PDX-1 interaction and regulation of the Pancreatic Derived Factor (PANDER, FAM3B) promoter.

Pancreatic Derived Factor (PANDER) is a novel cytokine-like protein dominantly expressed within the endocrine pancreas. Our previous study demonstrated that the PANDER promoter was both tissue-specific and glucose-responsive. Surrounding the PANDER transcriptional start site are several putative A- and E-Box elements that may bind to the various pancreatic transcriptional factors of MafA, BETA2/NeuroD, and Pancreatic Duodenal Homeobox-1 (PDX-1). To characterize the transcriptional regulatory factors involved in PANDER gene expression, we performed co-transfection reporter gene analysis and demonstrated upregulation by all three transcription factors, with the greatest individual increase stemming from PDX-1. Potential binding of PDX-1 to A box (TAAT) regions of the PANDER promoter was demonstrated by chromatin immunoprecipitation (ChIP) and further corroborated by electrophoretic mobility shift assay (EMSA). Binding of PDX-1 to the A box regions was inhibited by mutagenized (TAGT) oligonucleotides. Site-directed mutagenesis of the three PDX-1 A box binding motifs revealed that A box sites 2 and 3 in combination were critical for maximal gene expression and deletion resulted in a 82% reduction in promoter activity. Furthermore, deletion of A box sites 2 and 3 completely diminished the glucose-responsiveness of the PANDER promoter. Our findings demonstrate that PANDER is a potential PDX-1 target gene and the A box sites within the promoter region are critical for basal and glucose-stimulated PANDER expression.

Overview of the Standing Operating Procedure (SOP) for the development of Provisional Advisory Levels (PALs).

Provisional Advisory Levels (PALs) are concentrations in air and drinking water for priority toxic chemicals. This article summarizes the Standing Operating Procedure (SOP) currently in place for the data-driven development of chemical-specific PALs. To provide consistency and transparency, and to avoid faults of arbitrariness, the SOP was developed for guidance in deriving PAL values. Three levels (PAL 1, PAL 2, and PAL 3), distinguished by severity of toxic effects, are developed for 24-hour, 30-day, 90-day, and 2-year durations of potential drinking water and inhalation exposures for the general public. The SOP for PAL development focuses on (1) data acquisition and analysis, (2) identification of a chemical-specific critical effect, (3) selection of a quantitative point-of-departure (POD), (4) uncertainty analysis and adjustments, (5) exposure duration adjustment and extrapolation, (6) identification of special concerns and issues, and (7) verification, documentation, and dissemination of PALs. To avoid uncompromising rigidity in deriving PAL values and to allow for incorporation of new or refined methodologies, the overall procedure is fluid and subject to modification. The purpose of this publication is to provide a summary of this SOP.

Mechanisms of glucose-induced secretion of pancreatic-derived factor (PANDER or FAM3B) in pancreatic beta-cells.

Pancreatic-derived factor (PANDER) is an islet-specific cytokine present in both pancreatic alpha- and beta-cells, which, in vitro, induces beta-cell apoptosis of primary islet and cell lines. In this study, we investigated whether PANDER is secreted by pancreatic alpha- and beta-cells and whether PANDER secretion is regulated by glucose and other insulin secretagogues. In mouse-derived insulin-secreting beta-TC3 cells, PANDER secretion in the presence of stimulatory concentrations of glucose was 2.8 +/- 0.4-fold higher (P < 0.05) than without glucose. Insulin secretion was similarly increased by glucose in the same cells. The total concentration of secreted PANDER in the medium was approximately 6-10 ng/ml (0.3-0.5 nmol/l) after a 24-h culture with glucose. L-Glucose failed to stimulate PANDER secretion in beta-TC3 cells. KCl stimulated PANDER secretion 2.1 +/- 0.1-fold compared with control without glucose. An L-type Ca2+ channel inhibitor, nifedipine, completely blocked both glucose- or KCl-induced insulin and PANDER secretion. In rat-derived INS-1 cells, glucose (20 mmol/l) stimulated PANDER secretion 4.4 +/- 0.9-fold, while leucine plus glutamine stimulated 4.4 +/- 0.7-fold compared with control without glucose. In mouse islets overexpressing PANDER, glucose (20 mmol/l) stimulated PANDER secretion 3.2 +/- 0.5-fold (P < 0.05) compared with basal (3 mmol/l glucose). PANDER was also secreted by alpha-TC3 cells but was not stimulated by glucose. Mutations of cysteine 229 or of cysteines 91 and 229 to serine, which may form one disulfide bond, and truncation of the COOH-terminus or NH2-terminus of PANDER all resulted in failure of PANDER secretion, even though these mutant or truncated PANDERs were highly expressed within the cells. In conclusion, we found that 1) PANDER is secreted from both pancreatic alpha- and beta-cells, 2) glucose stimulates PANDER secretion dose dependently in beta-cell lines and primary islets but not in alpha-cells, 3) PANDER is likely cosecreted with insulin via the same regulatory mechanisms, and 4) structure and conformation is vital for PANDER secretion.

Insulin receptor signaling and sarco/endoplasmic reticulum calcium ATPase in beta-cells.

Glucose is the main physiological secretagogue for insulin secretion by pancreatic beta-cells, and the major biochemical mechanisms involved have been elucidated. In particular, an increase in intracellular calcium is important for insulin exocytosis. More recently, it has become apparent that the beta-cell also has many of the elements of the insulin receptor signal transduction pathway, including the insulin receptor and insulin receptor substrate (IRS) proteins 1 and 2. Studies with transgenic models have shown that the beta-cell-selective insulin receptor knockout and the IRS-1 knockout lead to reduced glucose-induced insulin secretion. Overexpression of the insulin receptor and IRS-1 in beta-cells results in increased insulin secretion and increased cytosolic Ca(2+). We have thus postulated the existence of a novel autocrine-positive feedback loop of insulin on its own secretion involving interaction with the insulin receptor signal transduction pathway and regulation of intracellular calcium homeostasis. Our current working hypothesis is that this glucose-dependent interaction occurs at the level of IRS-1 and the sarco(endo)plasmic reticulum calcium ATPase, the calcium pump of the endoplasmic reticulum.

Distinguishing features of leucine and alpha-ketoisocaproate sensing in pancreatic beta-cells.

Culturing rat islets in high glucose (HG) increased 1-(14)C-alpha-ketoisocaproate (KIC) oxidation compared with culturing them in low glucose. Leucine caused insulin secretion (IS) in low glucose but not in HG rat islets, whereas KIC did so in both. Pretreatment with HG for 40 min abolished leucine stimulation of IS by mouse islets and prevented the cytosolic Ca(2+) rise without inhibiting IS and Ca(2+) increments caused by KIC. When islets were pretreated without glucose and glutamine, aminooxyacetic acid (AOA) markedly decreased KIC effects. When islets were pretreated without glucose and with glutamine, AOA potentiated leucine effects but attenuated KIC effects. AOA stimulated glutamine oxidation in the presence but not the absence of +/-2-amino-2-norbornane-carboxylic acid, a nonmetabolized leucine analog. Pretreatment with HG and glutamine partially reversed AOA inhibition of KIC effects. Glucose increased intracellular ATP and GTP, whereas it decreased ADP and GDP in beta HC9 cells. Glutamate dehydrogenase activity of beta HC9 cell extracts was increased by leucine and attenuated by GTP, but it was potentiated by ADP. In conclusion, leucine and KIC stimulated beta-cells via distinct mechanisms. Glutamate dehydrogenase is the sensor of leucine, whereas transamination plays an important role in KIC stimulation of pancreatic beta-cells.

Liver-specific overexpression of pancreatic-derived factor (PANDER) induces fasting hyperglycemia in mice.

The pancreas-derived hormones, insulin and glucagon, are the two main regulators of glucose homeostasis. However, their actions can be modulated by the presence of other circulating factors including cytokines. Pancreatic-derived factor (PANDER) is a novel cytokine-like molecule secreted from the endocrine pancreas, but its biological function is currently unknown. To address this, we employed adenoviral gene delivery to develop a novel murine model of PANDER overexpression, which we used to study PANDER's effect on glucose homeostasis. Although serum metabolites in fed mice were unaffected by PANDER overexpression, fasting glucose, insulin, and corticosterone levels were significantly elevated. Additionally, PANDER-overexpressing mice displayed elevated glucose and insulin levels during a glucose tolerance test, indicating that glucose tolerance was impaired. However, there were no defects in glucose-stimulated insulin secretion or peripheral insulin sensitivity. Elevated transcription of hepatic gluconeogenic genes, PEPCK and G6Pase accompanied the fasting hyperglycemia observed in PANDER-overexpressing animals. Similarly, treatment of primary hepatocytes with PANDER-expressing adenovirus or PANDER-enriched conditioned medium elevated gluconeogenic gene expression and glucose output. PANDER treatment also resulted in higher levels of Ser133-phosphorylated cAMP-response element-binding protein in hepatocytes stimulated with 8-bromo-cAMP and dexamethasone and higher levels of intracellular cAMP upon stimulation with forskolin. In summary, we provide the first report that identifies PANDER as a regulator of hepatic glucose metabolism, where it serves as a novel factor that amplifies hepatic cAMP and cAMP-response element-binding protein signaling to induce gluconeogenic gene expression and glucose output.

Characterization of the expression, localization, and secretion of PANDER in alpha-cells.

The novel islet-specific protein PANcreatic DERived Factor (PANDER; FAM3B) has been extensively characterized with respect to the beta-cell, and these studies suggest a potential function for PANDER in the regulation of glucose homeostasis. Little is known regarding PANDER in pancreatic -cells, which are critically involved in maintaining euglycemia. Here we present the first report elucidating the expression and regulation of PANDER within the alpha-cell. Pander mRNA and protein are detected in alpha-cells, with primary localization to a glucagon-negative granular cytosolic compartment. PANDER secretion from alpha-cells is nutritionally and hormonally regulated by l-arginine and insulin, demonstrating similarities and differences with glucagon. Signaling via the insulin receptor (IR) through the PI3K and Akt/PKB node is required for insulin-stimulated PANDER release. The separate localization of PANDER and glucagon is consistent with their differential regulation, and the effect of insulin suggests a paracrine/endocrine effect on PANDER release. This provides further insight into the potential glucose-regulatory role of PANDER.

Targeted disruption of pancreatic-derived factor (PANDER, FAM3B) impairs pancreatic beta-cell function.

OBJECTIVE: Pancreatic-derived factor (PANDER, FAM3B) is a pancreatic islet-specific cytokine-like protein that is secreted from beta-cells upon glucose stimulation. The biological function of PANDER is unknown, and to address this we generated and characterized a PANDER knockout mouse. RESEARCH DESIGN AND METHODS: To generate the PANDER knockout mouse, the PANDER gene was disrupted and its expression was inhibited by homologous recombination via replacement of the first two exons, secretion signal peptide and transcriptional start site, with the neomycin gene. PANDER(-/-) mice were then phenotyped by a number of in vitro and in vivo tests to evaluate potential effects on glucose regulation, insulin sensitivity, and beta-cell morphology and function. RESULTS: Glucose tolerance tests demonstrated significantly higher blood glucose levels in PANDER(-/-) versus wild-type male mice. To identify the mechanism of the glucose intolerance, insulin sensitivity and pancreatic beta-cell function were examined. Hyperinsulinemic-euglycemic clamps and insulin tolerance testing showed similar insulin sensitivity for both the PANDER(-/-) and wild-type mice. The in vivo insulin response following intraperitoneal glucose injection surprisingly produced significantly higher insulin levels in the PANDER(-/-) mice, whereas insulin release was blunted with arginine administration. Islet perifusion and calcium imaging studies showed abnormal responses of the PANDER(-/-) islets to glucose stimulation. In contrast, neither islet architecture nor insulin content was impacted by the loss of PANDER. Interestingly, the elevated insulin levels identified in vivo were attributed to decreased hepatic insulin clearance in the PANDER(-/-) islets. Taken together, these results demonstrated decreased pancreatic beta-cell function in the PANDER(-/-) mouse. CONCLUSIONS: These results support a potential role of PANDER in the pancreatic beta-cell for regulation or facilitation of insulin secretion.

Epigenetic events, remodelling enzymes and their relationship to chromatin organization in prostatic intraepithelial neoplasia and prostatic adenocarcinoma.

OBJECTIVE: To explore the nuclear chromatin phenotype, overall epigenetic mechanisms, chromatin remodelling enzymes and their role as diagnostic biomarkers in prostate lesions, using high-resolution computerized quantitative digital image analysis (DIA). MATERIALS AND METHODS: A tissue microarray (TMA) was constructed using paraffin wax-embedded prostatic tissues from 78 patients, containing 30 cores of benign prostatic hyperplasia (BPH), 10 of low-grade prostatic intraepithelial neoplasia (LGPIN), 38 of prostate adenocarcinoma, 20 of BPH tissue excised at 0.6-1 mm from LGPIN lesions, and 10 of BPH prostatic tissues obtained 0.6-1 mm from high-grade PIN (HGPIN) lesions. Chromatin phenotype was assessed on haematoxylin-stained sections using high-resolution texture analysis. For quantitative immunohistochemistry, antibodies raised against acetylated histone H3 lysine 9 (AcH3K9), 5'methylcytidine (5MeC) and the chromatin remodelling ATPase ISWI (SNF2H and SNF2L) were used. The immunodensity was measured using DIA to determine the epigenetic profile of the cases. At least 60 nuclei were measured from each case. RESULTS: There were many statistically significant differences in staining intensity and nuclear distribution patterns in chromatin phenotype and immunostaining (p

Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells.

PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.

Protein kinase A translocation and insulin secretion in pancreatic beta-cells: studies with adenylate cyclase toxin from Bordetella pertussis.

Activation of protein kinase A (cAMP-dependent protein kinase; PKA) triggers insulin secretion in the beta-cell. Adenylate cyclase toxin (ACT), a bacterial exotoxin with adenylate cyclase activity, and forskolin, an activator of adenylate cyclase, both dose-dependently increased insulin secretion in the presence, but not the absence, of glucose in insulin-secreting betaTC3 cells. The stimulation of cAMP release by either agent was dose-dependent but glucose-independent. Omission of extracellular Ca(2+) totally abolished the effects of ACT on insulin secretion and cytosolic cAMP accumulation. ACT and forskolin caused rapid and dramatic increases in cytosolic Ca(2+), which were blocked by nifedipine and the omission of extracellular Ca(2+). Omission of glucose completely blocked the effects of forskolin and partially blocked the effects of ACT on cytosolic Ca(2+). PKA alpha, beta and gamma catalytic subunits (Calpha, Cbeta and Cgamma respectively) were identified in betaTC6 cells by confocal microscopy. Glucose and glucagon-like polypeptide-1 (GLP-1) caused translocation of Calpha to the nucleus and of Cbeta to the plasma membrane and the nucleus, but did not affect the distribution of Cgamma. In conclusion, glucose and GLP-1 amplify insulin secretion via cAMP production and PKAbeta activation.