NFAT regulates the expression of AIF-1 and IRT-1: Yin and yang splice variants of neointima formation and atherosclerosis.

Aims Alternative transcription and splicing of the allograft inflammatory factor-1 (AIF-1) gene results in the expression of two different proteins: AIF-1 and interferon responsive transcript-1 (IRT-1). Here we explore the impact of AIF-1 and IRT-1 on vascular smooth muscle cell (VSMC) activation and neointima formation, the mechanisms underlying their alternative splicing, and associations of AIF-1 and IRT-1 mRNA with parameters defining human atherosclerotic plaque phenotype.Methods and results Translation of AIF-1 and IRT-1 results in different products with contrasting cellular distribution and functions. Overexpression of AIF-1 stimulates migration and proliferation of human VSMCs, whereas IRT-1 exerts opposite effects. Adenoviral infection of angioplasty-injured rat carotid arteries with AdAIF-1 exacerbates intima hyperplasia, whereas infection with AdIRT-1 reduces neointima. Expression of these variants is modulated by changes in nuclear factor of activated T-cells (NFAT) activity. Pharmacological inhibition of NFAT or targeting of NFATc3 with siRNA lowers the AIF-1/IRT-1 ratio and favors an anti-proliferative outcome. NFAT acts as a repressor on the IRT-1 transcriptional start site, which is also sensitive to interferon-γ stimulation. Expression of AIF-1 mRNA in human carotid plaques associates with less extracellular matrix and a more pro-inflammatory plaque and plasma profile, features that may predispose to plaque rupture. In contrast, expression of IRT-1 mRNA associates with a less aggressive phenotype and less VSMCs at the most stenotic region of the plaque.Conclusions Inhibition of NFAT signaling, by shifting the AIF-1/IRT-1 ratio, may be an attractive target to regulate the VSMC response to injury and manipulate plaque stability in atherosclerosis

Two novel type 2 diabetes loci revealed through integration of TCF7L2 DNA occupancy and SNP association data

BACKGROUND: The transcription factor 7-like 2 (TCF7L2) locus is strongly implicated in the pathogenesis of type 2 diabetes (T2D). We previously mapped the genomic regions bound by TCF7L2 using ChIP (chromatin immunoprecipitation)-seq in the colorectal carcinoma cell line, HCT116, revealing an unexpected highly significant over-representation of genome-wide association studies (GWAS) loci associated primarily with endocrine (in particular T2D) and cardiovascular traits. METHODS: In order to further explore if this observed phenomenon occurs in other cell lines, we carried out ChIP-seq in HepG2 cells and leveraged ENCODE data for five additional cell lines. Given that only a minority of the predicted genetic component to most complex traits has been identified to date, plus our GWAS-related observations with respect to TCF7L2 occupancy, we investigated if restricting association analyses to the genes yielded from this approach, in order to reduce the constraints of multiple testing, could reveal novel T2D loci. RESULTS: We found strong evidence for the continued enrichment of endocrine and cardiovascular GWAS categories, with additional support for cancer. When investigating all the known GWAS loci bound by TCF7L2 in the shortest gene list, derived from HCT116, the coronary artery disease-associated variant, rs46522 at the UBE2Z-GIP-ATP5G1-SNF8 locus, yielded significant association with T2D within DIAGRAM. Furthermore, when we analyzed tag-SNPs (single nucleotide polymorphisms) in genes not previously implicated by GWAS but bound by TCF7L2 within 5 kb, we observed a significant association of rs4780476 within CPPED1 in DIAGRAM. CONCLUSIONS: ChIP-seq data generated with this GWAS-implicated transcription factor provided a biologically plausible method to limit multiple testing in the assessment of genome-wide genotyping data to uncover two novel T2D-associated loci

Ser649 and Ser650 Are the Major Determinants of Protein Kinase A-Mediated Activation of Human Hormone-Sensitive Lipase against Lipid Substrates

BACKGROUND: Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In this study we employed site-directed mutagenesis, in vitro phosphorylation and mass spectrometry to show that in vitro phosphorylation of human HSL by PKA occurs primarily on Ser649 and Ser650 (Ser659 and Ser660 in rat HSL). The wild type enzyme and four mutants were expressed in C-terminally His-tagged form in Sf9 insect cells and purified to homogeneity. HSL variants in which Ser552 and/or Ser554 were mutated to Ala or Glu retained both lipolytic and non-lipolytic activity and were phosphorylated by PKA and activated to a similar extent as the wild type enzyme. (32)P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor phosphorylation of Ser552 and Ser554. MS/MS analysis demonstrated that the peptide containing Ser649 and Ser650 was primarily phosphorylated on Ser650. The mutant lacking all four serines had severely reduced lipolytic activity, but a lesser reduction in non-lipolytic activity, had S(0.5) values for p-nitrophenol butyrate and triolein comparable to those of wild type HSL and was not phosphorylated by PKA. PKA phosphorylation of the wild type enzyme resulted in an increase in both the maximum turnover and S(0,5) using the TO substrate. CONCLUSIONS: Our results demonstrate that PKA activates human HSL against lipid substrates in vitro primarily through phosphorylation of Ser649 and Ser650. In addition the results suggest that Ser649 and Ser650 are located in the vicinity of a lipid binding region and that PKA phosphorylation controls the accessibility of this region

Tissue-specific alternative splicing of TCF7L2

Common variants in the transcription factor 7-like 2 (TCF7L2) gene have been identified as the strongest genetic risk factors for type 2 diabetes (T2D). However, the mechanisms by which these non-coding variants increase risk for T2D are not well-established. We used 13 expression assays to survey mRNA expression of multiple TCF7L2 splicing forms in up to 380 samples from eight types of human tissue (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscle, subcutaneous adipose tissue and lymphoblastoid cell lines) and observed a tissue-specific pattern of alternative splicing. We tested whether the expression of TCF7L2 splicing forms was associated with single nucleotide polymorphisms (SNPs), rs7903146 and rs12255372, located within introns 3 and 4 of the gene and most strongly associated with T2D. Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146). Expression of this form in glucose-stimulated pancreatic islets correlated with expression of proinsulin (r2 = 0.84–0.90, P < 0.00063). In summary, we identified a tissue-specific pattern of alternative splicing of TCF7L2. After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant. Alternative splicing of TCF7L2 in pancreatic islets warrants future studies. GenBank Accession Numbers: FJ010164–FJ010174

Type 2 Diabetes Susceptibility Gene Expression in Normal or Diabetic Sorted Human Alpha and Beta Cells: Correlations with Age or BMI of Islet Donors

BACKGROUND: Genome-wide association studies have identified susceptibility genes for development of type 2 diabetes. We aimed to examine whether a subset of these (comprising FTO, IDE, KCNJ11, PPARG and TCF7L2) were transcriptionally restricted to or enriched in human beta cells by sorting islet cells into alpha and beta - specific fractions. We also aimed to correlate expression of these transcripts in both alpha and beta cell types with phenotypic traits of the islet donors and to compare diabetic and non-diabetic cells. METHODOLOGY/PRINCIPAL FINDINGS: Islet cells were sorted using a previously published method and RNA was extracted, reverse transcribed and used as the template for quantitative PCR. Sorted cells were also analysed for insulin and glucagon immunostaining and insulin secretion from the beta cells as well as insulin, glucagon and GLP-1 content. All five genes were expressed in both alpha and beta cells, with significant enrichment of KCNJ11 in the beta cells and of TCF7L2 in the alpha cells. The ratio of KCNJ11 in beta to alpha cells was negatively correlated with BMI, while KCNJ11 expression in alpha cells was negatively correlated with age but not associated with BMI. Beta cell expression of glucagon, TCF7L2 and IDE was increased in cells from islets that had spent more time in culture prior to cell sorting. In beta cells, KCNJ11, FTO and insulin were positively correlated with each other. Diabetic alpha and beta cells had decreased expression of insulin, glucagon and FTO. CONCLUSIONS/SIGNIFICANCE: This study has identified novel patterns of expression of type 2 diabetes susceptibility genes within sorted islet cells and suggested interactions of gene expression with age or BMI of the islet donors. However, expression of these genes in islets is less associated with BMI than has been found for other tissues

A Central Role for GRB10 in Regulation of Islet Function in Man

Peer reviewe

Due Diligence - Skyldighet eller möjlighet? : Om målbolagsstyrelsens eventuella skyldighet att tillåta en due diligence-undersökning i samband med ett offentligt uppköpserbjudande

Ett offentligt uppköpserbjudande definieras i 1 kap. 2 § 1p. Lag (2006:451) om offentliga uppköpserbjudanden på aktiemarknaden (LUA) så som ett offentligt erbjudande till innehavare av aktier som har getts ut av ett svenskt eller utländskt aktiebolag att överlåta samtliga eller en del av dessa aktier. Innan ett offentligt uppköpserbjudande lämnas vill den potentiella budgivaren som regel genomföra en due diligence-undersökning på målbolaget. Syftet med denna uppsats är att söka fastslå (1) om det föreligger en skyldighet för målbolaget att medge en due diligence-undersökning, (2) under vilka omständigheter denna eventuella skyldighet föreligger samt (3) vilka sanktioner som står till buds för aktieägarna då en eventuell skyldighet inte efterföljs. I arbetet med uppsatsen har en traditionell rättsdogmatisk metod använts. Frågan om styrelsens eventuella skyldighet att medge en due diligence-undersökning undersöks utifrån både ett aktiebolagsrättsligt och ett aktiemarknadsrättsligt perspektiv. Ur ett aktiemarknadsrättsligt perspektiv förutsätts som regel att en due diligence-undersökning kan och får genomföras. Due diligence-undersökningar är dock inte oproblematiska ur ett aktiebolagsrättsligt perspektiv. Det finns en risk att den information som lämnas ut till budgivaren kan leda till skada för målbolaget. Dessutom kan målbolaget lida skada genom de kostnader som uppkommer i samband med en due diligence-undersökning. Vad avser frågan om de sanktioner som står till buds för enskilda aktieägare har, utöver aktiebolagslagens bestämmelser om skadestånd, eventuell rätt till skadestånd enligt allmänna skadeståndsrättsliga principer undersökts.   Resultatet av undersökningen är att det föreligger en skyldighet för styrelsen att i vissa fall medger en begäran om en due diligence-undersökning. En huvudförutsättning för att så ska vara fallet är dock att en sådan undersökning anses ligga i de enskilda aktieägarnas intresse i form av ett bud som är mer förmånligt att acceptera än att förkasta. I praktiken är dock denna skyldighet i princip osanktionerad.