It has been suggested that pancreatic acinar cells can serve as progenitors for pancreatic islets, a concept with substantial implications for therapeutic efforts to increase insulin-producing β cell mass in patients with diabetes. We report what we believe to be the first in vivo lineage tracing approach to determine the plasticity potential of pancreatic acinar cells. We developed an acinar cell-specific inducible Cre recombinase transgenic mouse, which, when mated with a reporter strain and pulsed with tamoxifen, resulted in permanent and specific labeling of acinar cells and their progeny. During various time periods of observation and using several models to provoke injury, we failed to observe any chase of the labeled cells into the endocrine compartment, indicating that acinar cells do not normally transdifferentiate into islet β cells in vivo in adult mice. In contrast, we observed a substantial role for replication of preexisting acinar cells in the regeneration of new acinar cells after partial pancreatectomy. These results indicate that mature acinar cells harbor a facultative acinar but not endocrine progenitor capacity.

Uteroplacental insufficiency resulting in fetal growth retardation is a common complication of pregnancy and a significant cause of perinatal morbidity and mortality. Epidemiological studies show an increased incidence of type 2 diabetes in humans who were growth retarded at birth. The mechanisms by which an abnormal intrauterine milieu leads to the development of diabetes in adulthood are not known. Therefore, a rat model of uteroplacental insufficiency was developed; intrauterine growth-retarded (IUGR) rats develop diabetes with a phenotype similar to that observed in the human with type 2 diabetes. We show here that administration of a pancreatic ␤-cell trophic factor, exendin-4 (Ex-4), during the prediabetic neonatal period dramatically prevents the development of diabetes in this model. This occurs because neonatal Ex-4 prevents the progressive reduction in insulin-producing ␤-cell mass that is observed in IUGR rats over time. Expression of PDX, a critical regulator of pancreas development and islet differentiation, is restored to normal levels, and islet ␤-cell proliferation rates are normalized by the neonatal Ex-4 treatment. These results indicate that exposure to Ex-4 in the newborn period reverses the adverse consequences of fetal programming and prevents the development of diabetes in adulthood. Diabetes 52: 734 -740, 2003

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.

Hox factors are evolutionarily conserved homeodomain-containing transcription factors that activate and repress gene expression in a precise temporally and spatially regulated manner during development and differentiation. Pancreatic-duodenal homeobox 1 (PDX-1) is a Hox-type protein that is a critical requirement for normal pancreas development and for proper differentiation of the endocrine pancreas. In humans, PDX-1 gene mutation causes pancreatic agenesis and early-and late-onset type 2 diabetes. PDX-1 consists of an N-terminal transactivation domain, a homeodomain responsible for DNA binding and nuclear localization, and a conserved C terminus that is mutated in human diabetes but whose function is poorly understood. We have identified a novel POZ domain protein, PDX-1 C terminus-interacting factor 1 (PCIF1)/SPOP, that interacts with PDX-1 both in vitro and in vivo. PCIF1 is localized to the nucleus in a speckled pattern, and coexpression of PDX-1 alters the subnuclear distribution of PCIF1. Functionally, PCIF1 inhibits PDX-1 transactivation of established target gene promoters in a specific and dose-dependent manner that requires critical amino acids in the PDX-1 C terminus. PCIF1 is expressed in adult pancreatic insulin-producing ␤ cells, and overexpression of PCIF1 inhibits the rat insulin 1 and rat insulin 2 promoters in the MIN6 insulinoma ␤ cell line. The coexpression of PCIF1 with PDX-1 in ␤ cells and the ability of PCIF1 to repress PDX-1 transactivation suggest that modulation of PDX-1 function by PCIF1 may regulate normal ␤ cell differentiation.Hox factors are evolutionarily conserved homeodomain-containing transcription factors that are critical for the determination of the body plan during early development and for the regulation of key differentiation-specific genes that determine cellular specificity. Proper spatial and temporal expression of Hox factors is required to orchestrate anterior-posterior segmentation, limb development, and organogenesis. Hox proteins regulate gene transcription by a well-coordinated and tightly regulated balance of gene activation and repression. In particular, the addition and refinement of repression domains have contributed to the evolutionary diversification of body segmentation (15, 37).Pancreatic-duodenal homeobox 1 (PDX-1; also known as IPF-1, IDX-1, STF-1, Xlhbox8, GSF, and IUF-1) is a Hox-type homeodomain protein encoded by a gene in the ParaHox cluster that is expressed in the region of the developing foregut that later becomes the duodenum, distal stomach, and pancreas (5,24,28,29,32,46). As development proceeds, highlevel PDX-1 expression is eventually restricted to the hormone-producing ␤ and ␦ cells of the fully developed endocrine pancreas, whereas lower levels of expression are detected in certain ductal cells, the majority of exocrine acinar cells, and epithelial cells and Brunner's glands in the distal stomach and proximal duodenum. In the differentiated endocrine pancreas, PDX-1 regulates the transcription of important target genes, including the i...

Intrauterine growth restriction (IUGR) induced by uterine artery ligation in pregnant rats leads to low birth weight and early insulin secretory defects followed by the development of insulin resistance, decline in ␤-cell mass, and diabetes in adulthood. Neonatal administration of Exendin-4 (Ex-4) prevents the deterioration of ␤-cell mass and the onset of adult-onset diabetes. Our aim was to determine whether this effect occurs through preservation of islet vascularization. In 2 wk-old IUGR rats, endothelial-specific lectin staining revealed a 40% reduction in islet vascular density (p ϭ 0.027), which was normalized by neonatal Ex-4. VEGF-A protein expression was reduced in IUGR islets compared with controls at postnatal d 1 (P). Neonatal Ex-4 normalized islet VEGF protein expression at P7. Neither IUGR nor Ex-4 administration to IUGR rats affected relative VEGF splice isoform RNA levels. Together, the reduced vascularity in IUGR islets before the deterioration of ␤-cell mass, and the enhancement of VEGF expression and normalization of islet vascularity by neonatal Ex-4, suggest islet vascularity as an early determinant of ␤-cell mass and as a potential therapeutic target for diabetes prevention. Uteroplacental insufficiency, leading to reduced delivery of nutrients, oxygen, growth factors, and hormones to the developing fetus, is a common cause of fetal growth restriction. Epidemiologic studies in humans have established a correlation between intrauterine growth restriction (IUGR) and an increased risk of type 2 diabetes, obesity, and coronary artery disease in later life (1). This demonstration of the fetal origins hypothesis, the idea that the fetal environment has a crucial influence on development of adult-onset disease, has been reproduced in multiple human populations and in various animal models (1). In a rat model of uteroplacental insufficiency, bilateral uterine artery ligation in late gestation results in IUGR and low birth weight (2). IUGR rats develop a phenotype similar to that of type 2 diabetes in humans, with the development of obesity, insulin resistance, and insulinsecretory deficits. Although normal early in life, there is a progressive decline in ␤-cell mass of IUGR rats, preceding the development of hyperglycemia and diabetes by several weeks of age (2).Extensive islet remodeling occurs during the immediate postnatal period in rodent pancreatic development, with waves of ␤-cell proliferation, apoptosis, and neogenesis in the first weeks of life (3,4). Similar to the impact that a negative fetal environment has on future pancreatic function, therapeutic intervention within this critical neonatal period may permanently improve ␤-cell mass throughout adulthood. The incretin hormone glucagon-like peptide-1 (GLP-1) and the long-acting GLP-1 receptor agonist, Exendin-4 (Ex-4), augment insulin secretion and improve glucose tolerance in diabetes (5). GLP-1 also promotes expansion of ␤-cell mass by increasing proliferation and stimulating neogenesis (5). Importantly, Ex-4 when given in t...

RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC 50 = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC 50 = 10 nM) with reduced hERG activity (14 μM).

RIP2 kinase has been identified as a key signal transduction partner in the NOD2 pathway contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP2 kinase or its signaling partners on the NOD2 pathway that are suitable for advancement into the clinic have yet to be described. Herein, we report our discovery and profile of the prodrug clinical compound, inhibitor 3, currently in phase 1 clinical studies. Compound 3 potently binds to RIP2 kinase with good kinase specificity and has excellent activity in blocking many proinflammatory cytokine responses in vivo and in human IBD explant samples. The highly favorable physicochemical and ADMET properties of 3 combined with high potency led to a predicted low oral dose in humans.

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