Single-housing-induced islet epigenomic changes are related to polymorphisms in diabetic KK mice.

A lack of social relationships is increasingly recognized as a type 2 diabetes (T2D) risk. To investigate the underlying mechanism, we used male KK mice, an inbred strain with spontaneous diabetes. Given the association between living alone and T2D risk in humans, we divided the non-diabetic mice into singly housed (KK-SH) and group-housed control mice. Around the onset of diabetes in KK-SH mice, we compared H3K27ac ChIP-Seq with RNA-Seq using pancreatic islets derived from each experimental group, revealing a positive correlation between single-housing-induced changes in H3K27ac and gene expression levels. In particular, single-housing-induced H3K27ac decreases revealed a significant association with islet cell functions and GWAS loci for T2D and related diseases, with significant enrichment of binding motifs for transcription factors representative of human diabetes. Although these H3K27ac regions were preferentially localized to a polymorphic genomic background, SNVs and indels did not cause sequence disruption of enriched transcription factor motifs in most of these elements. These results suggest alternative roles of genetic variants in environment-dependent epigenomic changes and provide insights into the complex mode of disease inheritance.

Glucocorticoid receptor-NECAB1 axis can negatively regulate insulin secretion in pancreatic ß-cells.

The mechanisms of impaired glucose-induced insulin secretion from the pancreatic ß-cells in obesity have not yet been completely elucidated. Here, we aimed to assess the effects of adipocyte-derived factors on the functioning of pancreatic ß-cells. We prepared a conditioned medium using 3T3-L1 cell culture supernatant collected at day eight (D8CM) and then exposed the rat pancreatic ß-cell line, INS-1D. We found that D8CM suppressed insulin secretion in INS-1D cells due to reduced intracellular calcium levels. This was mediated by the induction of a negative regulator of insulin secretion-NECAB1. LC-MS/MS analysis results revealed that D8CM possessed steroid hormones (cortisol, corticosterone, and cortisone). INS-1D cell exposure to cortisol or corticosterone increased Necab1 mRNA expression and significantly reduced insulin secretion. The increased expression of Necab1 and reduced insulin secretion effects from exposure to these hormones were completely abolished by inhibition of the glucocorticoid receptor (GR). NECAB1 expression was also increased in the pancreatic islets of db/db mice. We demonstrated that the upregulation of NECAB1 was dependent on GR activation, and that binding of the GR to the upstream regions of Necab1 was essential for this effect. NECAB1 may play a novel role in the adipoinsular axis and could be potentially involved in the pathophysiology of obesity-related diabetes mellitus.

Synthesis and properties of oligonucleotides bearing thymidine derivatives with 1,6-dioxaspiro[4.5]decane skeleton.

Thymidine derivatives bearing spiroacetal moieties on the C4'-position (5'R-spiro-thymidine and 5'S-spiro-thymidine) were synthesized and incorporated into oligonucleotides. The duplex- and triplex-forming abilities of both the oligonucleotides were evaluated from UV melting experiments. Oligonucleotides with the 5'S-spiro modifications could form thermally stable duplexes with complementary RNA and DNA; however, the 5'R-spiro modification significantly decreased the thermal stabilities of the duplexes and triplexes. Oligonucleotides with these spiro-thymidines showed significantly high resistance towards enzymatic degradation.

Characterization of the taste receptor-related G-protein, α-gustducin, in pancreatic ß-cells.

AIMS/INTRODUCTION: Taste receptors, T1rs and T2rs, and the taste-selective G-protein, α-gustducin, are expressed outside the taste-sensing system, such as enteroendocrine L cells. Here, we examined whether α-gustducin also affects nutrition sensing and insulin secretion by pancreatic ß-cells. MATERIALS AND METHODS: The expression of α-gustducin and taste receptors was evaluated in ß-cell lines, and in rat and mouse islets either by quantitative polymerase chain reaction or fluorescence immunostaining. The effects of α-gustducin knockdown on insulin secretion and on cyclic adenosine monophosphate and intracellular Ca2+ levels in rat INS-1 cells were estimated. Sucralose (taste receptor agonist)-induced insulin secretion was investigated in INS-1 cells with α-gustducin suppression and in islets from mouse disease models. RESULTS: The expression of Tas1r3 and α-gustducin was confirmed in ß-cell lines and pancreatic islets. Basal levels of cyclic adenosine monophosphate, intracellular calcium and insulin secretion were significantly enhanced with α-gustducin knockdown in INS-1 cells. The expression of α-gustducin was decreased in high-fat diet-fed mice and in diabetic db/db mice. Sucralose-induced insulin secretion was not attenuated in INS-1 cells with α-gustducin knockdown or in mouse islets with decreased expression of α-gustducin. CONCLUSIONS: α-Gustducin is involved in the regulation of cyclic adenosine monophosphate, intracellular calcium levels and insulin secretion in pancreatic ß-cells in a manner independent of taste receptor signaling. α-Gustducin might play a novel role in ß-cell physiology and the development of type 2 diabetes.

Genome-wide profiling of histone H3K27 acetylation featured fatty acid signalling in pancreatic beta cells in diet-induced obesity in mice.

AIMS/HYPOTHESIS: Epigenetic regulation of gene expression has been implicated in the pathogenesis of obesity and type 2 diabetes. However, detailed information, such as key transcription factors in pancreatic beta cells that mediate environmental effects, is not yet available. METHODS: To analyse genome-wide cis-regulatory profiles and transcriptome of pancreatic islets derived from a diet-induced obesity (DIO) mouse model, we conducted chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) of histone H3 lysine 27 acetylation (histone H3K27ac) and high-throughput RNA sequencing. Transcription factor-binding motifs enriched in differential H3K27ac regions were examined by de novo motif analysis. For the predicted transcription factors, loss of function experiments were performed by transfecting specific siRNA in INS-1, a rat beta cell line, with and without palmitate treatment. Epigenomic and transcriptional changes of possible target genes were evaluated by ChIP and quantitative RT-PCR. RESULTS: After long-term feeding with a high-fat diet, C57BL/6J mice were obese and mildly glucose intolerant. Among 39,350 islet cis-regulatory regions, 13,369 and 4610 elements showed increase and decrease in ChIP-Seq signals, respectively, significantly associated with global change in gene expression. Remarkably, increased H3K27ac showed a distinctive genomic localisation, mainly in the proximal-promoter regions, revealing enriched elements for nuclear respiratory factor 1 (NRF1), GA repeat binding protein α (GABPA) and myocyte enhancer factor 2A (MEF2A) by de novo motif analysis, whereas decreased H3K27ac was enriched for v-maf musculoaponeurotic fibrosarcoma oncogene family protein K (MAFK), a known negative regulator of beta cells. By siRNA-mediated knockdown of NRF1, GABPA or MEF2A we found that INS-1 cells exhibited downregulation of fatty acid ß-oxidation genes in parallel with decrease in the associated H3K27ac. Furthermore, in line with the epigenome in DIO mice, palmitate treatment caused increase in H3K27ac and induction of ß-oxidation genes; these responses were blunted when NRF1, GABPA or MEF2A were suppressed. CONCLUSIONS/INTERPRETATION: These results suggest novel roles for DNA-binding proteins and fatty acid signalling in obesity-induced epigenomic regulation of beta cell function. DATA AVAILABILITY: The next-generation sequencing data in the present study were deposited at ArrayExpress. RNA-Seq: Dataset name: ERR2538129 (Control), ERR2538130 (Diet-induced obesity) Repository name and number: E-MTAB-6718 - RNA-Seq of pancreatic islets derived from mice fed a long-term high-fat diet against chow-fed controls. ChIP-Seq: Dataset name: ERR2538131 (Control), ERR2538132 (Diet-induced obesity) Repository name and number: E-MTAB-6719 - H3K27ac ChIP-Seq of pancreatic islets derived from mice fed a long-term high-fat diet (HFD) against chow-fed controls.

Comparative analysis of type 2 diabetes-associated SNP alleles identifies allele-specific DNA-binding proteins for the KCNQ1 locus.

Although recent genome-wide association studies (GWAS) have been extremely successful, it remains a big challenge to functionally annotate disease­associated single nucleotide polymorphisms (SNPs), as the majority of these SNPs are located in non­coding regions of the genome. In this study, we described a novel strategy for identifying the proteins that bind to the SNP­containing locus in an allele­specific manner and successfully applied this method to SNPs in the type 2 diabetes mellitus susceptibility gene, potassium voltage­gated channel, KQT­like subfamily Q, member 1 (KCNQ1). DNA fragments encompassing SNPs, and risk or non­risk alleles were immobilized onto the novel nanobeads and DNA­binding proteins were purified from the nuclear extracts of pancreatic ß cells using these DNA­immobilized nanobeads. Comparative analysis of the allele-specific DNA-binding proteins indicated that the affinities of several proteins for the examined SNPs differed between the alleles. Nuclear transcription factor Y (NF­Y) specifically bound the non­risk allele of the SNP rs2074196 region and stimulated the transcriptional activity of an artificial promoter containing SNP rs2074196 in an allele­specific manner. These results suggest that SNP rs2074196 modulates the affinity of the locus for NF­Y and possibly induces subsequent changes in gene expression. The findings of this study indicate that our comparative method using novel nanobeads is effective for the identification of allele­specific DNA­binding proteins, which may provide important clues for the functional impact of disease­associated non­coding SNPs.

MafA is required for postnatal proliferation of pancreatic ß-cells.

The postnatal proliferation and maturation of insulin-secreting pancreatic ß-cells are critical for glucose metabolism and disease development in adults. Elucidation of the molecular mechanisms underlying these events will be beneficial to direct the differentiation of stem cells into functional ß-cells. Maturation of ß-cells is accompanied by increased expression of MafA, an insulin gene transcription factor. Transcriptome analysis of MafA knockout islets revealed MafA is required for the expression of several molecules critical for ß-cell function, including Glut2, ZnT8, Granuphilin, Vdr, Pcsk1 and Urocortin 3, as well as Prolactin receptor (Prlr) and its downstream target Cyclin D2 (Ccnd2). Inhibition of MafA expression in mouse islets or ß-cell lines resulted in reduced expression of Prlr and Ccnd2, and MafA transactivated the Prlr promoter. Stimulation of ß-cells by prolactin resulted in the phosphorylation and translocation of Stat5B and an increased nuclear pool of Ccnd2 via Prlr and Jak2. Consistent with these results, the loss of MafA resulted in impaired proliferation of ß-cells at 4 weeks of age. These results suggest that MafA regulates the postnatal proliferation of ß-cells via prolactin signaling.

Quantitative assessment of Pdx1 promoter activity in vivo using a secreted luciferase reporter system.

The luciferase reporter system is useful for the assessment of various biological processes in vivo. The transcription factor pancreatic and duodenal homeobox 1 (Pdx1) is critical for the formation and the function of pancreatic ß-cells. A novel reporter system using secreted Gaussia princeps luciferase (GLuc) under the control of a Pdx1 promoter was generated and activated in rat and mouse ß-cell lines. This Pdx1-GLuc construct was used as a transgene for the generation of reporter mice to monitor Pdx1 promoter activity in vivo via the measurement of secreted GLuc activity in a small aliquot of blood. Significantly increased plasma GLuc activity was observed in Pdx1-GLuc mice. Analysis of Pdx1-GLuc mice by bioluminescence imaging, GLuc reporter assays using homogenates of various organs, and immunohistochemistry revealed that GLuc expression and activity were exponentially higher in pancreatic ß-cells than in pancreatic non-ß-cells, the duodenum, and other organs. In addition, GLuc activity secreted into the culture medium from islets isolated from Pdx1-GLuc mice correlated with the number of islets. The transplantation of Pdx1-GLuc islets into severe combined immunodeficiency mice elevated their plasma GLuc activity. Conversely, a partial pancreatectomy in Pdx1-GLuc mice reduced plasma GLuc activity. These results suggest that a secreted luciferase reporter system in vivo enables not only the monitoring of promoter activity but also a quantitative and minimally invasive assessment of physiological and pathological changes in small cell masses, such as pancreatic ß-cells.

Requirement for Runx proteins in IgA class switching acting downstream of TGF-beta 1 and retinoic acid signaling.

IgA is a specific isotype required for mucosal immunity and is the most abundant Ab produced in vivo. Recently, several inductive signals for IgA class switch recombination have been identified; however, the molecular details of the action of these signals and the specific factors acting in B cells remain elusive. In this study, we show that combination of retinoic acid (RA) and TGF-beta1 with other factors induced a much higher frequency of IgA-switched cells than reported previously. In addition, IgA production is severely impaired in Runx2-Runx3 double-deficient mice. In Runx2-Runx3-deficient B cells, both RA- and TGF-beta1-dependent inductions of alpha germline transcription are completely blocked. These data suggest that Runx proteins play an essential role in IgA class switching acting downstream of RA and TGF-beta1 signaling.

Essential role of ubiquitin-proteasome system in normal regulation of insulin secretion.

Insulin secretion from pancreatic beta-cells occurs by sequential cellular processes, including glucose metabolism, electrical activity, Ca2+ entry, and regulated exocytosis. Abnormalities in any of these functions can impair insulin secretion. In the present study, we demonstrate that inhibition of proteasome activity severely reduces insulin secretion in the mouse pancreatic beta-cell line MIN6-m9. Although no significant effects on glucose metabolism including ATP production were found in the presence of proteasome inhibitors, both glucose- and KCl-induced Ca2+ entry were drastically reduced. As Ca2+-ionophore-induced insulin secretion was unaffected by proteasome inhibition, a defect in Ca2+ entry through voltage-dependent calcium channels (VDCCs) is the likely cause of the impaired insulin secretion. We found that the pore-forming alpha-subunit of VDCCs undergoes ubiquitination, which does not decrease but slightly increases expression of the alpha-subunit protein at the plasma membrane. However, electrophysiological analysis revealed that treatment with proteasome inhibitors results in a severe reduction in VDCC activity in MIN6-m9 cells, indicating that VDCC function is suppressed by proteasome inhibition. Furthermore, insulin secretion in isolated mouse pancreatic islets was also decreased by proteasome inhibition. These results demonstrate that the ubiquitin-proteasome system plays a critical role in insulin secretion by maintaining normal function of VDCCs.

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells.

Although several studies have suggested that insulin-secreting cells can be generated in vitro from cells residing in adult exocrine pancreas, neither the origin of these cells nor their precise insulin secretory properties was obtained. We show here that insulin-secreting cells can be derived from adult mouse pancreatic exocrine cells by suspension culture in the presence of EGF and nicotinamide. The frequency of insulin-positive cells was only 0.01% in the initial preparation and increased to approximately 5% in the culture conditions. Analysis by the Cre/loxP-based direct cell lineage tracing system indicates that these newly made cells originate from amylase/elastase-expressing pancreatic acinar cells. Insulin secretion is stimulated by glucose, sulfonylurea, and carbachol, and potentiation by glucagon-like peptide-1 also occurs. Insulin-containing secretory granules are present in these cells. In addition, we found that the enzymatic dissociation of pancreatic acini itself leads to activation of EGF signaling, and that inhibition of EGF receptor kinase blocks the transdifferentiation. These data demonstrate that pancreatic acinar cells can transdifferentiate into insulin-secreting cells with secretory properties similar to those of native pancreatic beta cells, and that activation of EGF signaling is required in such transdifferentiation.

Cell permeable peptide of JNK inhibitor prevents islet apoptosis immediately after isolation and improves islet graft function.

Although application of the Edmonton protocol has markedly improved outcomes for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has proven to remain low. During the isolation process and subsequent clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. Pancreas preservation with the two-layer method (TLM) has proven to improve transplant results by protecting isolated islets against apoptosis through the mitochondrial pathway. However, pancreas storage with TLM cannot protect against activation of c-Jun NH2-terminal kinase (JNK) in isolated islets. This study investigated whether delivery of a JNK inhibitory peptide (JNKI) via the protein transduction system can prevent apoptosis of islet cells immediately after isolation. For efficient delivery of the (JNKI into isolated islets, we synthesized JNKI as a C-terminal fusion peptide with the 11-arginine protein transduction domain (11R-JNKI). 11R efficiently delivered the JNKI into isolated islets and 11R-JNKI prevented islet apoptosis immediately after isolation and improved islet graft function. These findings suggest that peptide drugs could be useful for the prevention of the impairment of islet cells and lead to improvement in the outcomes for pancreatic islet transplantation.

Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells.

Rab3 is a subfamily of the small GTP-binding protein Rab family and plays an important role in exocytosis. Several potential effectors of Rab3, including rabphilin3 and Rims (Rim1 and Rim2), have been isolated and characterized. Noc2 was identified originally in endocrine pancreas as a molecule homologous to rabphilin3, but its role in exocytosis is unclear. To clarify the physiological function of Noc2 directly, we have generated Noc2 knockout (Noc2(-/-)) mice. Glucose intolerance with impaired insulin secretion was induced in vivo by acute stress in Noc2(-/-) mice, but not in wild-type (Noc2(+/+)) mice. Ca(2+)-triggered insulin secretion from pancreatic isles of Noc2(-/-) mice was markedly impaired, but was completely restored by treatment with pertussis toxin, which inhibits inhibitory G protein Gi/o signaling. In addition, the inhibitory effect of clonidine, an alpha(2)-adrenoreceptor agonist, on insulin secretion was significantly greater in Noc2(-/-) islets than in Noc2(+/+) islets. Impaired Ca(2+)-triggered insulin secretion was rescued by adenovirus gene transfer of wild-type Noc2 but not by that of mutant Noc2, which does not bind to Rab3. Accordingly, Noc2 positively regulates insulin secretion from endocrine pancreas by inhibiting Gi/o signaling, and the interaction of Noc2 and Rab3 is required for the effect. Interestingly, we also found a marked accumulation of secretory granules in various exocrine cells of Noc2(-/-) mice, especially in exocrine pancreas with no amylase response to stimuli. Thus, Noc2, a critical effector of Rab3, is essential in normal regulation of exocytosis in both endocrine and exocrine cells.

Combined ventricular systolic and arterial stiffening in patients with heart failure and preserved ejection fraction: implications for systolic and diastolic reserve limitations.

BACKGROUND: Heart failure with preserved ejection fraction (HF-nlEF) is common in aged individuals with systolic hypertension and is frequently ascribed to diastolic dysfunction. We hypothesized that such patients also display combined ventricular-systolic and arterial stiffening that can exacerbate blood pressure lability and diastolic dysfunction under stress. METHODS AND RESULTS: Left ventricular pressure-volume relations were measured in patients with HF-nlEF (n=10) and contrasted with asymptomatic age-matched (n=9) and young (n=14) normotensives and age- and blood pressure-matched controls (n=25). End-systolic elastance (stiffness) was higher in patients with HF-nlEF (4.7+/-1.5 mm Hg/mL) than in controls (2.1+/-0.9 mm Hg/mL for normotensives and 3.3+/-1.0 mm Hg/mL for hypertensives; P<0.001). Effective arterial elastance was also higher (2.6+/-0.5 versus 1.9+/-0.5 mm Hg/mL) due to reduced total arterial compliance; the latter inversely correlated with end-systolic elastance (P=0.0001). Body size and stroke volumes were similar and could not explain differences in ventricular-arterial stiffening. HF-nlEF patients also displayed diastolic abnormalities, including higher left ventricular end-diastolic pressures (24.3+/-4.6 versus 12.9+/-5.5 mm Hg), caused by an upward-shifted diastolic pressure-volume curve. However, isovolumic relaxation and the early-to-late filling ratio were similar in age- and blood pressure-matched controls. Ventricular-arterial stiffening amplified stress-induced hypertension, which worsened diastolic function, and predicted higher cardiac energy costs to provide reserve output. CONCLUSION: Patients with HF-lnEF have systolic-ventricular and arterial stiffening beyond that associated with aging and/or hypertension. This may play an important pathophysiological role by exacerbating systemic load interaction with diastolic function, augmenting blood pressure lability, and elevating cardiac metabolic demand under stress.

Quantitation of basal dyssynchrony and acute resynchronization from left or biventricular pacing by novel echo-contrast variability imaging.

OBJECTIVES: This study sought to test a novel echocardiographic method based on contrast variability imaging (CVI), to quantify cardiac dyssynchrony and magnitude of resynchronization achieved by left ventricular (LV) and biventricular (BiV) pacing therapy. BACKGROUND: Left ventricular or BiV pacing is a promising new therapy for patients with heart failure and intraventricular conduction delay. However, precise quantitation of the extent of resynchronization achieved remains scant. METHODS: Ten patients treated with BiV or LV pacing therapy were studied. Echo-contrast was infused slowly, and gated images were acquired before and during contrast appearance. The temporally normalized variance derived from 30 to 50 sequential beats was determined at each pixel to yield the CVI image-displaying improved wall delineation. Systolic regional fractional area of radial sectors was calculated with active and temporarily suspended (AAI) pacing. All analyses were performed blinded to both patient and treatment. RESULTS: Pacing increased septal inward motion from -20.4 +/- 9.6% to -30.5 +/- 14.0%, whereas lateral wall motion occurred earlier with no net magnitude change. Both spatial and temporal dyssynchrony in the LV declined nearly 40% with LV or BiV pacing (p < or = 0.001), and this correlated with increasing ejection fraction (31% to 39%; p < 0.02; p < 0.004 for correlation with dyssynchrony). CONCLUSIONS: The new imaging and regional dyssynchrony analysis methods provide quantitative assessment of resynchronization analogous to that previously obtained only by tagged magnetic resonance imaging. This could provide a useful noninvasive method for both identifying candidates and following long-term therapy.