Daniel M. Kemp

Identification and functional characterization of melatonin Mel 1a receptors in pancreatic β cells: potential role in incretin-mediated cell function by sensitization of cAMP signaling

Melatonin receptors are expressed within the pancreatic islets of Langerhans, and melatonin induces a direct effect on insulin secretion ex-vivo. Here, we report the endogenous expression of the melatonin Mel 1a receptor in the INS-1 pancreatic beta cell line. Pharmacological characterization of the receptor using a CRE-luciferase reporter gene demonstrated its functional activity in INS-1 cells, displaying the characteristic signaling properties of the G(i/o) coupled receptor. Acute melatonin treatment of INS-1 cells in the presence of either forskolin or the incretin hormone glucagon-like peptide 1 (GLP-1) caused an attenuation of the responses in insulin secretion, insulin promoter activity, and CRE mediated gene expression, consistent with its effects in inhibiting cAMP mediated signal transduction. However, prolonged exposure (12 h) of INS-1 cells to melatonin treatment resulted in a sensitization of cAMP mediated responses to forskolin and GLP-1. Insulin secretion, insulin promoter activity and CRE mediated gene expression levels were augmented compared with responses without melatonin pre-treatment in INS-1 cells. In isolated rat islets, insulin secretion was enhanced following melatonin pre-treatment both in the absence and presence of GLP-1 or forskolin. This phenomenon reflects observations reported in other cell types expressing the melatonin Mel 1a receptor, and may represent the first evidence of a specific physiological role for melatonin-induced sensitization.

Developmental Aspects of the Endocrine Pancreas

A detailed morphological map of pancreatic development has evolved over many years of careful observation of embryonic growth, particularly in the mouse, providing a template for the more challenging feat of identifying the developmental pathways in the pancreas at the molecular level. Definition of the mechanisms that regulate pancreatic growth and the differentiation of the endocrine and exocrine cell lineages during embryonic development may yield a better understanding of the molecular determinants of diseases such as diabetes. These answers also may facilitate a more educated approach in generating viable islet cell lines from adult and embryonic cells for transplantation.

Heat shock protein 90 (HSP90) inhibitors activate the heat shock factor 1 (HSF1) stress response pathway and improve glucose regulation in diabetic mice

The cytoprotective stress response factor HSF1 regulates the transcription of the chaperone HSP70, which exhibits anti-inflammatory effects and improves insulin sensitivity. We tested the therapeutic potential of this pathway in rodent models of diabetes using pharmacological tools. Activation of the HSF1 pathway was achieved using potent inhibitors of the upstream regulatory protein, HSP90. Treatment with AUY922, a selective HSP90 inhibitor led to robust inhibition of JNK1 phosphorylation, cytoprotection and improved insulin signaling in cells, consistent with effects observed with HSP70 treatment. Chronic dosing with HSP90 inhibitors reversed hyperglycemia in the diabetic db/db mouse model, and improved insulin sensitivity in the diet-induced obese mouse model of insulin resistance, further supporting the concept that the HSF1 pathway is a potentially viable anti-diabetes target.

Adult tissue-specific expression of a Dppa3-derived retrogene represents a postnatal transcript of pluripotent cell origin

Processed pseudogenes emerge by reverse transcription of spliced mRNAs followed by incorporation of the resultant cDNA into the genome. Their genesis requires that retrotransposition occurs within the germ line, a provision that significantly limits random distribution of source genes. We previously identified embryonic stem cell-specific genes as an enriched source of retropseudogene origin. Nanog, Oct4, and Dppa3 (Stella/PGC7) presented as source genes for >30 processed pseudogenes within the human genome. In the current study, we extended our previous analysis and focused on the pluripotent cell-specific Dppa gene family. Of the five Dppa genes characterized, four were associated with putative retropseudogenes as determined by nucleotide BLAST (basic local alignment sequence tool) searches of the respective mRNA transcripts against the human genome. A subset of the 11 Dppa3-derived hits were then screened against a human adult tissue cDNA panel for evidence of transcriptional activity. One of the putative Dppa3-derived retropseudogenes, Dppa3(d), located on human chromosome 16p13, tested positive for mRNA transcript in bone marrow, peripheral blood, pancreas, adrenal gland, and thyroid gland. Specificity against the source Dppa3 gene expression was sequence verified, and independent human tissue samples were obtained to confirm Dppa3(d) expression. These data substantiate the existence of human adult tissue-specific transcripts that originate via retrotransposition of the pluripotent cell-specific gene, Dppa3. Further studies may reveal an evolutionary role for this example of genetic diversity, but in the short term our observations serve a cautionary purpose regarding the use of Dppa3 transcripts in adult tissue-derived cells as a potential marker of pluripotency.

Synergistic effect of dimethyl sulfoxide on glucagon-like peptide 1 (GLP-1)-stimulated insulin secretion and gene transcription in INS-1 cells: characterization and implications

Glucagon-like peptide 1 (GLP-1) is an incretin hormone that is secreted from the enteroendocrine L-cells of the gut in response to nutrient ingestion. GLP-1 enhances both insulin secretion and insulin gene expression in a glucose-dependent manner via activation of its putative G-protein-coupled receptor on pancreatic beta-cells. In the presence of DMSO (0.5-2.5%), these functional responses were enhanced significantly (2- to 2.5-fold) in a concentration-dependent manner in the beta-cell line INS-1, although basal levels were not affected. Rat insulin 1 (rINS1) promoter activity appeared to be augmented in a cAMP-response element (CRE)-dependent manner as the effect of DMSO was abolished following a mutation in the CRE of the rINS1 promoter. Also, expression of a generic cAMP-driven reporter gene was enhanced by 1.5% DMSO in response to GLP-1 (3.5-fold), forskolin (2-fold), and 3-isobutyl-1-methylxanthine (2-fold). Analysis of intracellular signaling components revealed that DMSO did not elevate cAMP levels, protein kinase A activity, or phosphorylated levels of CRE-binding protein (CREB), CRE-modulator (CREM), and activating transcription factor-1 (ATF-1). These data suggest that GLP-1 induces insulin gene transcription in a CREB, CREM, and ATF-1-independent manner in beta-cells. The mechanism by which DMSO imparts this amplifying action is unclear but may involve redistribution of intracellular compartments or a direct molecular interaction with a downstream target of the GLP-1 receptor signaling pathway in the beta-cell. These effects of DMSO on incretin action may provide novel applications with respect to further characterizing GLP-1 receptor signaling, identifying incretin-like compounds in screening assays, and as a therapeutic treatment in type 2 diabetes.

Uncovering mechanisms of transcriptional regulations by systematic mining of cis regulatory elements with gene expression profiles

BackgroundContrary to the traditional biology approach, where the expression patterns of a handful of genes are studied at a time, microarray experiments enable biologists to study the expression patterns of many genes simultaneously from gene expression profile data and decipher the underlying hidden biological mechanism from the observed gene expression changes. While the statistical significance of the gene expression data can be deduced by various methods, the biological interpretation of the data presents a challenge.ResultsA method, called CisTransMine, is proposed to help infer the underlying biological mechanisms for the observed gene expression changes in microarray experiments. Specifically, this method will predict potential cis-regulatory elements in promoter regions which could regulate gene expression changes. This approach builds on the MotifADE method published in 2004 and extends it with two modifications: up-regulated genes and down-regulated genes are tested separately and in addition, tests have been implemented to identify combinations of transcription factors that work synergistically. The method has been applied to a genome wide expression dataset intended to study myogenesis in a mouse C2C12 cell differentiation model. The results shown here both confirm the prior biological knowledge and facilitate the discovery of new biological insights.ConclusionThe results validate that the CisTransMine approach is a robust method to uncover the hidden transcriptional regulatory mechanisms that can facilitate the discovery of mechanisms of transcriptional regulation.

NRSF/REST confers transcriptional repression of the GPR10 gene via a putative NRSE/RE-1 located in the 5' promoter region.

The G protein‐coupled receptor GPR10 is highly localized to areas of the brain. In an effort to reveal transcriptional determinants of this tissue specificity, we recognized a putative NRSE (neuron‐restrictive silencer element) located in the 5′ promoter region of the gene. The cognate NRSE binding protein NRSF (neuron‐restrictive silencer factor) restricts gene expression to mature neurons and endocrine cells by repressing their transcription in non‐neuronal/‐endocrine cells. In cell lines where NRSF‐mediated gene repression has been functionally established, the activity of the GPR10 promoter was repressed in a manner consistent with NRSE‐dependent regulation. A specific point mutation to confer non‐functionality of the NRSE revealed a 10‐fold de‐repression of reporter gene expression. In contrast, in the GPR10‐expressing cell line GH3, mRNA transcripts of NRSF were undetectable and suppression of promoter activity was not observed. However, transfection of a rat NRSF expression vector resulted in significant repression of transcription, which was reversed by mutation of the NRSE. In conclusion, we demonstrate that the GPR10 gene is specifically regulated by NRSF, and suggest this to be a contributory factor in the tissue‐specific distribution of GPR10 in vivo.

Dynamic resolution of functionally related gene sets in response to acute heat stress

BackgroundUsing a gene clustering strategy we determined intracellular pathway relationships within skeletal myotubes in response to an acute heat stress stimuli. Following heat shock, the transcriptome was analyzed by microarray in a temporal fashion to characterize the dynamic relationship of signaling pathways.ResultsBioinformatics analyses exposed coordination of functionally-related gene sets, depicting mechanism-based responses to heat shock. Protein turnover-related pathways were significantly affected including protein folding, pre-mRNA processing, mRNA splicing, proteolysis and proteasome-related pathways. Many responses were transient, tending to normalize within 24 hours.ConclusionIn summary, we show that the transcriptional response to acute cell stress is largely transient and proteosome-centric.

Extending the pathway analysis framework with a test for transcriptional variance implicates novel pathway modulation during myogenic differentiation

MOTIVATION We describe an extension of the pathway-based enrichment approach for analyzing microarray data via a robust test for transcriptional variance. The use of a variance test is intended to identify additional patterns of transcriptional regulation in which many genes in a pathway are up- and down-regulated. Such patterns may be indicative of the reciprocal regulation of pathway activators and inhibitors or of the differential regulation of separate biological sub-processes and should extend the number of detectable patterns of transcriptional modulation. RESULTS We validated this new statistical approach on a microarray experiment that captures the temporal transcriptional profile of muscle differentiation in mouse C2C12 cells. Comparisons of the transcriptional state of myoblasts and differentiated myotubes via a robust variance test implicated several novel pathways in muscle cell differentiation previously overlooked by a standard enrichment analysis. Specifically, pathways involved in cell structure, calcium-mediated signaling and muscle-specific signaling were identified as differentially modulated based on their increased transcriptional variance. These biologically relevant results validate this approach and demonstrate the flexible nature of pathway-based methods of data analysis. AVAILABILITY The software is available as Supplementary Material.

Glucagon and Glucagon-Like Peptides

Glucagon and the glucagon-like peptides play a prominent role in controlling plasma glucose concentrations in the body by diverse signaling mechanisms. These peptide hormones are formed by specific enzymatic cleavages in the pancreas (glucagon) and the intestine (GLPs). Glucagon maintains plasma glucose levels during fasting by stimulating hepatic glucose production. GLP-1 stimulates glucose-dependent insulin secretion during feeding. GLP-2 promotes intestinal growth. Glucagon and the GLPs are important regulators in multiple distinct metabolic functions, facilitating the bodys capacity to “correctly” assimilate nutrients and are in use for the treatment of diabetes and bowel dysfunction.