The homeodomain protein IPF1 (also known as IDX1, STF1 and PDX1; see Methods) is critical for development of the pancreas in mice and is a key factor for the regulation of the insulin gene in the beta-cells of the endocrine pancreas. Targeted disruption of the Ipf1 gene encoding IPF1 in transgenic mice results in a failure of the pancreas to develop (pancreatic agenesis). Here, we report the identification of a single nucleotide deletion within codon 63 of the human IPF1 gene (13q12.1) in a patient with pancreatic agenesis. The patient is homozygous for the point deletion, whereas both parents are heterozygotes for the same mutation. The deletion was not found in 184 chromosomes from normal individuals, indicating that the mutation is unlikely to be a rare polymorphism. The point deletion causes a frame shift at the C-terminal border of the transactivation domain of IPF1 resulting in the translation of 59 novel codons before termination, aminoproximal to the homeodomain essential for DNA binding. Expression of mutant IPF1 in Cos-1 cells confirms the expression of a prematurely terminated truncated protein of 16 kD. Thus, the affected patient should have no functional IPF1 protein. Given the essential role of IPF1 in pancreas development, it is likely that this autosomal recessive mutation is the cause of the pancreatic agenesis phenotype in this patient. Thus, IPF1 appears to be a critical regulator of pancreas development in humans as well as mice.

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The homeodomain transcription factor insulin promoter factor-1 (IPF-1) is required for development of the pancreas and also mediates glucose-responsive stimulation of insulin gene transcription. Earlier we described a human subject with pancreatic agenesis attributable to homozygosity for a cytosine deletion in codon 63 of the IPF-1 gene (Pro63fsdelC). Pro63fsdelC resulted in the premature truncation of an IPF-1 protein which lacked the homeodomain required for DNA binding and nuclear localization. Subsequently, we linked the heterozygous state of this mutation with type 2 diabetes mellitus in the extended family of the pancreatic agenesis proband. In the course of expressing the mutant IPF-1 protein in eukaryotic cells, we detected a second IPF-1 isoform, recognized by COOH-but not NH 2 -terminal-specific antisera. This isoform localizes to the nucleus and retains DNA-binding functions. We provide evidence that internal translation initiating at an out-of-frame AUG accounts for the appearance of this protein. The reading frame crosses over to the wild-type IPF-1 reading frame at the site of the point deletion just carboxy proximal to the transactivation domain. Thus, the single mutated allele results in the translation of two IPF-1 isoproteins, one of which consists of the NH 2 -terminal transactivation domain and is sequestered in the cytoplasm and the second of which contains the COOH-terminal DNA-binding domain, but lacks the transactivation domain. Further, the COOH-terminal mutant IPF-1 isoform does not activate transcription and inhibits the transactivation functions of wild-type IPF-1. This circumstance suggests that the mechanism of diabetes in these individuals may be due not only to reduced gene dosage, but also to a dominant negative inhibition of transcription of the insulin gene and other ␤ cell-specific genes regulated by the mutant IPF-1. ( J. Clin. Invest. 1998. 102: 232-241.)

Aims/hypothesis The endogenous production of stromal cell-derived factor-1 (SDF-1) in beta cells in transgenic mice attenuates the development of diabetes in response to streptozotocin. Here we propose that beta cell injury induces SDF-1 production, and the SDF-1/chemokine (C-X-C motif) receptor 4 (CXCR4) interaction auto-activates Sdf1 expression, resulting in the autocrine production of SDF-1 by beta cells and the paracrine activation of glucagon-like peptide-1 (GLP-1) production by alpha cells. Methods SDF-1 production in adult mouse and human islets and rat INS-1 cells was measured in models of beta cell injury. The paracrine actions of SDF-1 on GLP-1 production in alpha cells were explored. The potential synergism between the growth-promoting actions of GLP-1 and the pro-survival actions of SDF-1 on the preservation of cell mass was evaluated by cell viability assays. Results In adult islets and INS-1 cells, Sdf1 expression was re-induced in response to injury. The interaction of SDF-1 with its receptor on alphaTC1 cells activated protein kinase Akt, stimulated cell proliferation and induced the expression of prohormone convertase 1/3 and the consequent production of GLP-1 in alpha cells. The combination of GLP-1 and SDF-1 additively enhanced both the growth and longevity of INS-1 beta cells. Conclusions/interpretation The results of these studies suggest that in response to beta cell injury and the ensuing induction of SDF-1, the biological function of alpha cells switches from the production of glucagon to the provision of the local growth factor GLP-1 which, in combination with SDF-1, promotes the growth, survival and viability of the beta cells.

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Type 2 diabetes, often associated with obesity, results from a deficiency of insulin production and action manifested in increased blood levels of glucose and lipids that further promote insulin resistance and impair insulin secretion. Glucolipotoxicity caused by elevated plasma glucose and lipid levels is a major cause of impaired glucose-stimulated insulin secretion from pancreatic β-cells, due to increased oxidative stress, and insulin resistance. Glucagon-like peptide-1 (GLP1), an insulinotropic glucoincretin hormone, is known to promote β-cell survival via its actions on its G-protein-coupled receptor on β-cells. Here, we report that a nonapeptide, GLP1(28–36)amide, derived from the C-terminal domain of the insulinotropic GLP1, exerts cytoprotective actions on INS-1 β-cells and on dispersed human islet cells in vitro in conditions of glucolipotoxicity and increased oxidative stress independently of the GLP1 receptor. The nonapeptide appears to enter preferably stressed, glucolipotoxic cells compared with normal unstressed cells. It targets mitochondria and improves impaired mitochondrial membrane potential, increases cellular ATP levels, inhibits cytochrome c release, caspase activation, and apoptosis, and enhances the viability and survival of INS-1 β-cells. We propose that GLP1(28–36)amide might be useful in alleviating β-cell stress and might improve β-cell functions and survival.

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The glucoincretin hormone glucagon-like peptide-1 (GLP-1) augments glucose-stimulated insulin secretion and is in use as an effective treatment for diabetes. However, after its secretion from the intestine, the insulinotropic GLP-1 (7-36) amide hormone is rapidly inactivated by enzymatic cleavage by the diaminopeptidyl peptidase-4 giving rise to GLP-1 (9-36) amide. Inasmuch as most of the circulating GLP-1 is in the form of the metabolite GLP-1 (9-36) amide it has been suggested that it has insulin-like actions on peripheral insulin-sensitive tissues. In earlier studies, infusions of GLP-1 (9-36) amide in obese insulin-resistant subjects showed a marked suppression of hepatic glucose production. However, it remained uncertain whether the effects on glucose production were due to direct effects on hepatocytes, involved central or portal vein-mediated actions, or were mediated by insulin secretion. Here we show that GLP-1 (9-36) amide directly suppresses glucose production in isolated mouse hepatocytes ex vivo independent of the GLP-1 receptor. These findings support direct insulinomimetic actions of the GLP-1 metabolite on gluconeogenesis in hepatocytes that are independent of insulin action and the GLP-1 receptor, and suggest that GLP-1 (9-36) amide-based peptides might present a novel therapy for the treatment of excessive hepatic glucose production in individuals with insulin-resistant diabetes.