin A10 (ANXA10), a member of the Annexin family, is a calcium­/phospholipid­binding protein; however, little is known concerning its functions. It is still unclear what molecule is involved in the induction of ANXA10. In the present study, we performed immunohistochemistry to evaluate the expression of ANXA10, pancreatic and duodenal homeobox­1 (PDX1) and mucin phenotype markers in 130 GC samples. ANXA10 was detected in 63 (48%) of the 130 GC cases and loss of ANXA10 was significantly correlated with disease progression and poor clinical outcomes in GC. PDX1 was significantly correlated with ANXA10 in GC cases and cell lines. Although PDX1 was not significantly correlated with the GC cases with any of the mucin phenotypes, ANXA10 was preferentially detected in the GC cases with the gastric mucin phenotype. As a further investigation, we generated organoids derived from human GC and identified the duplication of the mucin phenotypes of GC by immunohistochemistry. The repression effect on cell growth that was observed in the ANXA10­knockdown cell lines was also clearly observed in the human gastric organoids. We demonstrated that the expression of ANXA10 was correlated with the gastric mucin phenotype and ANXA10 was involved in the induction of PDX1 expression in GC. We also provided evidence that GC organoids represent a powerful tool for scrutinizing the biology of GC, especially with regard to the mucin phenotype.

Role and mechanism of PI3K/AKT/FoxO1/PDX-1 signaling pathway in functional changes of pancreatic islets in rats after severe burns.

AIMS: Studies on diabetes mellitus have shown that the phosphoinositide 3-kinase (PI3K)/serine threonine kinase (AKT)/forkhead box protein O1 (FoxO1) signaling pathway can regulate insulin secretion by modulating the expression of pancreatic and duodenal homeobox-1 (PDX-1). Therefore, it was hypothesized that the pathway also played an important role in functional abnormalities of pancreatic islets after severe burns. This study aimed to explore the role and mechanism of the PI3K/AKT/FoxO1/PDX-1 signaling pathway in functional changes of pancreatic islets in rats post severe burns. MAIN METHODS: Rats were grouped, subjected to full thickness burn injuries involving 50% total body surface area (TBSA), and injected intraperitoneally with BPV (HOpic) (0.6 mg/kg) or DMSO (0.55 mg/kg) once a day for three days. Glucose metabolism related indexes were measured by the glucometer, transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA). PI3K/AKT/FoxO1/PDX-1 signaling pathway related indexes were detected through immunofluorescence, western blot and RT-qPCR analyses. KEY FINDINGS: Dysglycemia and impaired insulin secretion occurred in rats, the activity of the PI3K/AKT/FoxO1/PDX-1 signaling pathway in the islets fell, and PDX-1 was translocated from the nucleus to the cytoplasm post severe burns. When BPV (HOpic) was used, glucose metabolism and insulin secretion were improved, the activity of the PI3K/AKT/FoxO1/PDX-1 signaling pathway in the islets was up-regulated, and PDX-1 was redistributed from the cytoplasm to the nucleus. SIGNIFICANCE: The activity of the PI3K/AKT/FoxO1/PDX-1 signaling pathway declined following severe burns. When its activity was up-regulated, insulin secretion could be improved, thus ameliorating hyperglycemia.

SIRT5 regulates pancreatic ß-cell proliferation and insulin secretion in type 2 diabetes.

Impaired insulin secretion and insulin resistance are the primary characteristics of type 2 diabetes (T2D). However, the mechanisms underlying insulin secretion failure have yet to be elucidated. The present study demonstrated that sirtuin 5 (SIRT5) is upregulated in patients with T2D and in pancreatic ß-cell lines. It was also revealed that elevated SIRT5 expression is positively associated with age and blood glucose levels, and negatively associated with pancreatic and duodenal homeobox 1 (PDX1) expression. Colony formation and Cell Counting Kit-8 assays demonstrated that SIRT5 suppressed the proliferation of pancreatic ß-cells in vitro. In addition, downregulation of SIRT5 promoted the secretion of insulin. Additionally, SIRT5 ectopic expression downregulated the expression of PDX1 and the inhibition of SIRT5 upregulated PDX1 expression. Chromatin immunoprecipitation assay analysis demonstrated that SIRT5 may regulate the transcription of PDX1 via H4K16 deacetylation. In conclusion, the results of the present study indicate that SIRT5 may serve an important role in the pathogenesis of T2D, and may present a novel therapeutic target for the treatment of patients with T2D.

Columnar-Lined Esophagus Develops via Wound Repair in a Surgical Model of Reflux Esophagitis.

Background & Aims: After esophagojejunostomy, rodents develop reflux esophagitis and a columnar-lined esophagus with features of Barrett's metaplasia. This rodent columnar-lined esophagus has been proposed to develop from cellular reprogramming of progenitor cells, but studies on early columnar-lined esophagus development are lacking. We performed a systematic, histologic, and immunophenotypic analysis of columnar-lined esophagus development in rats after esophagojejunostomy. Methods: At various times after esophagojejunostomy in 52 rats, the esophagus was removed and tissue sections were evaluated for type, location, and length of columnar lining. Molecular characteristics were evaluated by immunohistochemistry and immunofluorescence. Results: At week 2, ulceration was seen in esophageal squamous epithelium, starting distally at the esophagojejunostomy anastomosis. Re-epithelialization of the distal ulcer segment occurred via proliferation and expansion of immature-appearing glands budding directly off jejunal crypts, characteristic of wound healing. The columnar-lined esophagus's immunoprofile was similar to jejunal crypt epithelium, and columnar-lined esophagus length increased significantly from 0.15 mm (±0.1 SEM) at 2 weeks to 5.22 mm (±0.37) at 32 weeks. Neoglands were found within esophageal ulcer beds, and spindle-shaped cells expressing epithelial-mesenchymal transition markers were found at the columnar-lined esophagus's leading edge. Only proliferative squamous epithelium was found at the proximal ulcer border. Conclusions: After esophagojejunostomy in rats, metaplastic columnar-lined esophagus develops via a wound healing process that does not appear to involve cellular reprogramming of progenitor cells. This process involves EMT-associated migration of jejunal cells into the esophagus, where they likely have a competitive advantage over squamous cells in the setting of ongoing gastroesophageal reflux disease.

A multiple-funnels cell culture insert for the scale-up production of uniform cell spheroids.

INTRODUCTION: Formation of cell spheres is an important procedure in biomedical research. A large number of high-quality cell spheres of uniform size and shape are required for basic studies and therapeutic applications. Conventional approaches, including the hanging drop method and suspension culture, are used for cell sphere production. However, these methods are time consuming, cell spheres cannot be harvested easily, and it is difficult to control the size and geometry of cell spheres. To resolve these problems, a novel multiple-funnel cell culture insert was designed for size controlling, easy harvesting, and scale-up production of cell spheres. METHODS: The culture substrate has 680 micro-funnels with a 1-mm width top, 0.89 mm depth, and 0.5 mm square bottom. Mouse embryonic stem cells were used to test the newly developed device. The seeded embryonic stem cells settled at the downward medium surface toward the bottom opening and aggregated as embryoid bodies (EBs). For cell sphere harvest, the bottom of the culture insert was put in contact with the medium surface in another culture dish, and the medium in the device flowed down with cell spheres by hydrostatic pressure. RESULTS: Compact cell spheres with uniform size and shape were collected easily. The diameter of the spheres could be controlled by adjusting the seeding cell density. Spontaneous neural differentiation (nestin and Tju1) and retinoic acid-induced endodermal differentiation (Pdx-1 and insulin I) were improved in the EBs produced using the new insert compared to those in EBs produced by suspension culture. CONCLUSIONS: This novel cell culture insert shall improve future studies of cell spheres and benefit clinical applications of cell therapy.

PDX1 associated therapy in translational medicine.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely poor prognosis and a low median survival due to lack of the early and reliable detection and effective therapeutic options, despite improvements observed for many other cancers in last decade. Pancreatic and duodenal homeobox 1 (PDX1), which is a homeodomain-containing transcription factor and a key regulator for insulin gene expression, ß cell maturation and proper ß cell function maintenance in the pancreas. Our previous studies revealed that PDX1 promotes tumorigenesis and it is a promising therapeutic target for PDAC. For translational purposes, we developed three therapeutic platforms utilizing RNA interference (RNAi), gene therapy and small inhibitory drug targeting PDX1, and further validated them in PDAC preclinical models both in vitro and in vivo. These PDX1 targeted therapies significantly inhibited PDX1 expression in PDAC cells, ablated PDX1-expressing human PDAC xenograft tumor growth, and prolonged survival in the PDAC mouse models. The data from these preclinical studies proved the translational potentials of PDX1 targeted therapies in PDAC and suggest that the strategy of developing PDX1 targeted therapies would permit a rapid bench-to-bedside translation of other relevant gene therapies, which would eventually benefit the patients suffering from this deadly disease.

An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion.

OBJECTIVE: Transport of pyruvate into the mitochondrial matrix by the Mitochondrial Pyruvate Carrier (MPC) is an important and rate-limiting step in its metabolism. In pancreatic ß-cells, mitochondrial pyruvate metabolism is thought to be important for glucose sensing and glucose-stimulated insulin secretion. METHODS: To evaluate the role that the MPC plays in maintaining systemic glucose homeostasis, we used genetically-engineered Drosophila and mice with loss of MPC activity in insulin-producing cells. RESULTS: In both species, MPC deficiency results in elevated blood sugar concentrations and glucose intolerance accompanied by impaired glucose-stimulated insulin secretion. In mouse islets, ß-cell MPC-deficiency resulted in decreased respiration with glucose, ATP-sensitive potassium (KATP) channel hyperactivity, and impaired insulin release. Moreover, treatment of pancreas-specific MPC knockout mice with glibenclamide, a sulfonylurea KATP channel inhibitor, improved defects in islet insulin secretion and abnormalities in glucose homeostasis in vivo. Finally, using a recently-developed biosensor for MPC activity, we show that the MPC is rapidly stimulated by glucose treatment in INS-1 insulinoma cells suggesting that glucose sensing is coupled to mitochondrial pyruvate carrier activity. CONCLUSIONS: Altogether, these studies suggest that the MPC plays an important and ancestral role in insulin-secreting cells in mediating glucose sensing, regulating insulin secretion, and controlling systemic glycemia.

Engineering pancreatic tissues from stem cells towards therapy.

Pancreatic islet transplantation is performed as a potential treatment for type 1 diabetes mellitus. However, this approach is significantly limited due to the critical shortage of islet sources. Recently, a number of publications have developed protocols for directed ß-cell differentiation of pluripotent cells, such as embryonic stem (ES) or induced pluripotent stem (iPS) cells. Decades of studies have led to the development of modified protocols that recapitulate molecular developmental cues by combining various growth factors and small molecules with improved efficiency. However, the later step of pancreatic differentiation into functional ß-cells has yet to be satisfactory in vitro, highlighting alternative approach by recapitulating spatiotemporal multicellular interaction in three-dimensional (3D) culture. Here, we summarize recent progress in the directed differentiation into pancreatic ß-cells with a focus on both two-dimensional (2D) and 3D differentiation settings. We also discuss the potential transplantation strategies in combination with current bioengineering approaches towards diabetes therapy.

Epithelial Regeneration After Gastric Ulceration Causes Prolonged Cell-Type Alterations.

BACKGROUND & AIMS: The peptic ulcer heals through a complex process, although the ulcer relapse often occurs several years later after healing. Our hypothesis is that even after visual evidence of healing of gastric ulceration, the regenerated epithelium is aberrant for an extended interval, increasing susceptibility of the regenerated epithelium to damage and further diseases. METHODS: Gastric ulcers were induced in mice by serosal topical application of acetic acid. RESULTS: Gastric ulcers induced by acetic acid visually healed within 30 days. However, regenerated epithelial architecture was poor. The gene profile of regenerated tissue was abnormal, indicating increased stem/progenitor cells, deficient differentiated gastric cell types, and deranged cell homeostasis. Despite up-regulation of PDX1 in the regenerated epithelium, no mature antral cell type was observed. Four months after healing, the regenerated epithelium lacks parietal cells, trefoil factor 2 (TFF2) and (sex-determining region Y)-box 9 (SOX9) remain up-regulated deep in the gastric gland, and the Na/H exchanger 2 (a TFF2 effector in gastric healing) remains down-regulated. Gastric ulcer healing was strongly delayed in TFF2 knockout mice, and re-epithelialization was accompanied with mucous metaplasia. After Helicobacter pylori inoculum 30 days after ulceration, we observed that the gastric ulcer selectively relapses at the same site where it originally was induced. Follow-up evaluation at 8 months showed that the relapsed ulcer was not healed in H pylori-infected tissues. CONCLUSIONS: These findings show that this macroscopically regenerated epithelium has prolonged abnormal cell distribution and is differentially susceptible to subsequent damage by H pylori.

Down-regulation of pancreatic and duodenal homeobox-1 by somatostatin receptor subtype 5: a novel mechanism for inhibition of cellular proliferation and insulin secretion by somatostatin.

Somatostatin (SST) is a regulatory peptide and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells. SST's actions are mediated by a family of seven transmembrane domain G protein-coupled receptors that comprise five distinct subtypes (SSTR1-5). SSTR5 is one of the major SSTRs in the islets of Langerhans. Homeodomain-containing transcription factor pancreatic and duodenal homeobox-1 (PDX-1) is essential for pancreatic development, ß cell differentiation, maintenance of normal ß cell functions in adults and tumorigenesis. Recent studies show that SSTR5 acts as a negative regulator for PDX-1 expression and that SSTR5 mediates somatostatin's inhibitory effect on cell proliferation and insulin expression/excretion through down-regulating PDX-1 expression. SSTR5 exerts its inhibitory effect on PDX-1 expression at both the transcriptional level by down-regulating PDX-1 mRNA and the post-translational level by enhancing PDX-1 ubiquitination. Identification of PDX-1 as a transcriptional target for SSTR5 may help in guiding the choice of therapeutic cancer treatments.