Philippe Ravassard

The Ectopic Expression of Pax4 in the Mouse Pancreas Converts Progenitor Cells into α and Subsequently β Cells

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

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.

Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants

Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.

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.

Identification and characterization of a lysosomal transporter for small neutral amino acids

In eukaryotic cells, lysosomes represent a major site for macromolecule degradation. Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters. Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases. However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level. In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters. LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons. Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as γ-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport. Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker. LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles. Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family.

In Vitro Proliferation of Cells Derived From Adult Human β-Cells Revealed By Cell-Lineage Tracing

OBJECTIVE— Expansion of insulin-producing β-cells from adult human islets could alleviate donor shortage for cell-replacement therapy of diabetes. A major obstacle to development of effective expansion protocols is the rapid loss of β-cell markers in the cultured cells. Here, we report a genetic cell-lineage tracing approach for following the fate of cultured β-cells. RESEARCH DESIGN AND METHODS— Cells dissociated from isolated human islets were infected with two lentiviruses, one expressing Cre recombinase under control of the insulin promoter and the other, a reporter cassette with the structure cytomegalovirus promoter-loxP-DsRed2-loxP-eGFP. RESULTS— β-Cells were efficiently and specifically labeled by the dual virus system. Label+, insulin− cells derived from β-cells were shown to proliferate for a maximum of 16 population doublings, with an approximate doubling time of 7 days. Isolated labeled cells could be expanded in the absence of other pancreas cell types if provided with medium conditioned by pancreatic non–β-cells. Analysis of mouse islet cells by the same method revealed a much lower proliferation of labeled cells under similar culture conditions. CONCLUSIONS— Our findings provide direct evidence for survival and dedifferentiation of cultured adult human β-cells and demonstrate that the dedifferentiated cells significantly proliferate in vitro. The findings confirm the difference between mouse and human β-cell proliferation under our culture conditions. These findings demonstrate the feasibility of cell-specific labeling of cultured primary human cells using a genetic recombination approach that was previously restricted to transgenic animals.

Expression of vesicular glutamate transporters in rat lumbar spinal cord, with a note on dorsal root ganglia

Three vesicular glutamate transporters (VGLUTs) have been recently identified and their distribution has been mapped in various brain areas. In the present study, we used morphological approaches to investigate their expression in the rat lumbar spinal cord and dorsal root ganglia. Our results show a complementary distribution of VGLUT‐expressing fibers in the spinal cord, with no overlapping in nerve endings. In the dorsal horn, VGLUT1 is most abundant in mechanosensory/proprioceptive deep afferent fibers. VGLUT2 and VGLUT3 are expressed only at moderate levels in primary sensory afferent fibers and are not used by central projections of nociceptive neurons. VGLUT1 and VGLUT2 mRNAs are mainly segregated in superficial laminae but colocalized in deeper laminae. Weak expression of VGLUT3 mRNA is only detected in deep laminae. The colocalization of VGLUT1 and VGLUT2 transcripts in most sensory neurons of the dorsal root ganglia is not in agreement with the clear segregation between the proteins in their spinal projections. Such a discrepancy suggests targeting mechanisms specific for each transporter and/or a distinct regulation of their translation. In the ventral horn, the expression of VGLUT1 and VGLUT2 mRNAs in motoneuron perikarya suggests the possible unexpected role of glutamate in the vertebrate neuromuscular junction. These results demonstrate the existence of different subpopulations of glutamate nerve terminals in the rat lumbar spinal cord and suggest that functionally distinct subsets of excitatory glutamatergic neuronal networks are involved in sensory processing and motor control. J. Comp. Neurol. 468:380–394, 2004.

Adult Duct-Lining Cells Can Reprogram into β-like Cells Able to Counter Repeated Cycles of Toxin-Induced Diabetes

It was recently demonstrated that embryonic glucagon-producing cells in the pancreas can regenerate and convert into insulin-producing β-like cells through the constitutive/ectopic expression of the Pax4 gene. However, whether α cells in adult mice display the same plasticity is unknown. Similarly, the mechanisms underlying such reprogramming remain unclear. We now demonstrate that the misexpression of Pax4 in glucagon(+) cells age-independently induces their conversion into β-like cells and their glucagon shortage-mediated replacement, resulting in islet hypertrophy and in an unexpected islet neogenesis. Combining several lineage-tracing approaches, we show that, upon Pax4-mediated α-to-β-like cell conversion, pancreatic duct-lining precursor cells are continuously mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon(+) and a β-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition. Importantly, these processes can repeatedly regenerate the whole β cell mass and thereby reverse several rounds of toxin-induced diabetes, providing perspectives to design therapeutic regenerative strategies.

The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon+ cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.