Blocking endothelial lipase with monoclonal antibody MEDI5884 durably increases high density lipoprotein in nonhuman primates and in a phase 1 trial.

Cardiovascular disease (CVD) is the leading global cause of death, and treatments that further reduce CV risk remain an unmet medical need. Epidemiological studies have consistently identified low high-density lipoprotein cholesterol (HDL-C) as an independent risk factor for CVD, making HDL elevation a potential clinical target for improved CVD resolution. Endothelial lipase (EL) is a circulating enzyme that regulates HDL turnover by hydrolyzing HDL phospholipids and driving HDL particle clearance. Using MEDI5884, a first-in-class, EL-neutralizing, monoclonal antibody, we tested the hypothesis that pharmacological inhibition of EL would increase HDL-C by enhancing HDL stability. In nonhuman primates, MEDI5884 treatment resulted in lasting, dose-dependent elevations in HDL-C and circulating phospholipids, confirming the mechanism of EL action. We then showed that a favorable lipoprotein profile of elevated HDL-C and reduced low-density lipoprotein cholesterol (LDL-C) could be achieved by combining MEDI5884 with a PCSK9 inhibitor. Last, when tested in healthy human volunteers, MEDI5884 not only raised HDL-C but also increased HDL particle numbers and average HDL size while enhancing HDL functionality, reinforcing EL neutralization as a viable clinical approach aimed at reducing CV risk.

CREB coactivators CRTC2 and CRTC3 modulate bone marrow hematopoiesis.

Populations of circulating immune cells are maintained in equilibrium through signals that enhance the retention or egress of hematopoietic stem cells (HSCs) from bone marrow (BM). Prostaglandin E2 (PGE2) stimulates HSC renewal and engraftment through, for example, induction of the cAMP pathway. Triggering of PGE2 receptors increases HSC survival in part via the PKA-mediated induction of the cAMP response element-binding protein (CREB) signaling pathway. PKA stimulates cellular gene expression by phosphorylating CREB at Ser133 and by promoting the dephosphorylation of the cAMP- responsive transcriptional coactivators (CRTCs). We show here that disruption of both CRTC2 and CRTC3 causes embryonic lethality, and that a single allele of either CRTC2 or CRTC3 is sufficient for viability. CRTC2 knockout mice that express one CRTC3 allele (CRTC2/3m mice) develop neutrophilia and splenomegaly in adulthood due to the up-regulation of granulocyte-colony stimulating factor (G-CSF); these effects are reversed following administration of neutralizing anti-G-CSF antiserum. Adoptive transfer of CRTC2/3m BM conferred the splenomegaly/neutrophilia phenotype in WT recipients. Targeted disruption of both CRTC2 and CRTC3 in stromal cells with a mesenchymal Prx1-Cre transgene also promoted this phenotype. Depletion of CRTC2/3 was found to decrease the expression of Suppressor of Cytokine Signaling 3 (SOCS3), leading to increases in STAT3 phosphorylation and to the induction of CEBPß, a key regulator of the G-CSF gene. As small molecule inhibition of JAK activity disrupted CEBPß induction and reduced G-CSF expression in CRTC2/3m stromal cells, our results demonstrate how cross-coupling between the CREB/CRTC and JAK/STAT pathways contributes to BM homeostasis.

Neurturin and a GLP-1 Analogue Act Synergistically to Alleviate Diabetes in Zucker Diabetic Fatty Rats.

Neurturin (NRTN), a member of the glial-derived neurotrophic factor family, was identified from an embryonic chicken pancreatic cDNA library in a screen for secreted factors. In this study, we assessed the potential antidiabetic activities of NRTN relative to liraglutide, a glucagon-like peptide 1 receptor agonist, in Zucker diabetic fatty (ZDF) rats. Subcutaneous administration of NRTN to 8-week-old male ZDF rats prevented the development of hyperglycemia and improved metabolic parameters similar to liraglutide. NRTN treatment increased pancreatic insulin content and ß-cell mass and prevented deterioration of islet organization. However, unlike liraglutide-treated rats, NRTN-mediated improvements were not associated with reduced body weight or food intake. Acute NRTN treatment did not activate c-Fos expression in key feeding behavior and metabolic centers in ZDF rat brain or directly enhance glucose-stimulated insulin secretion from pancreatic ß-cells. Treating 10-week-old ZDF rats with sustained hyperglycemia with liraglutide resulted in some alleviation of hyperglycemia, whereas NRTN was not as effective despite improving plasma lipids and fasting glucose levels. Interestingly, coadministration of NRTN and liraglutide normalized hyperglycemia and other metabolic parameters, demonstrating that combining therapies with distinct mechanism(s) can alleviate advanced diabetes. This emphasizes that therapeutic combinations can be more effective to manage diabetes in individuals with uncontrolled hyperglycemia.

CREB pathway links PGE2 signaling with macrophage polarization.

Obesity is thought to promote insulin resistance in part via activation of the innate immune system. Increases in proinflammatory cytokine production by M1 macrophages inhibit insulin signaling in white adipose tissue. In contrast, M2 macrophages have been found to enhance insulin sensitivity in part by reducing adipose tissue inflammation. The paracrine hormone prostaglandin E2 (PGE2) enhances M2 polarization in part through activation of the cAMP pathway, although the underlying mechanism is unclear. Here we show that PGE2 stimulates M2 polarization via the cyclic AMP-responsive element binding (CREB)-mediated induction of Krupple-like factor 4 (KLF4). Targeted disruption of CREB or the cAMP-regulated transcriptional coactivators 2 and 3 (CRTC2/3) in macrophages down-regulated M2 marker gene expression and promoted insulin resistance in the context of high-fat diet feeding. As re-expression of KLF4 rescued M2 marker gene expression in CREB-depleted cells, our results demonstrate the importance of the CREB/CRTC pathway in maintaining insulin sensitivity in white adipose tissue via its effects on the innate immune system.

The BisPCR(2) method for targeted bisulfite sequencing.

BACKGROUND: DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification. Next-generation sequencing (NGS) techniques offer single base resolution of CpG methylation levels with high statistical significance, but are also high cost if performed genome-wide. Here, we describe a simplified targeted bisulfite sequencing approach in which DNA sequencing libraries are prepared following sodium bisulfite conversion and two rounds of PCR for target enrichment and sample barcoding, termed BisPCR(2). RESULTS: We have applied the BisPCR(2) technique to validate differential methylation at several type 2 diabetes risk loci identified in genome-wide studies of human islets. We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR(2) compared to prior array-based approaches. CONCLUSION: This study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illustrates its utility by reanalysis of prior findings from genome-wide studies.

The CREB/CRTC2 pathway modulates autoimmune disease by promoting Th17 differentiation.

Following their activation in response to inflammatory signals, innate immune cells secrete T-cell-polarizing cytokines that promote the differentiation of naive CD4 T cells into T helper (Th) cell subsets. Among these, Th17 cells play a prominent role in the development of a number of autoimmune diseases. Although regarded primarily as an immunosuppressant signal, cAMP has been found to mediate pro-inflammatory effects of macrophage-derived prostaglandin E2 (PGE2) on Th17 cells. Here we show that PGE2 enhances Th17 cell differentiation via the activation of the CREB co-activator CRTC2. Following its dephosphorylation, CRTC2 stimulates the expression of the cytokines IL-17A and IL-17F by binding to CREB over both promoters. CRTC2-mutant mice have decreased Th17 cell numbers, and they are protected from experimental autoimmune encephalitis, a model for multiple sclerosis. Our results suggest that small molecule inhibitors of CRTC2 may provide therapeutic benefit to individuals with autoimmune disease.

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts.

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator-like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics.

CREB mediates the insulinotropic and anti-apoptotic effects of GLP-1 signaling in adult mouse ß-cells.

OBJECTIVE: Glucagon-like peptide-1 (GLP-1) plays a major role in pancreatic ß-cell function and survival by increasing cytoplasmic cAMP levels, which are thought to affect transcription through activation of the basic leucine zipper (bZIP) transcription factor CREB. Here, we test CREB function in the adult ß-cell through inducible gene deletion. METHODS: We employed cell type-specific and inducible gene ablation to determine CREB function in pancreatic ß-cells in mice. RESULTS: By ablating CREB acutely in mature ß-cells in tamoxifen-treated Creb (loxP/loxP);Pdx1-CreERT2 mice, we show that CREB has little impact on ß-cell turnover, in contrast to what had been postulated previously. Rather, CREB is required for GLP-1 to elicit its full effects on stimulating glucose-induced insulin secretion and protection from cytokine-induced apoptosis. Mechanistically, we find that CREB regulates expression of the pro-apoptotic gene p21 (Cdkn1a) in ß-cells, thus demonstrating that CREB is essential to mediating this critical aspect of GLP-1 receptor signaling. CONCLUSIONS: In sum, our studies using conditional gene deletion put into question current notions about the importance of CREB in regulating ß-cell function and mass. However, we reveal an important role for CREB in the ß-cell response to GLP-1 receptor signaling, further validating CREB as a therapeutic target for diabetes.

The transcription factor CREB has no non-redundant functions in hepatic glucose metabolism in mice.

AIMS/HYPOTHESIS: Excessive hepatic glucose production is a hallmark of insulin resistance in type 2 diabetes. The cAMP responsive transcription factor cAMP responsive element binding protein (CREB), thought to be a key activator of the hepatic gluconeogenic gene regulation programme, has been suggested as a therapeutic target to reduce glucose output by the liver. Here, we test directly the requirement for hepatocytic CREB for the maintenance of glucose homeostasis. METHODS: We derived mice with a Creb (also known as Creb1) loxP allele for conditional, cell-type specific gene ablation. Hepatocyte-specific deletion of Creb was induced by injecting Creb (loxP/loxP) mice with Cre recombinase expression adeno-associated virus. RESULTS: Strikingly, we found no difference in fed and fasted glucose levels, or in glucose, insulin and glucagon tolerance in mice fed a normal chow or a high-fat diet. In addition, mRNA levels of liver-specific genes, including several CREB target genes involved in gluconeogenesis, were not affected by CREB deficiency in the liver. CONCLUSION/INTERPRETATION: Our data show that CREB has no non-redundant functions in hepatic glucose metabolism, and is therefore not likely to be a useful target for the development of glucose-lowering drugs.

Elevated mouse hepatic betatrophin expression does not increase human ß-cell replication in the transplant setting.

The recent discovery of betatrophin, a protein secreted by the liver and white adipose tissue in conditions of insulin resistance and shown to dramatically stimulate replication of mouse insulin-producing ß-cells, has raised high hopes for the rapid development of a novel therapeutic approach for the treatment of diabetes. At present, however, the effects of betatrophin on human ß-cells are not known. Here we use administration of the insulin receptor antagonist S961, shown to increase betatrophin gene expression and stimulate ß-cell replication in mice, to test its effect on human ß-cells. Although mouse ß-cells, in their normal location in the pancreas or when transplanted under the kidney capsule, respond with a dramatic increase in ß-cell DNA replication, human ß-cells are completely unresponsive. These results put into question whether betatrophin can be developed as a therapeutic approach for treating human diabetes.

CISH has no non-redundant functions in glucose homeostasis or beta cell proliferation during pregnancy in mice.

AIMS/HYPOTHESIS: Increased beta cell proliferation during pregnancy is mediated by the Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signalling pathway in response to increased lactogen levels. Activation of the pathway leads to transcriptional upregulation of Cish (encoding cytokine-inducible SH2 domain-containing protein), a member of the suppressor of cytokine signalling (SOCS) family of genes, forming a negative-feedback loop. Here, we examined whether conditional gene ablation of Cish in the pancreas improves beta cell proliferation and beta cell function during pregnancy in mice. METHODS: We derived mice with a novel, conditional loxP allele for Cish. Pancreas-specific ablation of Cish was achieved by crossing Cish (loxP/loxP) mice with Pdx1-Cre (Early) mice. Beta cell proliferation was quantified by BrdU labelling. Glucose homeostasis was examined with glucose tolerance tests and determination of plasma insulin levels. The expression of other Socs genes and target genes of p-STAT5 related to beta cell function and beta cell proliferation was determined by quantitative PCR. RESULTS: There was no difference in beta cell proliferation or glucose homeostasis between the Cish mutant group and the control group. The p-STAT5 protein level was the same in Cish mutant and control mice. Socs2 gene expression was higher in Cish mutant than control mice at pregnancy day 9.5. The expression of other Socs genes was the same between control and mutant mice. CONCLUSIONS/INTERPRETATION: Our results show that CISH has no non-redundant functions in beta cell proliferation or glucose homeostasis during pregnancy in mice. Socs2 might compensate for the loss of Cish during pregnancy.

Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver.

BACKGROUND: Metabolic homeostasis in mammals critically depends on the regulation of fasting-induced genes by CREB in the liver. Previous genome-wide analysis has shown that only a small percentage of CREB target genes are induced in response to fasting-associated signaling pathways. The precise molecular mechanisms by which CREB specifically targets these genes in response to alternating hormonal cues remain to be elucidated. RESULTS: We performed chromatin immunoprecipitation coupled to high-throughput sequencing of CREB in livers from both fasted and re-fed mice. In order to quantitatively compare the extent of CREB-DNA interactions genome-wide between these two physiological conditions we developed a novel, robust analysis method, termed the 'single sample independence' (SSI) test that greatly reduced the number of false-positive peaks. We found that CREB remains constitutively bound to its target genes in the liver regardless of the metabolic state. Integration of the CREB cistrome with expression microarrays of fasted and re-fed mouse livers and ChIP-seq data for additional transcription factors revealed that the gene expression switches between the two metabolic states are associated with co-localization of additional transcription factors at CREB sites. CONCLUSIONS: Our results support a model in which CREB is constitutively bound to thousands of target genes, and combinatorial interactions between DNA-binding factors are necessary to achieve the specific transcriptional response of the liver to fasting. Furthermore, our genome-wide analysis identifies thousands of novel CREB target genes in liver, and suggests a previously unknown role for CREB in regulating ER stress genes in response to nutrient influx.

Adiponectin suppresses gluconeogenic gene expression in mouse hepatocytes independent of LKB1-AMPK signaling.

The adipocyte-derived hormone adiponectin signals from the fat storage depot to regulate metabolism in peripheral tissues. Inversely correlated with body fat levels, adiponectin reduction in obese individuals may play a causal role in the symptoms of metabolic syndrome. Adiponectin lowers serum glucose through suppression of hepatic glucose production, an effect attributed to activation of AMPK. Here, we investigated the signaling pathways that mediate the effects of adiponectin by studying mice with inducible hepatic deletion of LKB1, an upstream regulator of AMPK. We found that loss of LKB1 in the liver partially impaired the ability of adiponectin to lower serum glucose, though other actions of the hormone were preserved, including reduction of gluconeogenic gene expression and hepatic glucose production as assessed by euglycemic hyperinsulinemic clamp. Furthermore, in primary mouse hepatocytes, the absence of LKB1, AMPK, or the transcriptional coactivator CRTC2 did not prevent adiponectin from inhibiting glucose output or reducing gluconeogenic gene expression. These results reveal that whereas some of the hormone's actions in vivo may be LKB1 dependent, substantial LKB1-, AMPK-, and CRTC2-independent signaling pathways also mediate effects of adiponectin.

Tissue-specific regulation of mouse microRNA genes in endoderm-derived tissues.

MicroRNAs fine-tune the activity of hundreds of protein-coding genes. The identification of tissue-specific microRNAs and their promoters has been constrained by the limited sensitivity of prior microRNA quantification methods. Here, we determine the entire microRNAome of three endoderm-derived tissues, liver, jejunum and pancreas, using ultra-high throughput sequencing. Although many microRNA genes are expressed at comparable levels, 162 microRNAs exhibited striking tissue-specificity. After mapping the putative promoters for these microRNA genes using H3K4me3 histone occupancy, we analyzed the regulatory modules of 63 microRNAs differentially expressed between liver and jejunum or pancreas. We determined that the same transcriptional regulatory mechanisms govern tissue-specific gene expression of both mRNA and microRNA encoding genes in mammals.

Foxa1 and Foxa2 maintain the metabolic and secretory features of the mature beta-cell.

Foxa1 and Foxa2 play both redundant and distinct roles in early pancreas development. We demonstrate here that inducible ablation of both transcription factors in mature mouse beta-cells leads to impaired glucose homeostasis and insulin secretion. The defects in both glucose-stimulated insulin secretion and intracellular calcium oscillation are more pronounced than those in beta-cells lacking only Foxa2. Unexpectedly, in contrast to the severe reduction of beta-cell-enriched factors contributing to metabolic and secretory pathways, expression of a large number of genes that are involved in neural differentiation and function is significantly elevated. We further demonstrate that expression of carbohydrate response element-binding protein (ChREBP or Mlxipl), an important transcriptional regulator of carbohydrate metabolism, is significantly affected in compound Foxa1/a2 mutant beta-cells. ChREBP expression is directly controlled by Foxa1 and Foxa2 in both the fetal endocrine pancreas as well as mature islets. These data demonstrate that Foxa1 and Foxa2 play crucial roles in the development and maintenance of beta-cell-specific secretory and metabolic pathways.

Genome-wide analysis of histone modifications in human pancreatic islets.

The global diabetes epidemic poses a major challenge. Epigenetic events contribute to the etiology of diabetes; however, the lack of epigenomic analysis has limited the elucidation of the mechanistic basis for this link. To determine the epigenetic architecture of human pancreatic islets we mapped the genome-wide locations of four histone marks: three associated with gene activation-H3K4me1, H3K4me2, and H3K4me3-and one associated with gene repression, H3K27me3. Interestingly, the promoters of the highly transcribed insulin and glucagon genes are occupied only sparsely by H3K4me2 and H3K4me3. Globally, we identified important relationships between promoter structure, histone modification, and gene expression. We demonstrated co-occurrences of histone modifications including bivalent marks in mature islets. Furthermore, we found a set of promoters that is differentially modified between islets and other cell types. We also use our histone marks to determine which of the known diabetes-associated single-nucleotide polymorphisms are likely to be part of regulatory elements. Our global map of histone marks will serve as an important resource for understanding the epigenetic basis of type 2 diabetes.

The Fox genes in the liver: from organogenesis to functional integration.

Formation and function of the liver are highly controlled, essential processes. Multiple signaling pathways and transcriptional regulatory networks cooperate in this complex system. The evolutionarily conserved FOX, for Forkhead bOX, class of transcriptional regulators is critical to many aspects of liver development and function. The FOX proteins are small, mostly monomeric DNA binding factors containing the so-called winged helix DNA binding motif that distinguishes them from other classes of transcription factors. We discuss the biochemical and genetic roles of Foxa, Foxl1, Foxm1, and Foxo, as these have been shown to regulate many processes throughout the life of the organ, controlling both formation and function of the liver.

CRTC2 (TORC2) contributes to the transcriptional response to fasting in the liver but is not required for the maintenance of glucose homeostasis.

The liver contributes to glucose homeostasis by promoting either storage or production of glucose, depending on the physiological state. The cAMP response element-binding protein (CREB) is a principal regulator of genes involved in coordinating the hepatic response to fasting, but its mechanism of gene activation remains controversial. We derived CRTC2 (CREB-regulated transcription coactivator 2, previously TORC2)-deficient mice to assess the contribution of this cofactor to hepatic glucose metabolism in vivo. CRTC2 mutant hepatocytes showed reduced glucose production in response to glucagon, which correlated with decreased CREB binding to several gluconeogenic genes. However, despite attenuated expression of CREB target genes, including PEPCK, G6Pase, and PGC-1alpha, no hypoglycemia was observed in mutant mice. Collectively, these results provide genetic evidence supporting a role for CRTC2 in the transcriptional response to fasting, but indicate only a limited contribution of this cofactor to the maintenance of glucose homeostasis.

Jagged1 is a competitive inhibitor of Notch signaling in the embryonic pancreas.

Pancreatic endocrine cells originate from precursors that express the transcription factor Neurogenin3 (Ngn3). Ngn3 expression is repressed by active Notch signaling. Accordingly, mice with Notch signaling pathway mutations display increased Ngn3 expression and endocrine cell lineage allocation. To determine how the Notch ligand Jagged1 (Jag1) functions during pancreas development, we deleted Jag1 in foregut endoderm and examined postnatal and embryonic endocrine cells and precursors. Postnatal Jag1 mutants display increased Ngn3 expression, alpha-cell mass, and endocrine cell percentage, similar to the early embryonic phenotype of Dll1 and Rbpj mutants. However, in sharp contrast to postnatal animals, Jag1-deficient embryos display increased expression of Notch transcriptional targets and decreased Ngn3 expression, resulting in reduced endocrine lineage allocation. Jag1 acts as an inhibitor of Notch signaling during embryonic pancreas development but an activator of Notch signaling postnatally. Expression of the Notch modifier Manic Fringe (Mfng) is limited to endocrine precursors, providing a possible explanation for the inhibition of Notch signaling by Jag1 during mid-gestation embryonic pancreas development.

Hepatic function is preserved in the absence of mature microRNAs.

UNLABELLED: MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression through partial or complete complementarity with target messenger RNAs. The function of miRNAs in normal liver physiology is largely unknown. We address the role of Dicer1 in the differentiated liver. We derived mice lacking Dicer1 function in hepatocytes and assessed the loss of mature miRNA via quantitative polymerase chain reaction. Gene expression microarray analysis was performed on liver RNA from mutant and control mice. Liver sections from mutant and control mice were examined and liver function tests were performed. Mice lacking Dicer1 function in hepatocytes appeared and behaved normally. Despite the loss of mature miRNAs, hepatic function was maintained, as reflected by normal blood glucose, albumin, cholesterol, and bilirubin. However, mutant mice between 2 and 4 months of age exhibited progressive hepatocyte damage with elevated serum alanine aminotransferase and aspartate aminotransferase. Liver mass was increased in mutant mice, as were cellular markers of both proliferation and apoptosis. Microarray analysis indicated large-scale changes in gene expression, with increased expression of many miRNA targets, particularly imprinted genes. CONCLUSIONS: Loss of miRNA processing in the liver at late gestation has a remarkably mild phenotype, suggesting that miRNAs do not play an essential role in hepatic function. However, miRNA deficiency results in hepatocyte apoptosis, hepatocyte regeneration, and portal inflammation. Finally, microarray analysis of gene expression in the mutant liver supports a previously hypothesized role for Dicer1 in the repression of imprinted genes.