Raphael Scharfmann

Transcription Factor TCF7L2 Genetic Study in the French Population: Expression in Human β-Cells and Adipose Tissue and Strong Association With Type 2 Diabetes

Recently, the transcription factor 7-like 2 (TCF7L2) gene has been associated with type 2 diabetes in subjects of European origin in the DeCode study. We genotyped the two most associated variants (rs7903146 and rs12255372) in 2,367 French type 2 diabetic subjects and in 2,499 control subjects. Both the T-allele of rs7903146 and the T-allele of rs12255372 significantly increase type 2 diabetes risk with an allelic odds ratio (OR) of 1.69 (95% CI 1.55–1.83) (P = 6.0 × 10−35) and 1.60 (1.47–1.74) (P = 7.6 × 10−28), respectively. In nonobese type 2 diabetic subjects (BMI <30 kg/m2, n = 1,346), the ORs increased to 1.89 (1.72–2.09) (P = 2.1 × 10−38) and 1.79 (1.62–1.97) (P = 5.7 × 10−31), respectively. The rs7903146 T at-risk allele associates with decreased BMI and earlier age at diagnosis in the type 2 diabetic subjects (P = 8.0 × 10−3 and P = 3.8 × 10−4, respectively), which is supported by quantitative family-based association tests. TCF7L2 is expressed in most human tissues, including mature pancreatic β-cells, with the exception of the skeletal muscle. In the subcutaneous and omental fat from obese type 2 diabetic subjects, TCF7L2 expression significantly decreased compared with obese normoglycemic individuals. During rat fetal β-cell differentiation, TCF7L2 expression pattern mimics the key marker NGN3 (neurogenin 3), suggesting a role in islet development. These data provide evidence that TCF7L2 is a major determinant of type 2 diabetes risk in European populations and suggests that this transcription factor plays a key role in glucose homeostasis.

Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants

OBJECTIVE Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS ZnT8−/− mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8−/− islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn2+ transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

A genetically engineered human pancreatic β cell line exhibiting glucose-inducible insulin secretion

Despite intense efforts over the past 30 years, human pancreatic β cell lines have not been available. Here, we describe a robust technology for producing a functional human β cell line using targeted oncogenesis in human fetal tissue. Human fetal pancreatic buds were transduced with a lentiviral vector that expressed SV40LT under the control of the insulin promoter. The transduced buds were then grafted into SCID mice so that they could develop into mature pancreatic tissue. Upon differentiation, the newly formed SV40LT-expressing β cells proliferated and formed insulinomas. The resulting β cells were then transduced with human telomerase reverse transcriptase (hTERT), grafted into other SCID mice, and finally expanded in vitro to generate cell lines. One of these cell lines, EndoC-βH1, expressed many β cell-specific markers without any substantial expression of markers of other pancreatic cell types. The cells secreted insulin when stimulated by glucose or other insulin secretagogues, and cell transplantation reversed chemically induced diabetes in mice. These cells represent a unique tool for large-scale drug discovery and provide a preclinical model for cell replacement therapy in diabetes. This technology could be generalized to generate other human cell lines when the cell type-specific promoter is available.

Human β Cell Transcriptome Analysis Uncovers lncRNAs That Are Tissue-Specific, Dynamically Regulated, and Abnormally Expressed in Type 2 Diabetes

A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and β cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the β cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a β cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to β cell programming and diabetes pathophysiology.

Expression of Neuronal Traits in Pancreatic Beta Cells IMPLICATION OF NEURON-RESTRICTIVE SILENCING FACTOR/REPRESSOR ELEMENT SILENCING TRANSCRIPTION FACTOR, A NEURON-RESTRICTIVE SILENCER

Pancreatic beta cells (insulin-producing cells) and neuronal cells share a large number of similarities. Here, we investigate whether the same mechanisms could control the expression of neuronal genes in both neurons and insulin-producing cells. For that purpose, we tested the role of the transcriptional repressor neuron-restrictive silencing factor/repressor element silencing transciption factor (NRSF/REST) in the expression of a battery of neuronal genes in insulin-producing cells. NRSF/REST is a negative regulator of the neuronal fate. It is known to silence neuronal-specific genes in non-neuronal cells. We demonstrate that, as in the case of the neuronal pheochromocytoma cell line PC12, mRNA coding for NRSF/REST is absent from the insulinoma cell line INS-1 and from three other insulin- and glucagon-producing cell lines. NRSF/REST activity is also absent from insulin-producing cell lines. Transient expression of REST in insulin-producing cell lines is sufficient to silence a reporter gene containing a NRSF/REST binding site, demonstrating the role of NRSF/REST in the expression of neuronal markers in insulin-producing cells. Finally, by searching for the expression of NRSF/REST-regulated genes in insulin-producing cells, we increased the list of the genes expressed in both neurons and insulin-producing cells.

Histone Deacetylase Inhibitors Modify Pancreatic Cell Fate Determination and Amplify Endocrine Progenitors

ABSTRACT During pancreas development, transcription factors play critical roles in exocrine and endocrine differentiation. Transcriptional regulation in eukaryotes occurs within chromatin and is influenced by posttranslational histone modifications (e.g., acetylation) involving histone deacetylases (HDACs). Here, we show that HDAC expression and activity are developmentally regulated in the embryonic rat pancreas. We discovered that pancreatic treatment with different HDAC inhibitors (HDACi) modified the timing and determination of pancreatic cell fate. HDACi modified the exocrine lineage via abolition and enhancement of acinar and ductal differentiation, respectively. Importantly, HDACi treatment promoted the NGN3 proendocrine lineage, leading to an increased pool of endocrine progenitors and modified endocrine subtype lineage choices. Interestingly, treatments with trichostatin A and sodium butyrate, two inhibitors of both class I and class II HDACs, enhanced the pool of β cells. These results highlight the roles of HDACs at key points in exocrine and endocrine differentiation. They show the powerful use of HDACi to switch pancreatic cell determination and amplify specific cellular subtypes, with potential applications in cell replacement therapies in diabetes.

Role for FGFR2IIIb-mediated signals in controlling pancreatic endocrine progenitor cell proliferation

Pancreatic development is a classic example of epithelium–mesenchyme interaction. During embryonic life, signals from the mesenchyme control the proliferation of precursor cells within the pancreatic epithelium and their differentiation into endocrine or acinar cells. It has been shown that signals from the mesenchyme activate epithelial cell proliferation but repress development of the pancreatic epithelium into endocrine cells. Here, experiments with specific inhibitors established that mesenchymal effects on epithelial cell development depended on the mitogen-activated protein kinase pathway. Then we demonstrated that these effects of the mesenchyme were mimicked by fibroblast growth factor 7 (FGF7), a specific ligand of FGFR2IIIb, which is a tyrosine kinase receptor of the FGF-receptor family. When pancreatic epithelium expressing FGFR2IIIb was grown with FGF7, epithelial cell growth occurred in a concentration-dependent manner, whereas endocrine tissue development was repressed. The epithelial cells that proliferated in response to FGF7 were endocrine pancreatic precursor cells, as shown by their differentiation en masse into endocrine cells on FGF7 removal. Thus, efficient propagation of pancreatic progenitor cells can be achieved in vitro by exposure to FGF7, which does not affect their ability to differentiate en masse into endocrine cells on FGF7 removal.

Heterozygous Missense Mutations in the Insulin Gene Are Linked to Permanent Diabetes Appearing in the Neonatal Period or in Early Infancy: A Report From the French ND (Neonatal Diabetes) Study Group

OBJECTIVE—Permanent neonatal diabetes (PND) is defined by chronic hyperglycemia due to severe nonautoimmune insulin deficiency diagnosed in the first months of life. Several genes, including KCNJ11 and ABCC8, which encode the two subunits of the ATP-sensitive K+ channel (KATP channel) can cause PND. Mutations in the insulin (INS) gene have been recently described in families with neonatal diabetes. Our study aimed to investigate the genetic anomalies and clinical heterogeneity in PND patients who are negative for a KATP channel mutation. RESEARCH DESIGN AND METHODS—We screened the INS gene by direct sequencing in 38 PND patients and in one child with nonautoimmune early-infancy diabetes, where no mutation in GCK, KCNJ11, and ABCC8 was identified. A detailed clinical phenotyping of the patients was carried out to specify the diabetes features in those found with an INS mutation. RESULTS—We identified three missense mutations in the INS gene in four probands. Two of four mutations were inherited in a dominant manner, and the familial description evidenced a marked variability in age of diagnosis and disease progression. In our cohort, the INS mutations may represent ∼10% of all permanent neonatal diabetes cases, having a later presentation of diabetes and no associated symptoms compared with cases with KATP channel mutations. CONCLUSIONS—Heterozygous INS gene mutations can cause isolated permanent early-infancy diabetes and should be assessed in neonatal as well as in childhood diabetes appearing like type 1, when autoimmune markers are absent. New pharmacogenomic strategies may be applicable, since residual β-cell function is still present in some patients.

Molecular Diagnosis of Neonatal Diabetes Mellitus Using Next-Generation Sequencing of the Whole Exome

Background Accurate molecular diagnosis of monogenic non-autoimmune neonatal diabetes mellitus (NDM) is critical for patient care, as patients carrying a mutation in KCNJ11 or ABCC8 can be treated by oral sulfonylurea drugs instead of insulin therapy. This diagnosis is currently based on Sanger sequencing of at least 42 PCR fragments from the KCNJ11, ABCC8, and INS genes. Here, we assessed the feasibility of using the next-generation whole exome sequencing (WES) for the NDM molecular diagnosis. Methodology/Principal Findings We carried out WES for a patient presenting with permanent NDM, for whom mutations in KCNJ11, ABCC8 and INS and abnormalities in chromosome 6q24 had been previously excluded. A solution hybridization selection was performed to generate WES in 76 bp paired-end reads, by using two channels of the sequencing instrument. WES quality was assessed using a high-resolution oligonucleotide whole-genome genotyping array. From our WES with high-quality reads, we identified a novel non-synonymous mutation in ABCC8 (c.1455G>C/p.Q485H), despite a previous negative sequencing of this gene. This mutation, confirmed by Sanger sequencing, was not present in 348 controls and in the patients mother, father and young brother, all of whom are normoglycemic. Conclusions/Significance WES identified a novel de novo ABCC8 mutation in a NDM patient. Compared to the current Sanger protocol, WES is a comprehensive, cost-efficient and rapid method to identify mutations in NDM patients. We suggest WES as a near future tool of choice for further molecular diagnosis of NDM cases, negative for chr6q24, KCNJ11 and INS abnormalities.

Control of early development of the pancreas in rodents and humans: implications of signals from the mesenchyme

Abstract During the last few years, progress has been made in the control of pancreatic development. Many transcription factors have been described in the pancreas and a genetic approach has been used to define their role in pancreatic development. Pancreatic development depends on mesodermic signals, with the initial steps controlled by signals from the notochord that is in close contact with the dorsal endoderm of the gut fated to become pancreas. Later signals from the mesenchyme that surrounds the embryonic pancreatic epithelium regulate the proliferation of immature pancreatic epithelial cells and their differentiation into endocrine or exocrine tissue. This review discusses recent data on the role the signals from the mesenchyme have in the development of the pancreas in rodents and humans. [Diabetologia (2000) 43: 1083–1092]