LGR signaling mediates muscle-adipose tissue crosstalk and protects against diet-induced insulin resistance.

Obesity impairs tissue insulin sensitivity and signaling, promoting type-2 diabetes. Although improving insulin signaling is key to reversing diabetes, the multi-organ mechanisms regulating this process are poorly defined. Here, we screen the secretome and receptome in Drosophila to identify the hormonal crosstalk affecting diet-induced insulin resistance and obesity. We discover a complex interplay between muscle, neuronal, and adipose tissues, mediated by Bone Morphogenetic Protein (BMP) signaling and the hormone Bursicon, that enhances insulin signaling and sugar tolerance. Muscle-derived BMP signaling, induced by sugar, governs neuronal Bursicon signaling. Bursicon, through its receptor Rickets, a Leucine-rich-repeat-containing G-protein coupled receptor (LGR), improves insulin secretion and insulin sensitivity in adipose tissue, mitigating hyperglycemia. In mouse adipocytes, loss of the Rickets ortholog LGR4 blunts insulin responses, showing an essential role of LGR4 in adipocyte insulin sensitivity. Our findings reveal a muscle-neuronal-fat-tissue axis driving metabolic adaptation to high-sugar conditions, identifying LGR4 as a critical mediator in this regulatory network.

Detection of the novel HLA allele, HLA-DRB1*08:112, identified in a Danish family.

HLA-DRB1*08:112 differs from HLA-DRB1*08:01 in exon 2 at amino acid 62; asparagine to lysine substitution.

Detection of the novel HLA allele, HLA-DQA1*01:65, identified in a Danish donor.

HLA-DQA1*01:65 differs from HLA-DQA1*01:03 in exon 1 at amino acid -7 a valine to methionine substitution.

Identification of the novel HLA allele, HLA-DPA1*01:46, identified in a man of Serbian origin.

HLA-DPA1*01:46 differs from HLA-DPA1*01:03 in exon 2 at amino acid 85; Aspartate to Asparagine substitution.

Identification of the novel HLA allele, HLA-C*07:780, identified in a Danish woman.

HLA-C*07:780 differs from HLA-C*07:04:01:01 in exon 2 at amino acid 49; alanine to threonine substitution.

The EndoC-ßH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates.

OBJECTIVE: To characterize the EndoC-ßH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. METHODS: EndoC-ßH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. RESULTS: Transplantation of EndoC-ßH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-ßH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. CONCLUSIONS: Overall, the EndoC-ßH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-ßH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates.

Fibroblast growth factor 21 (FGF21) and glucagon-like peptide 1 contribute to diabetes resistance in glucagon receptor-deficient mice.

Mice genetically deficient in the glucagon receptor (Gcgr(-/-)) show improved glucose tolerance, insulin sensitivity, and α-cell hyperplasia. In addition, Gcgr(-/-) mice do not develop diabetes after chemical destruction of ß-cells. Since fibroblast growth factor 21 (FGF21) has insulin-independent glucose-lowering properties, we investigated whether FGF21 was contributing to diabetes resistance in insulin-deficient Gcgr(-/-) mice. Plasma FGF21 was 25-fold higher in Gcgr(-/-) mice than in wild-type mice. FGF21 was found to be expressed in pancreatic ß- and α-cells, with high expression in the hyperplastic α-cells of Gcgr(-/-) mice. FGF21 expression was also significantly increased in liver and adipose tissue of Gcgr(-/-) mice. To investigate the potential antidiabetic actions of FGF21 in insulin-deficient Gcgr(-/-) mice, an FGF21-neutralizing antibody was administered prior to oral glucose tolerance tests (OGTTs). FGF21 neutralization caused a decline in glucose tolerance in insulin-deficient Gcgr(-/-) mice during the OGTT. Despite this decline, insulin-deficient Gcgr(-/-) mice did not develop hyperglycemia. Glucagon-like peptide 1 (GLP-1) also has insulin-independent glucose-lowering properties, and an elevated circulating level of GLP-1 is a known characteristic of Gcgr(-/-) mice. Neutralization of FGF21, while concurrently blocking the GLP-1 receptor with the antagonist Exendin 9-39 (Ex9-39), resulted in significant hyperglycemia in insulin-deficient Gcgr(-/-) mice, while blocking with Ex9-39 alone did not. In conclusion, FGF21 acts additively with GLP-1 to prevent insulinopenic diabetes in mice lacking glucagon action.

Role of the ductal transcription factors HNF6 and Sox9 in pancreatic acinar-to-ductal metaplasia.

OBJECTIVE: Growing evidence suggests that a phenotypic switch converting pancreatic acinar cells to duct-like cells can lead to pancreatic intraepithelial neoplasia and eventually to invasive pancreatic ductal adenocarcinoma. Histologically, the onset of this switch is characterised by the co-expression of acinar and ductal markers in acini, a lesion called acinar-to-ductal metaplasia (ADM). The transcriptional regulators required to initiate ADM are unknown, but need to be identified to characterise the regulatory networks that drive ADM. In this study, the role of the ductal transcription factors hepatocyte nuclear factor 6 (HNF6, also known as Onecut1) and SRY-related HMG box factor 9 (Sox9) in ADM was investigated. DESIGN: Expression of HNF6 and Sox9 was measured by immunostaining in normal and diseased human pancreas. The function of the factors was tested in cultured cells and in mouse models of ADM by a combination of gain and loss of function experiments. RESULTS: Expression of HNF6 and Sox9 was ectopically induced in acinar cells in human ADM as well as in mouse models of ADM. HNF6 and, to a lesser extent, Sox9 were required for repression of acinar genes, for modulation of ADM-associated changes in cell polarity and for activation of ductal genes in metaplastic acinar cells. CONCLUSIONS: HNF6 and Sox9 are new biomarkers of ADM and constitute candidate targets for preventive treatment in cases when ADM may lead to cancer. This work also shows that ectopic activation of transcription factors may underlie metaplastic processes occurring in other organs.

Huntingtin-interacting protein 14 is a type 1 diabetes candidate protein regulating insulin secretion and beta-cell apoptosis.

Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting ß-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease-causing genes in T1D, we performed an in silico "phenome-interactome analysis" on a genome-wide linkage scan dataset. This method prioritizes candidates according to their physical interactions at the protein level with other proteins involved in diabetes. A total of 11 genes were predicted to be likely disease genes in T1D, including the INS gene. An unexpected top-scoring candidate gene was huntingtin-interacting protein (HIP)-14/ZDHHC17. Immunohistochemical analysis of pancreatic sections demonstrated that HIP14 is almost exclusively expressed in insulin-positive cells in islets of Langerhans. RNAi knockdown experiments established that HIP14 is an antiapoptotic protein required for ß-cell survival and glucose-stimulated insulin secretion. Proinflammatory cytokines (IL-1ß and IFN-γ) that mediate ß-cell dysfunction in T1D down-regulated HIP14 expression in insulin-secreting INS-1 cells and in isolated rat and human islets. Overexpression of HIP14 was associated with a decrease in IL-1ß-induced NF-κB activity and protection against IL-1ß-mediated apoptosis. Our study demonstrates that the current network biology approach is a valid method to identify genes of importance for T1D and may therefore embody the basis for more rational and targeted therapeutic approaches.

G protein-coupled receptor 39 deficiency is associated with pancreatic islet dysfunction.

G protein-coupled receptor (GPR)-39 is a seven-transmembrane receptor expressed mainly in endocrine and metabolic tissues that acts as a Zn(++) sensor signaling mainly through the G(q) and G(12/13) pathways. The expression of GPR39 is regulated by hepatocyte nuclear factor (HNF)-1alpha and HNF-4alpha, and in the present study, we addressed the importance of GPR39 for glucose homeostasis and pancreatic islets function. The expression and localization of GPR39 were characterized in the endocrine pancreas and pancreatic cell lines. Gpr39(-/-) mice were studied in vivo, especially in respect of glucose tolerance and insulin sensitivity, and in vitro in respect of islet architecture, gene expression, and insulin secretion. Gpr39 was down-regulated on differentiation of the pluripotent pancreatic cell line AR42J cells toward the exocrine phenotype but was along with Pdx-1 strongly up-regulated on differentiation toward the endocrine phenotype. Immunohistochemistry demonstrated that GRP39 is localized selectively in the insulin-storing cells of the pancreatic islets as well as in the duct cells of the exocrine pancreas. Gpr39(-/-) mice displayed normal insulin sensitivity but moderately impaired glucose tolerance both during oral and iv glucose tolerance tests, and Gpr39(-/-) mice had decreased plasma insulin response to oral glucose. Islet architecture was normal in the Gpr39 null mice, but expression of Pdx-1 and Hnf-1alpha was reduced. Isolated, perifused islets from Gpr39 null mice secreted less insulin in response to glucose stimulation than islets from wild-type littermates. It is concluded that GPR39 is involved in the control of endocrine pancreatic function, and it is suggested that this receptor could be a novel potential target for the treatment of diabetes.

Generation and characterization of Ptf1a antiserum and localization of Ptf1a in relation to Nkx6.1 and Pdx1 during the earliest stages of mouse pancreas development.

Ptf1a and Pdx1 are critical transcription factors of early pancreatic development, as shown by loss of function studies where lack of each gene alone causes almost complete pancreas agenesis. Ptf1a is particularly interesting because it is linked to a recently reported signature gene expression profile associated with the multipotent condition. Few useful antibody reagents have been available for consistent and reliable immunohistochemical visualization of Ptf1a protein expression in the early developing pancreas in which the level of production of this critical regulator seems to be very low. We describe a novel rabbit antibody raised against the c-terminal portion of the mouse Ptf1a protein and report immunodetection, for the first time, as early as embryonic day (e) 8.5-e8.75 in the dorsal and ventral buds of the mouse pancreas as well as in the neural tube at e10.0. Detailed confocal analysis identifies an abundant triple-positive (Ptf1a(+)/Nkx6.1(+)/Pdx1(+)) putative early multipotent pancreatic progenitor cell that marks the e9.5 dorsal pancreas and e10.5 ventral pancreas. Furthermore, expression patterns of Nkx6.1 vs Ptf1a subsequently segregate during branching morphogenesis (trunk vs tip), ending up marking two distinct cell populations of progenitors at e12.5. From e15.5 (mouse) and in adult pancreas (mouse, rat, and human), the Ptf1a antibody marks only acinar cell nuclei, as expected for its subsequent role in committing/maintaining cells in this differentiated state. In summary, this antibody is a novel tool to further characterize important early steps of pancreas differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

An illustrated review of early pancreas development in the mouse.

Pancreas morphogenesis and cell differentiation are highly conserved among vertebrates during fetal development. The pancreas develops through simple budlike structures on the primitive gut tube to a highly branched organ containing many specialized cell types. This review presents an overview of key molecular components and important signaling sources illustrated by an extensive three-dimensional (3D) imaging of the developing mouse pancreas at single cell resolution. The 3D documentation covers the time window between embryonic days 8.5 and 14.5 in which all the pancreatic cell types become specified and therefore includes gene expression patterns of pancreatic endocrine hormones, exocrine gene products, and essential transcription factors. The 3D perspective provides valuable insight into how a complex organ like the pancreas is formed and a perception of ventral and dorsal pancreatic growth that is otherwise difficult to uncover. We further discuss how this global analysis of the developing pancreas confirms and extends previous studies, and we envisage that this type of analysis can be instrumental for evaluating mutant phenotypes in the future.

Preservation of proliferating pancreatic progenitor cells by Delta-Notch signaling in the embryonic chicken pancreas.

BACKGROUND: Genetic studies have shown that formation of pancreatic endocrine cells in mice is dependent on the cell autonomous action of the bHLH transcription factor Neurogenin3 and that the extent and timing of endocrine differentiation is controlled by Notch signaling. To further understand the mechanism by which Notch exerts this function, we have investigated pancreatic endocrine development in chicken embryos. RESULTS: In situ hybridization showed that expression of Notch signaling components and pro-endocrine bHLH factors is conserved to a large degree between chicken and mouse. Cell autonomous inhibition of Notch signal reception results in significantly increased endocrine differentiation demonstrating that these early progenitors are prevented from differentiating by ongoing Notch signaling. Conversely, activated Notch1 induces Hes5-1 expression and prevents endocrine development. Notably, activated Notch also prevents Ngn3-mediated induction of a number of downstream targets including NeuroD, Hes6-1, and MyT1 suggesting that Notch may act to inhibit both Ngn3 gene expression and protein function. Activated Notch1 could also block endocrine development and gene expression induced by NeuroD. Nevertheless, Ngn3- and NeuroD-induced delamination of endodermal cells was insensitive to activated Notch under these conditions. Finally, we show that Myt1 can partially overcome the repressive effect of activated Notch on endocrine gene expression. CONCLUSION: We conclude that pancreatic endocrine development in the chicken relies on a conserved bHLH cascade under inhibitory control of Notch signaling. This lays the ground for further studies that take advantage of the ease at which chicken embryos can be manipulated. Our results also demonstrate that Notch can repress Ngn3 and NeuroD protein function and stimulate progenitor proliferation. To determine whether Notch in fact does act in Ngn3-expressing cells in vivo will require further studies relying on conditional mutagenesis. Lastly, our results demonstrate that expression of differentiation markers can be uncoupled from the process of delamination of differentiating cells from the epithelium.

An improved method for three-dimensional reconstruction of protein expression patterns in intact mouse and chicken embryos and organs.

We have developed a wholemount immunofluorescence protocol for the simultaneous detection of up to three proteins in mouse and chicken embryos. Combined with Murray's clearing reagent (BABB) and microscope objectives with long working ranges and high numerical apertures mounted on a confocal microscope, cellular resolution can be obtained in depths offering the possibility of examining expression patterns in entire organs or embryos. Three-dimensional projections of the optical confocal sections can be computed with computer software allowing rotation around any axis. The protocol is robust and we find that most antibodies working on tissue sections also work with this protocol. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Activated Notch1 prevents differentiation of pancreatic acinar cells and attenuate endocrine development.

Mice carrying loss-of-function mutations in certain Notch pathway genes display increased and accelerated pancreatic endocrine development, leading to depletion of precursor cells followed by pancreatic hypoplasia. Here, we have investigated the effect of expressing a constitutively active form of the Notch1 receptor (Notch1(ICD)) in the developing pancreas using the pdx1 promoter. At e10.5 to e12.5, we observe a disorganized pancreatic epithelium with reduced numbers of endocrine cells, confirming a repressive activity of Notch1 upon the early differentiation program. Subsequent branching morphogenesis is impaired and the pancreatic epithelium forms cyst-like structures with ductal phenotype containing a few endocrine cells but completely devoid of acinar cells. The endocrine cells that do form show abnormal expression of cell type-specific markers. Our observations show that sustained Notch1 signaling not only significantly represses endocrine development, but also fully prevents pancreatic exocrine development, suggesting that a possible role of Notch1 is to maintain the undifferentiated state of common pancreatic precursor cells.