The expression of ob gene is not acutely regulated by insulin and fasting in human abdominal subcutaneous adipose tissue

The regulation of ob gene expression in abdominal subcutaneous adipose tissue was investigated using a reverse transcription-competitive PCR method to quantify the mRNA level of leptin. Leptin mRNA level was highly correlated with the body mass index of 26 subjects (12 lean, 7 non-insulin-dependent diabetic, and 7 obese patients). The effect of fasting on ob gene expression was investigated in 10 subjects maintained on a hypocaloric diet (1045 KJ/d) for 5 d. While their metabolic parameters significantly changed (decrease in insulinemia, glycemia, and resting metabolic rate and increase in plasma ketone bodies), the caloric restriction did not modify the leptin mRNA level in the adipose tissue. To verify whether insulin regulates ob gene expression, six lean subjects underwent a 3-h euglycemic hyperinsulinemic (846 +/- 138 pmol/liter) clamp. Leptin and Glut 4 mRNA levels were quantified in adipose tissue biopsies taken before and at the end of the clamp. Insulin infusion produced a significant threefold increase in Glut 4 mRNA while leptin mRNA was not affected. It is concluded that ob gene expression is not acutely regulated by insulin or by metabolic factors related to fasting in human abdominal subcutaneous adipose tissue

Sequentiality and processivity of nuclear receptor coregulators in regulation of target gene expression

A series of data has accumulated over the past five years that raises questions about our current understanding of the transcriptional process and its regulation. Following the discovery of coactivators for nuclear receptors (NRs), a large number of these molecules have been reported in the literature. This perspective will summarize some opinions on the significance of this large number of factors

Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

BACKGROUND: Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. METHODS: MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. RESULTS: We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. CONCLUSION: E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

The PPARγ Agonist Rosiglitazone Impairs Colonic Inflammation in Mice with Experimental Colitis

Various animal models showed that peroxisome proliferator-activated receptor (PPAR)γ agonists, when given as a gavage shortly preceding colitis induction, protect against inflammatory bowel disease (IBD). We have examined the effects of 16 days rosiglitazone treatment via the diet prior to dextran sodium sulphate (DSS)-induced colitis in mice. After 7 days DSS in the drinking water, rosiglitazone-fed mice had lost significantly more weight than control mice. Rosiglitazone-treated mice had more diarrhea, weight of colon and spleen were increased, and length of colon was decreased. Histology showed that rosiglitazone-treated mice had more severe colitis, mainly caused by more ulceration, crypt loss, and edema. Immunofluorescence showed a loss of tight junction structure Zonula Occludens protein 1 (ZO-1) in colons of rosiglitazone-treated mice as compared to control mice. Also, serum amyloid P component (SAP) concentrations in plasma were increased. However, concentrations of tumor necrosis factor (TNF)-α and interferon (IFN)-γ in colon homogenates, and TNF-α in spleen homogenates were significantly decreased, whereas interleukin (IL)-10 in spleen homogenates was increased. Other cytokines (IL-2, IL-4, IL-6, IL-12p70 and monocyte chemotactic protein (MCP)-1) and myeloperoxidase (MPO) concentrations showed no differences. In conclusion, 16 days pretreatment with rosiglitazone impaired DSS-induced colitis in mice

An unexpected, mild phenotype of glucocorticoid resistance associated with glucocorticoid receptor gene mutation case report and review of the literature

BACKGROUND: Glucocorticoid resistance is a rare, sporadic or familial condition caused by mutation of the gene encoding the glucocorticoid receptor (GR). Clinically it is characterized by symptoms developed due to local, tissue-specific, or generalized partial insensitivity to glucocorticoids. CASE PRESENTATION: A 31-year-old woman was evaluated because of infertility at the Endocrine Unit of the 2nd Department of Medicine, Semmelweis University. During her laboratory investigations, elevated serum and salivary cortisol were observed which failed to be suppressed after administration of 1 mg dexamethasone. 24 h urinary cortisol was increased, but a normal midnight serum cortisol was detected suggesting a maintained circadian rhythm. Plasma dehydroepiandrosterone-sulfate and androstendione levels were also elevated. Repeated plasma ACTH measurements indicated slightly elevated or normal values. Bone mineral density was normal. All laboratory results confirmed the diagnosis of glucocorticoid resistance. Genetic counseling followed by Sanger sequencing of the coding region of the gene encoding human glucocorticoid receptor was performed and a missense mutation (Arg714Gln, R714Q) in a heterozygous form was detected. Following family screening, the same mutation was found in her clinically-healthy 35-year-old sister who had no fertility problems.This variant was not detected in more than 60 patients and controls tested either for glucocorticoid resistance or Cushing's syndrome in our Laboratory and it was absent in Exome Variant Server, HumanGene Mutation Database and ExAC databases. CONCLUSIONS: Our case fulfils the diagnostic criteria of glucocorticoid resistance, also named Chrousos syndrome. The glucocorticoid receptor gene mutation detected in our patient has been already reported in a 2-year-old child with hypoglycaemia, hypokalaemia, hypertension and premature puberty. These distinct phenotypes may suggest that other factors may modify the functional consequences of the R714Q variant of GR

Expression of the ubiquitin-proteasome pathway and muscle loss in experimental cancer cachexia

Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E214k, has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E214k were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E214k mRNA and protein levels in gastrocnemius muscle with increases of 6–8-fold for C2 and two-fold for E214k between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25–27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased two-fold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18–20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E214k mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss

Attenuation of Colon Inflammation through Activators of the Retinoid X Receptor (Rxr)/Peroxisome Proliferator–Activated Receptor γ (Pparγ) Heterodimer: A Basis for New Therapeutic Strategies

The peroxisome proliferator–activated receptor γ (PPARγ) is highly expressed in the colon mucosa and its activation has been reported to protect against colitis. We studied the involvement of PPARγ and its heterodimeric partner, the retinoid X receptor (RXR) in intestinal inflammatory responses. PPARγ1/− and RXRα1/− mice both displayed a significantly enhanced susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis compared with their wild-type littermates. A role for the RXR/PPARγ heterodimer in the protection against colon inflammation was explored by the use of selective RXR and PPARγ agonists. TNBS-induced colitis was significantly reduced by the administration of both PPARγ and RXR agonists. This beneficial effect was reflected by increased survival rates, an improvement of macroscopic and histologic scores, a decrease in tumor necrosis factor α and interleukin 1β mRNA levels, a diminished myeloperoxidase concentration, and reduction of nuclear factor κB DNA binding activity, c-Jun NH2-terminal kinase, and p38 activities in the colon. When coadministered, a significant synergistic effect of PPARγ and RXR ligands was observed. In combination, these data demonstrate that activation of the RXR/PPARγ heterodimer protects against colon inflammation and suggest that combination therapy with both RXR and PPARγ ligands might hold promise in the clinic due to their synergistic effects

Alternative Promoters Influence Alternative Splicing at the Genomic Level

Background: More and more experiments have shown that transcription and mRNA processing are not two independent events but are tightly coupled to each other. Both promoter and transcription rate were found to influence alternative splicing. More than half of human genes have alternative promoters, but it is still not clear why there are so many alternative promoters and what their biological roles are. Methodology/Principal Findings: In this study, we explored whether there is a functional correlation between alternative promoters and alternative splicing by a genome-wide analysis of human and mouse genes. We constructed a large data set of genes with alternative promoter and alternative splicing annotations. By analyzing these genes, we showed that genes with alternative promoters tended to demonstrate alternative splicing compare to genes with single promoter, and, genes with more alternative promoters tend to have more alternative splicing variants. Furthermore, transcripts from different alternative promoters tended to splice differently. Conclusions/Significance: Thus at the genomic level, alternative promoters are positively correlated with alternativ

Alternate Splicing of Interleukin-1 Receptor Type II (IL1R2) In Vitro Correlates with Clinical Glucocorticoid Responsiveness in Patients with AIED

Autoimmune Inner Ear Disease (AIED) is poorly characterized clinically, with no definitive laboratory test. All patients suspected of having AIED are given glucocorticoids during periods of acute hearing loss, however, only half initially respond, and still fewer respond over time

Gene Profiling of Mta1 Identifies Novel Gene Targets and Functions

BACKGROUND: Metastasis-associated protein 1 (MTA1), a master dual co-regulatory protein is found to be an integral part of NuRD (Nucleosome Remodeling and Histone Deacetylation) complex, which has indispensable transcriptional regulatory functions via histone deacetylation and chromatin remodeling. Emerging literature establishes MTA1 to be a valid DNA-damage responsive protein with a significant role in maintaining the optimum DNA-repair activity in mammalian cells exposed to genotoxic stress. This DNA-damage responsive function of MTA1 was reported to be a P53-dependent and independent function. Here, we investigate the influence of P53 on gene regulation function of Mta1 to identify novel gene targets and functions of Mta1. METHODS: Gene expression analysis was performed on five different mouse embryonic fibroblasts (MEFs) samples (i) the Mta1 wild type, (ii) Mta1 knock out (iii) Mta1 knock out in which Mta1 was reintroduced (iv) P53 knock out (v) P53 knock out in which Mta1 was over expressed using Affymetrix Mouse Exon 1.0 ST arrays. Further Hierarchical Clustering, Gene Ontology analysis with GO terms satisfying corrected p-value<0.1, and the Ingenuity Pathway Analysis were performed. Finally, RT-qPCR was carried out on selective candidate genes. SIGNIFICANCE/CONCLUSION: This study represents a complete genome wide screen for possible target genes of a coregulator, Mta1. The comparative gene profiling of Mta1 wild type, Mta1 knockout and Mta1 re-expression in the Mta1 knockout conditions define "bona fide" Mta1 target genes. Further extensive analyses of the data highlights the influence of P53 on Mta1 gene regulation. In the presence of P53 majority of the genes regulated by Mta1 are related to inflammatory and anti-microbial responses whereas in the absence of P53 the predominant target genes are involved in cancer signaling. Thus, the presented data emphasizes the known functions of Mta1 and serves as a rich resource which could help us identify novel Mta1 functions