Pancreatic Duodenal Homeobox Research Articles

326 GENERATION OF A DOUBLE-TRANSGENIC PIG WITH PANCREAS-SPECIFIC GREEN AND LIVER-SPECIFIC RED FLUORESCENCE

Transgenic (Tg) pigs with organ/tissue-specific fluorescence expression provide invaluable research tools for many types of studies, such as organogenesis analysis, in vitro tissue generation from pluripotent cells, and progenitor/stem cell transplantation therapy. We aimed to develop a Tg pig characterised by pancreas- and liver-specific fluorescence expression. A 8.4 kb transgene construct expressing Venus (green fluorescence) under the control of the mouse Pdx1 (pancreatic duodenal homeobox-1) promoter and a BAC-derived construct (170 kb) consisting of the whole-length porcine albumin (Alb) promoter and humanized Kusabira-Orange (huKO, red fluorescence) was introduced into porcine in vitro-matured oocytes using the intracytoplasmic sperm injection (ICSI)-mediated gene transfer method. Injected embryos were transferred to the oviducts of oestrus-synchronized recipients after culture for 1 to 3 days. The transfer of 370 Pdx1-Venus embryos into 4 recipients produce 22 (5.9%) fetuses/piglets, and 9 (40.9%) Tg pigs exhibited pancreas-specific Venus expression. Two (1 male and 1 female) founder Pdx1-Venus-Tg pigs were mated with wild-type (WT) pigs and produced 32 offspring in 3 litters, of which 16 (50.0%) were transgenic. Pancreas-specific Venus expression was inherited in these Tg offspring. The transfer of 523 Alb-huKO embryos into 4 recipients resulted in 19 (3.6%) piglets including a Tg female, which showed liver-specific huKO fluorescent expression. Expression of huKO was detected by RT-PCR exclusively in liver, but not in 7 other organs/tissues examined, including heart, lung, stomach, small intestine, spleen, kidney and skin. This founder Tg female produced a total of 12 non-Tg and 5 Tg offspring (in 2 litters) after mating with a WT boar. Liver-specific huKO expression was inherited in these Tg offspring. Furthermore, the mating of a female Pdx1-Venus pig with an Alb-huKO boar yielded 7 non-Tg and 10 Tg pigs. Four of these Tg pigs carrying both of the transgenes exhibited both pancreas-specific Venus and liver-specific huKO expression in single individuals. Double-Tg pigs with pancreas-specific green fluorescence and liver-specific red fluorescence grew normally, and tests of their reproduction ability are currently underway. These data demonstrate that transgene introduction by ICSI-mediated gene transfer into in vitro-matured oocytes is a feasible option for generating pigs expressing fluorescent proteins in a tissue-specific manner.

PDX-1/Hes-1 interactions determine cholangiocyte proliferative response to injury in rodents: Possible implications for sclerosing cholangitis

Cholangiocyte proliferation plays a role in the progression of cholangiopathies, in particular in primary sclerosing cholangitis. The mechanisms regulating cholangiocyte proliferation are still undefined. Pancreatic Duodenal Homeobox protein 1 (PDX-1) is expressed by reactive cholangiocytes. In the adult pancreas, PDX-1 regulates the proliferative response to injury of ductal cells. Its effects can be counteracted by Hairy and enhancer of split 1 (Hes-1). We aimed at studying whether PDX-1/Hes-1 interactions regulate cholangiocyte proliferation in response to injury. The effect of the loss of PDX-1 on cholangiocyte proliferation was studied in vitro. In vivo PDX-1-heterozygous (+/-) mice were subjected to either DDC feeding (a model of sclerosing cholangitis) or to bile duct ligation (BDL). PDX-1/Hes-1 interactions on cell proliferation were determined by exposure to All-trans Retinoic Acid (At-RA), an inductor of Hes-1. In vitro, cholangiocyte proliferation was undetectable in cells pre-treated with PDX-1 siRNA. In vivo, increases in bile duct mass and collagen deposition observed after DDC feeding or BDL were significantly reduced in PDX-1(+/-) mice. Hes-1 expression is reduced in proliferating cholangiocytes; At-RA induced a dose-dependent increase in Hes-1 and a decrease in PDX-1 expression. At-RA neutralized the increases in PDX-1 expression and cell proliferation, both in vitro and in vivo in DDC mice. PDX-1 is overexpressed and Hes-1 downregulated in cholangiocytes isolated from PSC livers. Hes-1 downregulation allows PDX-1 to act as a major determinant of cholangiocyte proliferation in response to cholestatic injury. These findings provide novel mechanistic insights into the pathophysiology of cholangiopathies.

Characterization of insulin-producing cells derived from PDX-1-transfected neural stem cells

Islet cell transplantation is a promising treatment strategy for type-1 diabetes. However, functional islet cells are hard to obtain for transplantation and are in short supply. Directing the differentiation of stem cells into insulin‑producing cells, which serve as islet cells, would overcome this shortage. Bone marrow contains hematopoietic stem cells and mesenchymal stem cells. The present study used bone marrow cells isolated from rats and neural stem cells (NSCs) that were derived from bone marrow cells in culture. Strong nestin staining was detected in NSCs, but not in bone marrow stromal cells (BMSCs). In vitro transfection of the pancreatic duodenal homeobox-1 (PDX-1) gene into NSCs generated insulin‑producing cells. Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analysis confirmed that PDX-1-transfected NSCs expressed insulin mRNA and released insulin protein. However, insulin release from PDX-1-transfected NSCs did not respond to the challenge of glucose and glucagon-like peptide-1. These results support the use of bone marrow-derived NSCs as a renewable source of insulin-producing cells for autologous transplantation to treat type-1 diabetes.

Depletion of homeodomain-interacting protein kinase 3 impairs insulin secretion and glucose tolerance in mice

Insufficient insulin secretion and reduced pancreatic beta cell mass are hallmarks of type 2 diabetes. Here, we focused on a family of serine-threonine kinases known as homeodomain-interacting protein kinases (HIPKs). HIPKs are implicated in the modulation of Wnt signalling, which plays a crucial role in transcriptional activity, and in pancreas development and maintenance. The aim of the present study was to characterise the role of HIPKs in glucose metabolism. We used RNA interference to characterise the role of HIPKs in regulating insulin secretion and transcription activity. We conducted RT-PCR and western blot analyses to analyse the expression and abundance of HIPK genes and proteins in the islets of high-fat diet-fed mice. Glucose-induced insulin secretion and beta cell proliferation were measured in islets from Hipk3 ( -/- ) mice, which have impaired glucose tolerance owing to an insulin secretion deficiency. The abundance of pancreatic duodenal homeobox (PDX)-1 and glycogen synthase kinase (GSK)-3β phosphorylation in Hipk3 ( -/- ) islets was determined by immunohistology and western blot analyses. We found that HIPKs regulate insulin secretion and transcription activity. Hipk3 expression was most significantly increased in the islets of high-fat diet-fed mice. Furthermore, glucose-induced insulin secretion and beta cell proliferation were decreased in the islets of Hipk3 ( -/- ) mice. Levels of PDX1 and GSK-3β phosphorylation were significantly decreased in Hipk3 ( -/- ) islets. Depletion of HIPK3 impairs insulin secretion and glucose tolerance. Decreased levels of HIPK3 may play a substantial role in the pathogenesis of type 2 diabetes.

Immune Regulatory Properties of Allogeneic Adipose-Derived Mesenchymal Stem Cells in the Treatment of Experimental Autoimmune Diabetes

Adipose-derived mesenchymal stem cells (ADMSCs) display immunosuppressive properties, suggesting a promising therapeutic application in several autoimmune diseases, but their role in type 1 diabetes (T1D) remains largely unexplored. The aim of this study was to investigate the immune regulatory properties of allogeneic ADMSC therapy in T cell–mediated autoimmune diabetes in NOD mice. ADMSC treatment reversed the hyperglycemia of early-onset diabetes in 78% of diabetic NOD mice, and this effect was associated with higher serum insulin, amylin, and glucagon-like peptide 1 levels compared with untreated controls. This improved outcome was associated with downregulation of the CD4+ Th1-biased immune response and expansion of regulatory T cells (Tregs) in the pancreatic lymph nodes. Within the pancreas, inflammatory cell infiltration and interferon-γ levels were reduced, while insulin, pancreatic duodenal homeobox-1, and active transforming growth factor-β1 expression were increased. In vitro, ADMSCs induced the expansion/proliferation of Tregs in a cell contact–dependent manner mediated by programmed death ligand 1. In summary, ADMSC therapy efficiently ameliorates autoimmune diabetes pathogenesis in diabetic NOD mice by attenuating the Th1 immune response concomitant with the expansion/proliferation of Tregs, thereby contributing to the maintenance of functional β-cells. Thus, this study may provide a new perspective for the development of ADMSC-based cellular therapies for T1D.

Downregulation of pancreatic-duodenal homeobox 1 expression in breast cancer patients: A mechanism of proliferation and apoptosis in cancer

Pancreatic-duodenal homeobox 1 (PDX-1) is a transcription factor that regulates embryological pancreas development and insulin expression in adult islets. The current study investigated the expression profile and potential role of PDX-1 in breast cancer. Immunohistochemistry was performed to determine the expression pattern of PDX-1 in breast cancer and adjacent benign breast tissues. In addition, cell proliferation and the cell cycle were evaluated following the transient inhibition of PDX-1 with antisense oligonucleotides in MCF-7 human breast cancer cells. Real-time PCR and western blotting were conducted to investigate the correlation between PDX-1, P53, Ki-67, Caspase 3 and Caspase 8. These experiments demonstrated that PDX-1 was downregulated in human breast cancer tissue compared with adjacent normal breast tissue. Knockdown of PDX-1 expression in vitro in MCF-7 breast cancer cells promoted cell proliferation and disrupted the cell cycle, as demonstrated by the overexpression of P53 and Ki-67 at the mRNA and protein levels. In conclusion, the current study shows that PDX-1 regulates cell proliferation and the cell cycle in human breast cancer cells by altering the expression of the cell cycle-related genes, P53 and Ki-67. These data suggest that PDX-1 is a putative tumor suppressor in breast cancer.

β-Cell Regeneration Mediated by Human Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) have been shown to ameliorate diabetes in animal models. The mechanism, however, remains largely unknown. An unanswered question is whether BMSCs are able to differentiate into β-cells in vivo, or whether BMSCs are able to mediate recovery and/or regeneration of endogenous β-cells. Here we examined these questions by testing the ability of hBMSCs genetically modified to transiently express vascular endothelial growth factor (VEGF) or pancreatic-duodenal homeobox 1 (PDX1) to reverse diabetes and whether these cells were differentiated into β-cells or mediated recovery through alternative mechanisms. Human BMSCs expressing VEGF and PDX1 reversed hyperglycemia in more than half of the diabetic mice and induced overall improved survival and weight maintenance in all mice. Recovery was sustained only in the mice treated with hBMSCs-VEGF. However, de novo β-cell differentiation from human cells was observed in mice in both cases, treated with either hBMSCs-VEGF or hBMSCs- PDX1, confirmed by detectable level of serum human insulin. Sustained reversion of diabetes mediated by hBMSCs-VEGF was secondary to endogenous β-cell regeneration and correlated with activation of the insulin/IGF receptor signaling pathway involved in maintaining β-cell mass and function. Our study demonstrated the possible benefit of hBMSCs for the treatment of insulin-dependent diabetes and gives new insight into the mechanism of β-cell recovery after injury mediated by hBMSC therapy.

Effects of long-term treatment with the dipeptidyl peptidase-4 inhibitor vildagliptin on islet endocrine cells in non-obese type 2 diabetic Goto-Kakizaki rats

Reduced β cell mass is a characteristic feature of type 2 diabetes and incretin therapy is expected to prevent this condition. However, it is unknown whether dipeptidyl peptidase-4 inhibitors influence β and α cell mass in animal models of diabetes that can be translated to humans. Therefore, we examined the long-term effects of treatment with the dipeptidyl peptidase-4 inhibitor vildagliptin on islet morphology in Goto-Kakizaki (GK) rats, a spontaneous, non-obese model of type 2 diabetes, and explored the underlying mechanisms. Four-week-old GK rats were orally administered with vildagliptin (15mg/kg) twice daily for 18 weeks. Glucose tolerance was monitored during the study. After 18 weeks, β and α cell morphology and the expression of molecules involved in cell proliferation and cell death were examined by immunohistochemistry and morphometric analysis. We found that vildagliptin improved glucose tolerance and insulin secretion, and suppressed hyperglucagonemia by increasing plasma active glucagon-like peptide-1 concentrations. β cell mass was reduced in GK rats to 40% of that in Wistar rats, but was restored to 80% by vildagliptin. Vildagliptin enhanced β and α cell proliferation, and increased the number of small neogenetic islets. Vildagliptin also reduced the number of 8-hydroxy-2′-deoxyguanosine-positive cells and forkhead box protein O1 expression, inhibited macrophage infiltration, and enhanced S6 ribosomal protein, molecule of target of rapamycin, and pancreatic duodenal homeobox 1 expression. These results indicate that starting vildagliptin treatment from an early age improved glucose tolerance and preserved islet β cell mass in GK rats by facilitating the proliferation of islet endocrine cells.

Decreased expression of insulin and increased expression of pancreatic transcription factor PDX-1 in islets in patients with liver cirrhosis: a comparative investigation using human autopsy specimens

Glucose intolerance in patients with liver cirrhosis (LC), known as hepatogenous diabetes, is thought to be distinct from type 2 diabetes (T2DM) in some aspects. Hyperinsulinemia and/or insulin resistance in liver disease is associated with hepatocarcinogenesis, growth of hepatocellular carcinoma, and poor prognosis. However, the pathophysiological processes in islets that are responsible for hyperinsulinemia in LC are still not precisely known. Therefore, we investigated the histopathological differences in islets of Langerhans cells between LC and T2DM. A total of 35 human autopsy pancreatic tissue samples were used in this study (control, n = 18; T2DM, n = 6; LC, n = 11). The expression of insulin, glucagon, somatostatin, pancreatic duodenal homeobox-1 (PDX-1), proliferating cell nuclear antigen (PCNA), and Ki-67 was examined using immunohistochemistry and quantitated by image analysis. Islet hypertrophy and a significant increase in PCNA-positive cells in islets were observed in the tissues from LC cases. The insulin-positive areas in islets were significantly decreased in LC cases compared with control and T2DM cases (P = 0.001, P = 0.035, respectively), whereas the PDX-1-positive area was significantly increased in LC cases (P = 0.001) compared with the control. Furthermore, disorganization of pancreatic endocrine cells and nucleocytoplasmic translocation of PDX-1 were both seen in the LC subjects. In LC, islets undergo hypertrophy and exhibit paradoxical expression of insulin and PDX-1. In the subjects autopsied, insulin expression was decreased, whereas expression of the pancreatic transcription factor PDX-1 was increased in LC. These results point to important distinctions between LC and T2DM.

A Beautiful Cell (or Two or Three?)

The intestinal tract is the largest endocrine organ of the body, containing at least 15 different cells types that release more than 100 biologically active peptides and hormones [Fig. 1 (1–3)]. The vast majority of these enteroendocrine cells have been identified using immunohistochemical techniques, an approach that is inherently limited by the specificity of the antisera/antibodies, particularly because they are used in high concentrations with this technique (1). The current study by Habib et al. (4) takes advantage of two newly developed fluorescent enteroendocrine cell models (5, 6) to purify specific cells of interest, thereby permitting deeper interrogation of the gene profiles of these cells. The findings both confirm and extend previous observations that different enteroendocrine cell types have more features in common than originally believed. These findings have important implications for current investigations into the possible use of enteroendocrine cell stimulators for the treatment of diseases such as type 2 diabetes. The enteroendocrine cell of focus in the present study by Habib et al. (4) is the L cell, originally described on the basis of enteroglucagon immunoreactivity, using the K cell that produces glucose-dependent insulinotropic peptide (GIP) as a comparator. Enteroglucagon is a generic term for the intestinal proglucagon-derived peptides, including glucagon-like peptide (GLP)-1, an incretin hormone that stimulates glucose-dependent insulin secretion; both GLP-1 receptor agonists and degradation inhibitors are currently used in the clinic to treat patients with type 2 diabetes (7, 8). The other main L cell proglucagon-derived peptides are the gut growth factor GLP-2 and the anorexigenic hormone oxyntomodulin, whereas GIP also serves a role as an incretin hormone (7). Based on the expression of GIP vs. GLP-1, the highest numbers of K cells are found in the duodenum with decreasing numbers in the aboral direction, whereas L cells demonstrate the opposite gradient, with the greatest numbers in the ileum/colon (9, 10). However, the presence of GLP-1 has been demonstrated in subsets of duodenal K cells, whereas immunoreactive GIP has been detected in subpopulations of proximal L cells (5, 9, 11, 12). To further complicate the issue, numerous studies have also demonstrated the expression of peptide YY (PYY) in some ileal and colonic L cells (5, 10, 13, 14) as well as coexpression of GLP-1 with cholecystokinin (CCK) and with neurotensin (14, 15). Thus, it has long been suspected that different types of K and L cells may exist in the intestine. Studies on enteroendocrine cell development have elucidated the transcription factors required for lineage progression from early stem cells to differentiated enteroendocrine cells (Fig. 1). Thus, the development of related enteroendocrine cell populations requires sequential expression of Math1 (mammalian atonal homolog-1), Ngn3 (neurogenin 3), NeuroD (neurogenic differentiation), followed by the expression of paired box transcription factor (Pax)-4 and/or ARX (aristaless related homeobox) (16– 21). The specific transcription factors required for final differentiation of these progenitor cells into specific enteroendocrine cells are not well characterized but include both Pax6 and pancreatic duodenal homeobox-1 (Pdx1) for K cells as well as Pax6 for L cells: in this regard, expression of Pdx1 in L cells appears to be permissive for their coexpression of GIP (22). Recent findings of Dtx1 (deltex1),Egr2 (earlygrowth responseprotein2),Tbx3 (T box 3) in duodenal but not colonic L cells and Etv1 (ETS translocation variant 1) and Prox1 (prospero-related homeobox 1) in proximal L cells but not in K cells remain of uncertain significance (4). In the current study by Habib et al. (4), intestinal L cells from transgenic mice expressing a fluorescent marker (Ve-

Protective effect of rat pancreatic progenitors cells expressing Pdx1 and nestin on islets survival and function in vitro and in vivo

To maintain islets survival and function is critical in successful pancreatic transplantation. Pancreatic progenitors cells (PPCs) with lineage potentials, giving rise to exocrine, endocrine, and duct cells, reside in developing and adult pancreas. As tissue-specific stem cells, they can produce pancreatic tissue-specific matrix factors to promote islets survival and function. The aim of our research was to investigate the protective effect of rat pancreatic-duodenal homeobox 1 (Pdx1)(+)/nestin(+) PPCs on islets. In vitro, co-culturing islets with Pdx1(+)/nestin(+) PPCs prolonged the former survival from 7 to 14 days. Furthermore, with high glucose (300.8 mg/dl) stimuli, the yield of insulin in co-cultures was significantly higher than that in control group (single islets group). In vivo, co-transplanting islets and Pdx1(+)/nestin(+) PPCs for 3 days, the blood glucose of diabetic rat was significantly decreased to normal level and sustained for 2 weeks. Without Pdx1(+)/nestin(+) PPCs in islets transplantation, hyperglycemia was reversed at day 7 and recovered at day 15. Pathology analysis showed that islets had remnants in co-transplantation at day 21, as complete graft rejection in alone islets transplantation. Our study showed that Pdx1(+)/nestin(+) PPCs displayed the ability of preserving islets viability and function in vitro and prolonging their survival in vivo.

Increased DNA Methylation and Decreased Expression of PDX-1 in Pancreatic Islets from Patients with Type 2 Diabetes

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.

PDX-1, CDX-2, TTF-1, and CK7

Neuroendocrine tumors (NETs) occur in virtually all sites of the body. As NETs arising in different organs share similar morphologic features, distinguishing metastatic from primary NETs on the basis of morphologic grounds alone is difficult. Pancreatic duodenal homeobox 1 (PDX-1) is a Hox-type transcription factor that is essential for both exocrine and endocrine pancreatic differentiation and maintenance of β-cell function. We investigated PDX-1 as an immunohistochemical (IHC) marker in primary pancreatic NETs. Eighty primary NETs [25 pancreatic, 29 bronchopulmonary, and 26 in the gastrointestinal (GI) tract] and 13 metastatic NETs in the liver were studied. Clinical and radiologic data were reviewed to confirm the stated primary sites. IHC analysis for PDX-1, CDX-2, thyroid transcription factor-1 (TTF-1), keratin 7 (CK7), and keratin 20 (CK20) was performed, and the results were based on review blinded to the primary sites. PDX-1 was seen in 18 of 25 (72%) pancreatic NETs; in contrast, only 3 of 29 (10%) bronchopulmonary NETs and 1 of 26 (4%) GI NETs were positive. PDX-1 was therefore 93% specific and 72% sensitive for pancreatic NETs. TTF-1 was expressed only in bronchopulmonary NETs; all other NETs were negative for TTF-1. CK7 was also very specific (92%) and moderately sensitive (66%) for bronchopulmonary NETs. CDX-2 was seen in 22 of 26 (85%) cases of GI NETs and in only 1 of 51 (2%) cases of extra-GI NETs. Thus, CDX-2 was 98% specific and 85% sensitive for GI NETs. In terms of metastatic NETs found in the liver, PDX-1 was positive in 5 of 5 cases of metastatic pancreatic NETs and 2 of 2 cases of metastatic duodenal NETs. PDX-1 is highly specific, with very good overall diagnostic accuracy for pancreatic NETs. An IHC panel including PDX-1, CDX-2, TTF-1, CK7, and CK20 may be useful in distinguishing NETs of pancreatic origin from other primaries.

Loss of HGF/c-Met Signaling in Pancreatic β-Cells Leads to Incomplete Maternal β-Cell Adaptation and Gestational Diabetes Mellitus

Hepatocyte growth factor (HGF) is a mitogen and insulinotropic agent for the β-cell. However, whether HGF/c-Met has a role in maternal β-cell adaptation during pregnancy is unknown. To address this issue, we characterized glucose and β-cell homeostasis in pregnant mice lacking c-Met in the pancreas (PancMet KO mice). Circulating HGF and islet c-Met and HGF expression were increased in pregnant mice. Importantly, PancMet KO mice displayed decreased β-cell replication and increased β-cell apoptosis at gestational day (GD)15. The decreased β-cell replication was associated with reductions in islet prolactin receptor levels, STAT5 nuclear localization and forkhead box M1 mRNA, and upregulation of p27. Furthermore, PancMet KO mouse β-cells were more sensitive to dexamethasone-induced cytotoxicity, whereas HGF protected human β-cells against dexamethasone in vitro. These detrimental alterations in β-cell proliferation and death led to incomplete maternal β-cell mass expansion in PancMet KO mice at GD19 and early postpartum periods. The decreased β-cell mass was accompanied by increased blood glucose, decreased plasma insulin, and impaired glucose tolerance. PancMet KO mouse islets failed to upregulate GLUT2 and pancreatic duodenal homeobox-1 mRNA, insulin content, and glucose-stimulated insulin secretion during gestation. These studies indicate that HGF/c-Met signaling is essential for maternal β-cell adaptation during pregnancy and that its absence/attenuation leads to gestational diabetes mellitus.

Hyperthermia With Mild Electrical Stimulation Protects Pancreatic β-Cells From Cell Stresses and Apoptosis

Induction of heat shock protein (HSP) 72 improves metabolic profiles in diabetic model mice. However, its effect on pancreatic β-cells is not known. The current study investigated whether HSP72 induction can reduce β-cell stress signaling and apoptosis and preserve β-cell mass. MIN6 cells and db/db mice were sham-treated or treated with heat shock (HS) and mild electrical stimulation (MES) (HS+MES) to induce HSP72. Several cellular markers, metabolic parameters, and β-cell mass were evaluated. HS+MES treatment or HSP72 overexpression increased HSP72 protein levels and decreased tumor necrosis factor (TNF)-α–induced Jun NH2-terminal kinase (JNK) phosphorylation, endoplasmic reticulum (ER) stress, and proapoptotic signal in MIN6 cells. In db/db mice, HS+MES treatment for 12 weeks significantly improved insulin sensitivity and glucose homeostasis. Upon glucose challenge, a significant increase in insulin secretion was observed in vivo. Compared with sham treatment, levels of HSP72, insulin, pancreatic duodenal homeobox-1, GLUT2, and insulin receptor substrate-2 were upregulated in the pancreatic islets of HS+MES-treated mice, whereas JNK phosphorylation, nuclear translocation of forkhead box class O-1, and nuclear factor-κB p65 were reduced. Apoptotic signals, ER stress, and oxidative stress markers were attenuated. Thus, HSP72 induction by HS+MES treatment protects β-cells from apoptosis by attenuating JNK activation and cell stresses. HS+MES combination therapy may preserve pancreatic β-cell volume to ameliorate glucose homeostasis in diabetes.

Mediation of glucolipotoxicity in INS-1 rat insulinoma cells by small heterodimer partner interacting leucine zipper protein (SMILE)

Sustained elevations of glucose and free fatty acid concentration have deleterious effects on pancreatic beta cell function. One of the hallmarks of such glucolipotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Sterol regulatory element binding protein-1c (SREBP-1c), a lipogenic transcription factor, is related to the development of beta cell dysfunction caused by elevated concentrations of glucose and free fatty acid. Small heterodimer partner (SHP) interacting leucine zipper protein (SMILE), also known as Zhangfei, is a novel protein which interacts with SHP that mediates glucotoxicity in INS-1 rat insulinoma cells. Treatment of INS-1 cells with high concentrations of glucose and palmitate increased SREBP-1c and SMILE expression, and decreased insulin gene expression. Adenovirus-mediated overexpression of SREBP-1c in INS-1 cells induced SMILE expression. Moreover, adenovirus-mediated overexpression of SMILE (Ad-SMILE) in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. Ad-SMILE overexpression also inhibited the expression of beta-cell enriched transcription factors including pancreatic duodenal homeobox factor-1, beta cell E box transactivator 2 and RIPE3b1/MafA, in INS-1 cells. Finally, in COS-1 cells, expression of SMILE inhibited the insulin promoter activity induced by these same beta-cell enriched transcription factors. These results collectively suggest that SMILE plays an important role in the development of beta cell dysfunction induced by glucolipotoxicity.

The Expression of the Class 1 Glucose Transporter Isoforms in Human Embryonic Stem Cells, and the Potential Use of GLUT2 as a Marker for Pancreatic Progenitor Enrichment

Even before the first appearance of the developing pancreas, glucose is the major substrate in the growing embryo. The transport of glucose across cell membranes is facilitated by a family of membranal glucose transporters (GLUT). We analyzed changes in expression of class 1 glucose transporters (GLUT1-4) during human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) differentiation, from undifferentiated cells to 28-day-old embryoid bodies (EBs). We also examined the potential use of GLUT2 as a marker for differentiating pancreatic progenitor cells. Using quantitative real time polymerase chain reaction (qPCR), western blot, and immunofluorescence, we observed enhanced expression of GLUT1 and GLUT2 during differentiation, but only minor change in GLUT3 expression. GLUT4 expression was found to be very low both at the RNA and in the protein levels. Expression of the early pancreatic transcription factor, pancreatic duodenal homeobox gene 1 (PDX1), correlated with GLUT2 expression, suggesting the potential use of GLUT2 as a surface marker for tracking pancreatic precursor cells. After sorting EBs according to their membranal GLUT2 expression, GLUT2 and PDX1 expression were found elevated, as was expression of other endodermal markers such as PAX4, NGN3, CXCR4, and SOX17. This simple method may be used to differentiate embryonic stem cells and to isolate from them, using GLUT2 as a surface marker, an enriched pancreatic progenitor cell population in order to achieve insulin-producing cells. The sorted GLUT2 cells may potentially be used in the future as insulin-producing cells for beta cell therapies.

Age-Related Impairment in Insulin Release

In this study, we investigated the significance of β2-adrenergic receptor (β2AR) in age-related impaired insulin secretion and glucose homeostasis. We characterized the metabolic phenotype of β2AR-null C57Bl/6N mice (β2AR−/−) by performing in vivo and ex vivo experiments. In vitro assays in cultured INS-1E β-cells were carried out in order to clarify the mechanism by which β2AR deficiency affects glucose metabolism. Adult β2AR−/− mice featured glucose intolerance, and pancreatic islets isolated from these animals displayed impaired glucose-induced insulin release, accompanied by reduced expression of peroxisome proliferator–activated receptor (PPAR)γ, pancreatic duodenal homeobox-1 (PDX-1), and GLUT2. Adenovirus-mediated gene transfer of human β2AR rescued these defects. Consistent effects were evoked in vitro both upon β2AR knockdown and pharmacologic treatment. Interestingly, with aging, wild-type (β2AR+/+) littermates developed impaired insulin secretion and glucose tolerance. Moreover, islets from 20-month-old β2AR+/+ mice exhibited reduced density of β2AR compared with those from younger animals, paralleled by decreased levels of PPARγ, PDX-1, and GLUT2. Overexpression of β2AR in aged mice rescued glucose intolerance and insulin release both in vivo and ex vivo, restoring PPARγ/PDX-1/GLUT2 levels. Our data indicate that reduced β2AR expression contributes to the age-related decline of glucose tolerance in mice.

T-37 Impaired pancreatic duodenal homeobox protein 1 (PDX-1) expression promotes diabetes and hepatocellular carcinoma (HCC) development in mice

T-37 Impaired pancreatic duodenal homeobox protein 1 (PDX-1) expression promotes diabetes and hepatocellular carcinoma (HCC) development in mice

COUP-TFII Controls Mouse Pancreatic β-Cell Mass through GLP-1-β-Catenin Signaling Pathways

BackgroundThe control of the functional pancreatic β-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how β-cell mass is determined.Methodology/Principal FindingsConditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1) gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal β-cell proliferation and apoptosis, this suggests decreased β-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured β-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the β-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1) via β-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced β-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2) in human islets and rat β-cells providing a feedback loop.Conclusions/SignificanceOur findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases β-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the β-catenin-dependent pathway and its expression is under the control of TCF7L2.