C/EBP␤ Reprograms White 3T3-L1 Preadipocytes to a Brown Adipocyte Pattern of Gene Expression *

cAMP-dependent protein kinase induction of PPAR␥ coactivator-1␣ (PGC-1␣) and uncoupling protein 1 (UCP1) expression is an essential step in the commitment of preadipocytes to the brown adipose tissue (BAT) lineage. We studied the molecular mechanisms responsible for differential expression of PGC-1␣ in HIB1B (BAT) and 3T3-L1 white adipose tissue (WAT) precursor cell lines. In HIB1B cells PGC-1␣ and UCP1 expression is cAMP-inducible, but in 3T3-L1 cells, expression is reduced and is cAMP-insensitive. A proximal 264-bp PGC-1␣ reporter construct was cAMP-inducible only in HIB1B cells and was suppressed by site-directed mutagenesis of the proximal cAMP response element (CRE). In electrophoretic mobility shift assays, the transcription factors CREB and C/EBP␤, but not C/EBP␣ and C/EBP␦, bound to the CRE on the PGC-1␣ promoter region in HIB1B and 3T3-L1 cells. Chromatin immunoprecipitation studies demonstrated that C/EBP␤ and CREB bound to the CRE region in HIB1B and 3T3-L1 cell lysates. C/EBP␤ expression was induced by cAMP only in HIB1B cells, and overexpression of C/EBP␤ rescued cAMP-inducible PGC-1␣ and UCP1 expression in 3T3-L1 cells. These data demonstrate that differentiation of preadipocytes toward the BAT rather than the WAT phenotype is controlled in part by the action of C/EBP␤ on the CRE in PGC-1␣ proximal promoter

Construction of a doxycycline inducible adipogenic lentiviral expression system

a b s t r a c t To provide a tool for research on regulating adipocyte differentiation, tetracycline inducible (Tet on) lentiviral expression vectors under the control of an adipose-specific promoter were constructed. The lowest basal expression in the absence of doxycycline and most efficient dose-dependent, doxycycline-induced transient overexpression was observed using vectors constructed with a combination of Tetracycline Responsive Element (TRE) and reverse tetracycline-controlled TransActivator advanced (rtTAadv), transfected in white (3T3-L1) and brown (HIB-1B) preadipocytes cell lines. The results demonstrate that doxycycline adipogenic inducible expression can be achieved using a pLenti TRE / rtTA adv under the control of the truncated aP2 promoter in HIB-1B preadipocytes

Construction of a doxycycline inducible adipogenic lentiviral expression system

To provide a tool for research on regulating adipocyte differentiation, tetracycline inducible (Tet on) lentiviral expression vectors under the control of an adipose-specific promoter were constructed. The lowest basal expression in the absence of doxycycline and most efficient dose-dependent, doxycycline-induced transient overexpression was observed using vectors constructed with a combination of Tetracycline Responsive Element (TRE) and reverse tetracycline-controlled TransActivator advanced (rtTAadv), transfected in white (3T3-L1) and brown (HIB-1B) preadipocytes cell lines. The results demonstrate that doxycycline adipogenic inducible expression can be achieved using a pLenti TRE / rtTA adv under the control of the truncated aP2 promoter in HIB-1B preadipocytes

Cold-induced changes in gene expression in brown adipose tissue, white adipose tissue and liver

Cold exposure imposes a metabolic challenge to mammals that is met by a coordinated response in different tissues to prevent hypothermia. This study reports a transcriptomic analysis in brown adipose tissue (BAT), white adipose (WAT) and liver of mice in response to 24 h cold exposure at 8°C. Expression of 1895 genes were significantly (P<0.05) up- or down-regulated more than two fold by cold exposure in all tissues but only 5 of these genes were shared by all three tissues, and only 19, 14 and 134 genes were common between WAT and BAT, WAT and liver, and BAT and liver, respectively. We confirmed using qRT-PCR, the increased expression of a number of characteristic BAT genes during cold exposure. In both BAT and the liver, the most common direction of change in gene expression was suppression (496 genes in BAT and 590 genes in liver). Gene ontology analysis revealed for the first time significant (P<0.05) down regulation in response to cold, of genes involved in oxidoreductase activity, lipid metabolic processes and protease inhibitor activity, in both BAT and liver, but not WAT. The results reveal an unexpected importance of down regulation of cytochrome P450 gene expression and apolipoprotein, in both BAT and liver, but not WAT, in response to cold exposure. Pathway analysis suggests a model in which down regulation of the nuclear transcription factors HNF4α and PPARα in both BAT and liver may orchestrate the down regulation of genes involved in lipoprotein and steroid metabolism as well as Phase I enzymes belonging to the cytochrome P450 group in response to cold stress in mice. We propose that the response to cold stress involves decreased gene expression in a range of cellular processes in order to maximise pathways involved in heat production

Role of Ucp1 enhancer methylation and chromatin remodelling in the control of Ucp1 expression in murine adipose tissue

Aims/hypothesis Increasing the expression of the brown adipose tissue-specific gene uncoupling protein-1 (Ucp1) is a potential target for treating obesity. We investigated the role of DNA methylation and histone modification in Ucp1 expression in adipose cell lines and ex vivo murine adipose tissues. Methods Methylation state of the Ucp1 enhancer was studied using bisulphite mapping in murine adipose cell lines, and tissue taken from cold-stressed mice, coupled with functional assays of the effects of methylation and demethylation of the Ucp1 promoter on gene expression and nuclear protein binding. Results We show that demethylation of the Ucp1 promoter by 5-aza-deoxycytidine increases Ucp1 expression while methylation of Ucp1 promoter–reporter constructs decreases expression. Brown adipose tissue-specific Ucp1 expression is associated with decreased CpG dinucleotide methylation of the Ucp1 enhancer. The lowest CpG dinucleotide methylation state was found in two cyclic AMP response elements (CRE3, CRE2) in the Ucp1 promoter and methylation of the CpG in CRE2, but not CRE3 decreased nuclear protein binding. Chromatin immunoprecipitation assays revealed the presence of the silencing DiMethH3K9 modification on the Ucp1 enhancer in white adipose tissue and the appearance of the active TriMethH3K4 mark at the Ucp1 promoter in brown adipose tissue in response to a cold environment. Conclusions/interpretation The results demonstrate that CpG dinucleotide methylation of the Ucp1 enhancer exhibits tissue-specific patterns in murine tissue and cell lines and suggest that adipose tissue-specific Ucp1 expression involves demethylation of CpG dinucleotides found in regulatory CREs in the Ucp1 enhancer, as well as modification of histone tails

Development and Validation of a Knowledge Management Questionnaire for Hospitals and Other Healthcare Organizations

Sustainable societies need to consider the connection between knowledge management (KM) and healthcare as a critical issue for social development. They need to investigate how to create knowledge and identify possible predictors of knowledge-sharing behavior that can support a hospital&rsquo;s sustainable knowledge-management strategy. KM strategies could help managers to increase the performance of hospitals and other healthcare organizations. The purpose of this paper is to present a valid and reliable questionnaire about KM in healthcare organizations. We develop a new knowledge-management questionnaire based on the use of an extensive literature review and health professionals&rsquo; consensus. The Applied Knowledge Management Instrument (AKMI) questionnaire was pilot tested and retested on a small group of employees of healthcare organizations (n = 31). After the pilot process, a larger group of health professionals (n = 261) completed the questionnaire. Further investigation resulted in item reduction and verification of the dimensions of AKMI. Finally, we explore the psychometric properties of the developed tool. The developed questionnaire seems to be reliable, valid, and suitable to be used for studying the suggested nine dimensions of KM: perceptions of KM, intrinsic and extrinsic motivations, knowledge synthesis and sharing, cooperation, leadership, organizational culture, and barriers. The developed questionnaire can help policymakers and hospital administrators collect information about KM processes in healthcare organizations and this can result in higher performance of health organizations

Health-related quality of life of prisoners with diabetes mellitus

The study explored health related quality of life of prisoners with diabetes mellitus (DM).48 male inmates incarcerated in a Greek prison, completed a demographic and clinical traits questionnaire. Prisoners with DM and those with additional health problems had worse physical, mental health and psychological well-being outcomes (p = 0.002, p < 0.001 and p = 0.014 respectively). People with DM in correctional facilities should receive specialized primary health care services either health services within the correctional institution or community liaison. The implementation of services targeting DM should be evaluated with the target of improving offenders’ physical and mental health

GO Lipid Metabolic Process list of genes up regulated and down regulated genes all tissues.

<p>GO Lipid Metabolic Process list of genes up regulated and down regulated genes all tissues.</p

Expression of (A) UCP1, (B) PGC-1α, (C ) C/EBPβ, (D) PPARα and (E) HNF4α in brown adipose tissue (BAT), white adipose tissue (WAT) and liver of acutely cold stressed mice.

<p>Animals were maintained at 22°C ±2°C (room temperature) or at 8°C ±2°C for 24 h (cold). Tissue samples were collected, mRNA prepared and analyzed by qRT-PCR. Values are normalised against either 18S rRNA or 36B4 expression and represent means +/− SEM of four animals in each group.</p