Statins are beneficial in the treatment of cardiovascular disease (CVD), but these lipid-lowering drugs are associated with increased incidence of new on-set diabetes. The cellular mechanisms behind the development of diabetes by statins are elusive. Here we have treated mice on normal diet (ND) and high fat diet (HFD) with rosuvastatin. Under ND rosuvastatin lowered blood glucose through improved insulin sensitivity and increased glucose uptake in adipose tissue. In vitro rosuvastatin reduced insulin secretion and insulin content in islets. In the beta cell Ca(2+) signaling was impaired and the density of granules at the plasma membrane was increased by rosuvastatin treatment. HFD mice developed insulin resistance and increased insulin secretion prior to administration of rosuvastatin. Treatment with rosuvastatin decreased the compensatory insulin secretion and increased glucose uptake. In conclusion, our data shows dual effects on glucose homeostasis by rosuvastatin where insulin sensitivity is improved, but beta cell function is impaired.
MicroRNAs are small non-coding RNAs, which negatively regulate the expression of target genes.They have emerged as important modulators in beta cell compensation upon increased metabolic demand, failure of which leads to reduced insulin secretion and type 2 diabetes.To elucidate the function of miRNAs in beta cells, insulin-secreting cell lines, such as the rat insulinoma INS-1 832/13 and the human EndoC-βH1, are widely used.Previous studies in the cancer field have suggested that miRNA expression is influenced by confluency of adherent cells.We therefore aimed to investigate whether one of the most enriched miRNAs in the pancreatic endocrine cells, miR-375, and two of its validated targets in mouse, Cav1 and Aifm1, were differentially-expressed in cell cultures with different confluences.Additionally, we measured the expression of other miRNAs, such as miR-152, miR-130a, miR-132, miR-212 and miR-200a, with known roles in beta cell function.We did not see any significant expression changes of miR-375 nor any of the two targets, in both the rat and human beta cell lines at different confluences.Interestingly, among the other miRNAs measured, the expression of miR-132 and miR-212 positive correlated with confluence but only in the INS-1 832/13 cells.Our results show that the expression of miR-375 and other miRNAs with known roles in beta cell function is independent of, or at least minimally influenced by the density of proliferating adherent cells, especially within the confluence range optimal for functional assays to elucidate miRNA-dependent regulatory mechanisms in the beta cell.
U2 snRNP auxiliary factor (U2AF) is an essential heterodimeric splicing factor composed of two subunits, U2AF 65 and U2AF 35 . During the past few years, a number of proteins related to both U2AF 65 and U2AF 35 have been discovered. Here, we review the conserved structural features that characterize the U2AF protein families and their evolutionary emergence. We perform a comprehensive database search designed to identify U2AF protein isoforms produced by alternative splicing, and we discuss the potential implications of U2AF protein diversity for splicing regulation.
Lipids are central to the development of atherosclerotic plaques. Specifically, which lipids are culprits remains controversial, and promising targets have failed in clinical studies. Sphingolipids are bioactive lipids present in atherosclerotic plaques, and they have been suggested to have both proatherogenic and antiatherogenic. However, the biological effects of these lipids remain unknown in the human atherosclerotic plaque. The aim of this study was to assess plaque levels of sphingolipids and investigate their potential association with and contribution to plaque vulnerability.Glucosylceramide, lactosylceramide, ceramide, dihydroceramide, sphingomyelin, and sphingosine-1-phosphate were analyzed in homogenates from 200 human carotid plaques using mass spectrometry. Inflammatory activity was determined by analyzing plaque levels of cytokines and plaque histology. Caspase-3 was analyzed by ELISA technique. Expression of regulatory enzymes was analyzed with RNA sequencing. Human coronary artery smooth muscle cells were used to analyze the potential role of the 6 sphingolipids as inducers of plaque inflammation and cellular apoptosis in vitro. All sphingolipids were increased in plaques associated with symptoms and correlated with inflammatory cytokines. All sphingolipids, except sphingosine-1-phosphate, also correlated with histological markers of plaque instability. Lactosylceramide, ceramide, sphingomyelin, and sphingosine-1-phosphate correlated with caspase-3 activity. In vitro experiments revealed that glucosylceramide, lactosylceramide, and ceramide induced cellular apoptosis. All analyzed sphingolipids induced an inflammatory response in human coronary artery smooth muscle cells.This study shows for the first time that sphingolipids and particularly glucosylceramide are associated with and are possible inducers of plaque inflammation and instability, pointing to sphingolipid metabolic pathways as possible novel therapeutic targets.
Nonsense-mediated decay (NMD) is a quality- control mechanism that degrades RNA transcripts harbouring premature stop codons and consequently reduces production of truncated proteins. Inhibition of NMD is being evaluated as a therapeutic approach for pulmonary vascular diseases caused by pathogenic nonsense substitutions causing hereditary haemorrhagic telangiectasia (HHT). As this non-specific approach might also affect the expression of transcripts that are naturally regulated by NMD, our aim was to identify exons that are controlled by NMD and the biological processes they are involved in.
Methods
Primary human microvascular endothelial cells (HMEC) were cultured to confluence in antibiotic-free medium before treatment for 1 hour with 100µg/ml cycloheximide to inhibit NMD, or fresh media. Ribosomal (r)-RNA-depleted total RNA was used to prepare strand-specific whole transcriptome libraries which were sequenced on an Illumina Genome Analyser II, aligned to hg18, counted using custom scripts, and normalized to total valid reads and exon size. Further scripts were written to identify exons present in HMEC treated with cycloheximide but not media-treated HMEC. Separately, blood outgrowth endothelial cells (BOECs) were established from 23 HHT patients with pathogenic nonsense substitutions in ENG, ACVRL1 and SMAD4.
Results
In the cycloheximide and media-treated normal HMEC, there were alignments to 15,756 RefSeq genes, and 113 micro (mi)RNAs. The 419 most differentially expressed RefSeq genes (p<0.15), clustered to Gene Ontology (GO) biological process compatible with the observed induction of membrane proteolysis in cycloheximide-treated cells, validating the methodological approach. There were overlaps between miRNAs that were differentially expressed, and their mRNA targets predicted by Targetscan. The approach also identified candidate alternate exons observed only in the cycloheximide-treated HMEC, including 333 alternate first exons, 662 mid exons, 275 terminal exons and 59 exon extensions. Candidate exons that introduced a premature stop codon into transcripts of genes involved in GO biological processes other than protein translation were validated by reverse transcriptase PCR, prior to selection as a panel to quantitatively evaluate NMD inhibition in BOECs from HHT patients.
Conclusion
Natural targets of nonsense-mediated decay in HMEC were identified. Further investigation should provide new insights into the role of NMD in cellular physiology.
The U2 snRNP auxiliary factor (U2AF) is an essential splicing factor composed of two subunits, a large, 65-kDa subunit (U2AF65) and a small subunit, U2AF35. U2AF65 binds to the polypyrimidine tract upstream from the 3′ splice site and promotes U2 snRNP binding to the pre-mRNA. Based on in vitro studies, it has been proposed that U2AF35 plays a role in assisting U2AF65 recruitment to nonconsensus polypyrimidine tracts. Here we have analyzed in vivo the roles of the two subunits of U2AF in the selection between alternative 3′ splice sites associated with polypyrimidine tracts of different strengths. Our results reveal a feedback mechanism by which RNA interference (RNAi)-mediated depletion of U2AF65 triggers the downregulation of U2AF35. We further show that the knockdown of each U2AF subunit inhibits weak 3′ splice site recognition, while overexpression of U2AF65 alone is sufficient to activate the selection of this splice site. A variant of U2AF65 lacking the interaction domain with U2AF35 shows a reduced ability to promote this splicing event, suggesting that recognition of the weak 3′ splice site involves the U2AF heterodimer. Furthermore, our data suggest that, rather than being required for splicing of all pre-mRNA substrates containing a weak polypyrimidine tract, U2AF35 regulates the selection of weak 3′ splice sites in a specific subset of cellular transcripts.
To improve our understanding of the dynamic interplay between non-coding and coding RNA species, we developed solution-based methods to capture the entire endothelial transcriptome. In contrast to microarrays and CHIP-based methodologies, there was no pre-specification of RNA target sequences, and the identity of the DNA strand of origin was preserved.
Methods
Triplicate confluent primary human microvascular endothelial cells (Promocell GmbH) were cultured in the presence and absence of TGF-beta stimuli relevant to pulmonary arteriovenous malformations and hereditary haemorrhagic telangiectasia (HHT). Following rRNA-depletion of total RNA, seven libraries were prepared using Illumina reagents, and 8 pM of each library used for cluster generation and sequencing on Genome Analyser II. Algorithms for aligning reads to NCBI36/hg18 and GRCh37/hg19 included Bowtie, TopHat and Seqmap. Validations were performed using quantitative rt-PCR.
Results
More than 2 Gigabases of sequence was generated. Transcriptome-wide profiles were similar between libraries, with sixteen types of RNA species detected including 146 micro (mi)RNA families (47 broadly conserved), and 10,749 protein-encoding mRNAs representing ~5.5% of mapped reads. Alignments to endothelial mRNAs/miRNAs were substantially higher than to gene loci for non-endothelial mRNAs/miRNAs. mRNA exon alignments demonstrated sharp exon boundary delineation, but replicate alignments to non-repetitive intronic regions involved in multi-exon deletions in HHT patients. There was an inverse relationship between alignments depths and qt-PCT cycle thresholds (Ct), where single alignments were detectable, and Ct values of 20 generated by 0.02 nM spiked RNA. Across all experiments in replicate donor/treatment RNAs, for a panel of single open reading frame miRNA genes, RNASeq alignments (gene strand read counts normalised to the total number of valid reads and exon/locus size) explained 72% of the variance of qt-PCR cycle threshold (p < 0.0001). Dynamic whole transcriptome profiling is in progress.
Conclusions
These novel directional next generation RNA sequencing methods provide new insights for mutational mechanisms, and a systems approach to dissection of regulatory and target RNA networks relevant to human disease.
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