Integrative analysis of a cross-loci regulation network identifies App as a gene regulating insulin secretion from pancreatic islets.

Complex diseases result from molecular changes induced by multiple genetic factors and the environment. To derive a systems view of how genetic loci interact in the context of tissue-specific molecular networks, we constructed an F2 intercross comprised of >500 mice from diabetes-resistant (B6) and diabetes-susceptible (BTBR) mouse strains made genetically obese by the Leptin(ob/ob) mutation (Lep(ob)). High-density genotypes, diabetes-related clinical traits, and whole-transcriptome expression profiling in five tissues (white adipose, liver, pancreatic islets, hypothalamus, and gastrocnemius muscle) were determined for all mice. We performed an integrative analysis to investigate the inter-relationship among genetic factors, expression traits, and plasma insulin, a hallmark diabetes trait. Among five tissues under study, there are extensive protein-protein interactions between genes responding to different loci in adipose and pancreatic islets that potentially jointly participated in the regulation of plasma insulin. We developed a novel ranking scheme based on cross-loci protein-protein network topology and gene expression to assess each gene's potential to regulate plasma insulin. Unique candidate genes were identified in adipose tissue and islets. In islets, the Alzheimer's gene App was identified as a top candidate regulator. Islets from 17-week-old, but not 10-week-old, App knockout mice showed increased insulin secretion in response to glucose or a membrane-permeant cAMP analog, in agreement with the predictions of the network model. Our result provides a novel hypothesis on the mechanism for the connection between two aging-related diseases: Alzheimer's disease and type 2 diabetes.

A survey of the genetics of stomach, liver, and adipose gene expression from a morbidly obese cohort.

To map the genetics of gene expression in metabolically relevant tissues and investigate the diversity of expression SNPs (eSNPs) in multiple tissues from the same individual, we collected four tissues from approximately 1000 patients undergoing Roux-en-Y gastric bypass (RYGB) and clinical traits associated with their weight loss and co-morbidities. We then performed high-throughput genotyping and gene expression profiling and carried out a genome-wide association analyses for more than 100,000 gene expression traits representing four metabolically relevant tissues: liver, omental adipose, subcutaneous adipose, and stomach. We successfully identified 24,531 eSNPs corresponding to about 10,000 distinct genes. This represents the greatest number of eSNPs identified to our knowledge by any study to date and the first study to identify eSNPs from stomach tissue. We then demonstrate how these eSNPs provide a high-quality disease map for each tissue in morbidly obese patients to not only inform genetic associations identified in this cohort, but in previously published genome-wide association studies as well. These data can aid in elucidating the key networks associated with morbid obesity, response to RYGB, and disease as a whole.

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.

Pharmacological AMPK activation induces transcriptional responses congruent to exercise in skeletal and cardiac muscle, adipose tissues and liver.

Physical activity promotes metabolic and cardiovascular health benefits that derive in part from the transcriptional responses to exercise that occur within skeletal muscle and other organs. There is interest in discovering a pharmacologic exercise mimetic that could imbue wellness and alleviate disease burden. However, the molecular physiology by which exercise signals the transcriptional response is highly complex, making it challenging to identify a single target for pharmacological mimicry. The current studies evaluated the transcriptome responses in skeletal muscle, heart, liver, and white and brown adipose to novel small molecule activators of AMPK (pan-activators for all AMPK isoforms) compared to that of exercise. A striking level of congruence between exercise and pharmacological AMPK activation was observed across the induced transcriptome of these five tissues. However, differences in acute metabolic response between exercise and pharmacologic AMPK activation were observed, notably for acute glycogen balances and related to the energy expenditure induced by exercise but not pharmacologic AMPK activation. Nevertheless, intervention with repeated daily administration of short-acting activation of AMPK was found to mitigate hyperglycemia and hyperinsulinemia in four rodent models of metabolic disease and without the cardiac glycogen accretion noted with sustained pharmacologic AMPK activation. These findings affirm that activation of AMPK is a key node governing exercise mediated transcription and is an attractive target as an exercise mimetic.

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.

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

BACKGROUND: Using 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. RESULTS: Bioinformatics 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. CONCLUSION: In summary, we show that the transcriptional response to acute cell stress is largely transient and proteosome-centric.

Systemic pan-AMPK activator MK-8722 improves glucose homeostasis but induces cardiac hypertrophy.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.

Minireview: transcriptional regulation in pancreatic development.

Considerable progress has been made in the understanding of the sequential activation of signal transduction pathways and the expression of transcription factors during pancreas development. Much of this understanding has been obtained by analyses of the phenotypes of mice in which the expression of key genes has been disrupted (knockout mice). Knockout of the genes for Pdx1, Hlxb9, Isl1, or Hex results in an arrest of pancreas development at a very early stage (embryonic d 8-9). Disruption of genes encoding components of the Notch signaling pathway, e.g. Hes1 or neurogenin-3, abrogates development of the endocrine pancreas (islets of Langerhans). Disruption of transcription factor genes expressed more downstream in the developmental cascade (Beta2/NeuroD, Pax4, NKx2.2, and Nkx6.1) curtails the formation of insulin-producing beta-cells. An understanding of the importance of transcription factor genes during pancreas development has provided insights into the pathogenesis of diabetes, in which the mass of insulin-producing beta-cells is reduced.

Glucose-induced expression of the cyclin-dependent protein kinase 5 activator p35 involved in Alzheimer's disease regulates insulin gene transcription in pancreatic beta-cells.

The deposition of amyloid within the insulin-producing islets of Langerhans in the pancreas is a common pathological finding in patients with type 2 diabetes. Its relationship with age and the progression of the disease resembles the pathological deposition of beta-amyloid in the brains of Alzheimer's patients. Endocrine cells of pancreatic islets and cells of neuronal lineages express a shared subset of specialized genes. The hyperactivity of the cyclin-dependent protein kinase CDK5, involved in the development and differentiation of the nervous system, is associated with Alzheimer's disease. Overactivity of CDK5 occurs by proteolytic cleavage and cellular mislocalization of its activator, p35. These alterations in p35/CDK5 signaling pathway may mediate, at least in part, the functional abnormalities characteristic of Alzheimer's disease. In this study we report that both the p35 and CDK5 genes are expressed in insulin-producing beta-cells of the pancreas. We detect in beta-cells the formation of an active p35/CDK5 complex with specific kinase activity. Notably, elevations of the extracellular concentration of glucose result in increases in p35 mRNA and protein levels that parallel elevations of p35/CDK5 activity. Functional studies show that p35 stimulates the activity of the insulin promoter and that the stimulation requires CDK5 because stimulation is blocked by roscovitine, an inhibitor of CDK5 activity, a dominant negative form of CDK5, and small interfering RNAs against p35. Our findings indicate that the expression of p35 and CDK5 in insulin-producing beta-cells ensembles a new signaling pathway, the activity of which is controlled by glucose, and its functional role may comprise the regulation of various biological processes in beta-cells, such as is the case for expression of the insulin gene.

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.

The effect of ICER on screening methods involving CRE-mediated reporter gene expression.

We describe a mechanism whereby increasing levels of cAMP in Chinese hamster ovary (CHO) and other cell lines lead to a significant repression in cAMP response element (CRE)-mediated luciferase reporter gene expression. This effect was shown to be mediated by a modulatory factor located downstream of cyclic AMP (cAMP), which displayed the temporal regulation pattern of an immediate early gene. The expression of this inducible cAMP early repressor (ICER) was shown to be coincident with the time and concentration dependency of the repression of CRE-mediated luciferase gene expression on the treatment of CHO cells with forskolin. Furthermore, this phenomenon was also observed in JEG and GH3 cell lines (both previously reported to express ICER), but not in COS-7 cells, which do not express ICER. These studies suggest that, in certain cell lines, expression of ICER can be induced at pharmacologically elevated cAMP levels, leading to a potent inhibition of CRE-mediated gene expression. We therefore conclude that screening methodologies employing such CRE-linked reporter genes (particularly in high-throughput screening assays) may produce false functional responses in certain cell lines. Moreover, such effects are likely to be exacerbated in screening assays in which receptors either are overexpressed or high concentrations of potent cAMP-elevating compounds are used.

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.

Identification and functional characterization of melatonin Mel 1a receptors in pancreatic beta 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.

Does chronic low-grade endotoxemia define susceptibility of obese humans to insulin resistance via dietary effects on gut microbiota?

Recent studies, including one from our own lab, report that different subpopulations of obese individuals display a variable inflammatory signature in their visceral adipose tissue that may contribute significantly to their risk for developing insulin resistance, type 2 diabetes, and other metabolic diseases. Understanding the molecular mechanisms and signaling pathways that lead to these differences in susceptibility to insulin resistance will equip us with important targets to help stem the tide of such debilitating diseases. Here we discuss an emerging theory that chronic, low-grade endotoxemia may represent a causal factor in obesity-related inflammatory states, and that diet-induced changes in the gut microbiome may be a key regulator of metabolic health. The implications to both disease prevention and to therapeutic intervention are also highlighted.

Inverse regulation of inflammation and mitochondrial function in adipose tissue defines extreme insulin sensitivity in morbidly obese patients.

Obesity is associated with insulin resistance, a major risk factor for type 2 diabetes and cardiovascular disease. However, not all obese individuals are insulin resistant, which confounds our understanding of the mechanistic link between these conditions. We conducted transcriptome analyses on 835 obese subjects with mean BMI of 48.8, on which we have previously reported genetic associations of gene expression. Here, we selected ~320 nondiabetic (HbA(1c) <7.0) subjects and further stratified the cohort into insulin-resistant versus insulin-sensitive subgroups based on homeostasis model assessment-insulin resistance. An unsupervised informatics analysis revealed that immune response and inflammation-related genes were significantly downregulated in the omental adipose tissue of obese individuals with extreme insulin sensitivity and, to a much lesser extent, in subcutaneous adipose tissue. In contrast, genes related to ß-oxidation and the citric acid cycle were relatively overexpressed in adipose of insulin-sensitive patients. These observations were verified by querying an independent cohort of our published dataset of 37 subjects whose subcutaneous adipose tissue was sampled before and after treatment with thiazolidinediones. Whereas the immune response and inflammation pathway genes were downregulated by thiazolidinedione treatment, ß-oxidation and citric acid cycle genes were upregulated. This work highlights the critical role that omental adipose inflammatory pathways might play in the pathophysiology of insulin resistance, independent of body weight.

Dissecting cis regulation of gene expression in human metabolic tissues.

Complex diseases such as obesity and type II diabetes can result from a failure in multiple organ systems including the central nervous system and tissues involved in partitioning and disposal of nutrients. Studying the genetics of gene expression in tissues that are involved in the development of these diseases can provide insights into how these tissues interact within the context of disease. Expression quantitative trait locus (eQTL) studies identify mRNA expression changes linked to proximal genetic signals (cis eQTLs) that have been shown to affect disease. Given the high impact of recent eQTL studies, it is important to understand what role sample size and environment plays in identification of cis eQTLs. Here we show in a genotyped obese human population that the number of cis eQTLs obey precise scaling laws as a function of sample size in three profiled tissues, i.e. omental adipose, subcutaneous adipose and liver. Also, we show that genes (or transcripts) with cis eQTL associations detected in a small population are detected at approximately 90% rate in the largest population available for our study, indicating that genes with strong cis acting regulatory elements can be identified with relatively high confidence in smaller populations. However, by increasing the sample size we allow for better detection of weaker and more distantly located cis-regulatory elements. Yet, we determined that the number of tissue specific cis eQTLs saturates in a modestly sized cohort while the number of cis eQTLs common to all tissues fails to reach a maximum value. Understanding the power laws that govern the number and specificity of eQTLs detected in different tissues, will allow a better utilization of genetics of gene expression to inform the molecular mechanism underlying complex disease traits.

Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions.

Here, we enriched a human cell population from adipose tissue that exhibited both mesenchymal plasticity, self-renewal capacity, and a cell-surface marker profile indistinguishable from that of bone marrow-derived mesenchymal stem cells. In addition to adipogenic and osteogenic differentiation, these adipose-derived stem cells displayed skeletal myogenic potential when co-cultured with mouse skeletal myocytes in reduced serum conditions. Physical incorporation of stem cells into multinucleated skeletal myotubes was determined by genetic lineage tracing, whereas human-specific antibody staining was employed to demonstrate functional contribution of the stem cells to a myogenic lineage. To investigate the effects of hypoxia, cells were maintained and differentiated at 2% O(2). In contrast with reports on bone marrow-derived stem cells, both osteogenic and adipogenic differentiation were significantly attenuated. In summary, the relative accessibility of adipose-derived mesenchymal stem cells from human donors provides opportunity for molecular investigation of mechanistic dysfunction in disease settings and may introduce new prospects for cell-based therapy.

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.

Identification of novel pathway regulation during myogenic differentiation.

Stem cell differentiation is governed by extracellular signals that activate intracellular networks (or pathways) to drive phenotypic specification. Using a novel gene clustering strategy we determined pathway relationships from a genome-wide transcriptional dataset of skeletal myoblast differentiation. Established myogenic pathways, including cell contractility and cell-cycle arrest, were predicted with extreme statistical significance (p approximately 0). In addition, gene sets associated with angiogenesis, neuronal activity, and mRNA splicing were regulated, exposing developmental and therapeutic implications. Acquisition of transcriptional data spanning the entire differentiation time course provided context for a dynamic landscape of functional pathway regulation. This novel perspective on myogenic cell differentiation revealed previously unrecognized patterns of regulation. We predict that similar analyses will facilitate ongoing efforts to define molecular mechanisms in other stem cell and developmental paradigms. Finally, by combining an iterative process of analysis with supplementation of novel pathways, this application may evolve into a powerful discovery tool.

Resveratrol inhibits firefly luciferase.

The potential therapeutic value of resveratrol in age-related disease settings including cancer, diabetes, and Alzheimer's has emerged from a rapidly growing body of experimental evidence. Protection from oxidative stress appears to be a common feature of resveratrol that may be mediated through SirT1, though more specific molecular mechanisms by which resveratrol mediates its effects remain unclear. This has prompted an upsurge in cell-based mechanistic studies, often incorporating reporter assays for pathway elucidation in response to resveratrol treatment. Here, we report that resveratrol potently inhibits firefly luciferase with a K(i) value of 2microM, and caution that this confounding element may lead to compromised data interpretation.