Over-expression of Akt1 Induces Skin Hypertrophy and Generates an Obesity Model in Zebrafish
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE21756 | GEO | 2010/12/31
SECONDARY ACCESSION(S): PRJNA127321
REPOSITORIES: GEO
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Project description:Recent evidences have demonstrated phosphatidylinositol3-kinase (PI3-K)/Akt signaling pathway participates in cell growth, cell survival, and adipocyte differentiation in vitro. However, the in vivo evidence to support Akt function on adipogenesis is limited. To achieve this goal, we generated the stable zebrafish transgenics of Tg(krt4:myrAkt1)cy18 carrying human constitutive active form of Akt1 (myrAkt1) that driven by a skin-specific krt4 promoter. The Tg(krt4:myrAkt1)cy18 display severely skin hypertrophy at embryonic stages, and support Akt1 function as a key gene on cell size control. When transgenics reached sexual maturation, they display obese phenotype due to adipocyte hypertrophy and up-regulation of adipogenesis-related genes. Collectively, our findings provided a direct evidence to support Akt play provital roles on cell size control as well as adipogenesis in vivo. In addition, the obese zebrafish line of Tg(krt4:myrAkt1)cy18 provide a new platform to study obesity and its related chronic diseases mechanism in vivo. The AB strain of Zebrafish (Danio rerio) were obtained from ZIRC (http://zebrafish.org/zirc/home/guide.php), and kept in the stock of Chung Yuan Christian University. Zebrafish were raised in a local tap water at 28.0±0.5℃ and under a constant photoperiodism of 14 hour light cycle/10 hour dark cycle. Zebrafish embryos were collected in 10 cm diameter Petri dishes containing 20 mL fish water after spawning and raised at 28.0±0.5℃. To prevent the disease of Zebrafish embryo, few drops methylen blue were added into fish water. At 5 to 7 dpf, larvae were transferred to 10 liter tanks containing 8.0 liter of fish water and fed with living Paramecium. After 14 dpf, larvae were feed with living artemia (OSI, USA) until adulthood. We dissected two tail tissues (as a pooled sample) from six month-old wild-type and Tg(krt4:myrAkt1)cy18 (n=5), and homogenized in Trizol reagent (Ambion) to isolate total RNA according to the manufacture instruction. The RNA was treated with DNase I at 25℃ for 10 minutes to remove DNA contamination and then cleanup by RNase-free spin columns (Qiagen). The total RNA concentration was determined by spectrophotometry (ND-1000 ; NanoDrop Technol, Wilmington, DE), and the RNA quality was checked by running electrophoresis in RNA-denatured gels. All total RNA were stored at -20℃. For real-time quantitative RT-PCR, 1μg of total RNA were reverse-transcribed with reverse transcriptase and the excess RNA were digested E.coli RNase H to enhance the cDNA purity. The commercial Zebrafish 14K oligo microarray chip was obtained from Institute of Cellular and Organismic Biology at Academia Sinica and contained 14067 oligonucleotides represent 9666 unique genes with a redundancy of 31%. The detail oligonucleotide description can be obtained from Ocimun Biosolution (http://www.ocimumbio.com/web/default.asp). We used SuperScriptTM Indirect cDNA Labeling System kit (Invitrogen) to generate the fluorescently labeled probes. Total RNA isolated from WT and Tg(krt4:myrAkt1)cy18 were reversed transcribed into cDNA and coupling for Alexa Fluor 555 and Alexa Fluor 647 fluorescent dyes (Invitrogen), respectively. Before hybridization, the Zebrafish microarray chips were pretreated with 1% bovine serum albumin, 4X SCC, and 1% sodium dodecylsulfate (SDS) for 45min at 42℃, and then hybridized in SlideHybTM buffer (Ambion) for overnight at 42℃. After hybridization, chips were washed with 2X SSC and 0.5% SDS for 15min at 25℃ and finally with 0.5X SSC and 0.5% SDS for 15min at 25℃. The fluorescence intensities of Alexa Fluor 555 and Alexa Fluor 647 targets were scanned by Genepix scanner (Molecular Devices, Sunnyvale, CA, USA) and the acquired date were analyzed by Genepix and Genespring software (Aglient Technologies, Foster City, CA, USA).
2010-12-31 | E-GEOD-21756 | biostudies-arrayexpress
Project description:To investigate molecular mechanisms involved in the development of cardiac hypertrophy and heart failure, a tetracycline-regulated transgenic system to conditionally switch a constitutively-active form of the Akt1 protein kinase on or off in the adult heart was developed. Short-term activation (2 weeks) of Akt1 resulted in completely reversible hypertrophy with maintained contractility. In contrast, chronic Akt1 activation (6 weeks) induced extensive cardiac hypertrophy, severe contractile dysfunction, and massive interstitial fibrosis. The focus of this study was to create a transcript expression profile of the heart as it undergoes reversible Akt1-mediated hypertrophy and during the transition from compensated hypertrophy to heart failure. Heart tissue was analyzed before transgene induction, 2 weeks after transgene induction, 2 weeks of transgene induction followed by 2 days of repression, 6 weeks after transgene induction and 6 weeks of transgene induction followed by 2 weeks of repression. Acute over expression of Akt1 (2 weeks) leads to changes in the expression of 826 transcripts relative to non-induced hearts, whereas chronic induction (6 weeks) led to changes in the expression of 1611, of which 65% represented transcripts that were regulated during the pathological phase of heart growth. Another set of genes identified were uniquely regulated during heart regression but not growth, indicating that non-overlapping transcription programs participate in the processes of cardiac hypertrophy and atrophy. These data define the gene regulatory programs downstream of Akt that control heart size and contribute to the transition from compensatory hypertrophy to heart failure. Experiment Overall Design: Gene expression data from DTG-positive mouse hearts were examined before the induction of the transgene Akt1 (time point 1), 2 weeks after the induction of the transgene Akt1(time point 2), 2 weeks after the induction of the transgene Akt1 and 2 days after the repression of the transgene Akt1 (time point 3), 6 weeks after the induction of the transgene Akt1(time point 4) and 6 weeks after the induction of the transgene Akt1 and 2 weeks after the repression of the transgene Akt1 (time point 5). N = 3 to 4 sets of independent hybridizations from individual mice were performed for each time point.
2007-12-01 | E-GEOD-3383 | biostudies-arrayexpress
Project description:To investigate molecular mechanisms involved in the development of cardiac hypertrophy and heart failure, a tetracycline-regulated transgenic system to conditionally switch a constitutively-active form of the Akt1 protein kinase on or off in the adult heart was developed. Short-term activation (2 weeks) of Akt1 resulted in completely reversible hypertrophy with maintained contractility. In contrast, chronic Akt1 activation (6 weeks) induced extensive cardiac hypertrophy, severe contractile dysfunction, and massive interstitial fibrosis. The focus of this study was to create a transcript expression profile of the heart as it undergoes reversible Akt1-mediated hypertrophy and during the transition from compensated hypertrophy to heart failure. Heart tissue was analyzed before transgene induction, 2 weeks after transgene induction, 2 weeks of transgene induction followed by 2 days of repression, 6 weeks after transgene induction and 6 weeks of transgene induction followed by 2 weeks of repression. Acute over expression of Akt1 (2 weeks) leads to changes in the expression of 826 transcripts relative to non-induced hearts, whereas chronic induction (6 weeks) led to changes in the expression of 1611, of which 65% represented transcripts that were regulated during the pathological phase of heart growth. Another set of genes identified were uniquely regulated during heart regression but not growth, indicating that non-overlapping transcription programs participate in the processes of cardiac hypertrophy and atrophy. These data define the gene regulatory programs downstream of Akt that control heart size and contribute to the transition from compensatory hypertrophy to heart failure. Keywords: transgenic mice, Akt1, time course, cardiac hypertrophy and contractile dysfunction, DNA microarrays
2006-08-25 | GSE3383 | GEO
Project description:White adipose tissue (WAT) is a key regulator of systemic energy metabolism, and impaired WAT plasticity characterized by enlargement of preexisting adipocytes associates with WAT dysfunction, obesity and metabolic complications. However, the mechanisms that retain proper adipose tissue plasticity required for metabolic fitness are unclear. Here, we comprehensively showed that adipocyte-specific DNA methylation, manifested in enhancers and CTCF sites, directs distal enhancer-mediated transcriptomic features required to conserve metabolic functions of white adipocytes. Particularly, genetic ablation of adipocyte Dnmt1, the major methylation writer, led to increased adiposity characterized by increased adipocyte hypertrophy along with reduced expansion of adipocyte precursors (APs). These effects of Dnmt1 deficiency provoked systemic hyperlipidemia and impaired energy metabolism both in lean and obese mice. Mechanistically, Dnmt1 deficiency abrogated mitochondrial bioenergetics by inhibiting mitochondrial fission and promoted aberrant lipid metabolism in adipocytes, rendering adipocyte hypertrophy and WAT dysfunction. Dnmt1-dependent DNA methylation prevented aberrant CTCF binding and, in turn, sustained the proper chromosome architecture to permit interactions between enhancer and dynamin-related protein gene Drp1 in adipocytes. Also, adipose DNMT1 expression inversely correlated with adiposity and markers of metabolic health, but positively correlated with AP-specific markers in obese human subjects. Thus, these findings support strategies utilizing Dnmt1 action on mitochondrial bioenergetics in adipocytes to combat obesity and related metabolic pathology.
2021-03-24 | GSE158944 | GEO
Project description:Obesity is linked to the development of metabolic disorders. Expansion of white adipose tissue (WAT) from hypertrophy of pre-existing adipocytes and/or differentiation of precursors into new mature adipocytes contributes to obesity. We found that Nck2 expression is largely restricted to WAT, raising the hypothesis that it may play a unique function in that tissue. Using mice lacking Nck2, we found that Nck2 regulates adipocyte hypertrophy thus contributing to increased adiposity and progressive glucose intolerance, insulin resistance and hepatic steatosis. These findings were recapitulated in humans such that Nck2 expression in omental WAT was inversely correlated with the degree of obesity. Mechanistically, Nck2 deficiency promoted the induction of an adipocyte differentiation program and signaling by the PERK-eIF2α-ATF4 pathway in agreement with a role for the unfolded protein response in adipogenesis. These findings uncover Nck2 as a novel regulator of adipogenesis and that perturbation in its functionality contributes to adiposity-related metabolic disorders. Differential gene expression profile between epididymal white adipose tissue of Nck2-/- and Nck2+/+ mice by RNA sequencing (Illumina HiSEq 2000)
2016-07-01 | E-GEOD-63510 | biostudies-arrayexpress
Project description:First-degree relatives (FDRs) of type 2 diabetics (T2D) feature dysfunction of subcutaneous adipose tissue (SAT) long before T2D onset. miRNAs have a role in adipocyte precursor cells (APC) differentiation and in adipocyte identity. Thus, impaired miRNA expression may contribute to SAT dysfunction in FDRs. In the present work, we have explored changes of miRNA expression associated with T2D family history which may affect gene expression in SAT APCs from FDRs. Small RNA-seq was performed in APCs from healthy FDRs and matched controls and omics data were validated by qPCR. Integrative analyses of APC miRNome and transcriptome from FDRs revealed down-regulated hsa-miR-23a-5p, -193a-5p and -193b-5p accompanied by up-regulated Insulin-like Growth Factor 2 (IGF2) gene which proved to be their direct target. The expression changes of these marks were associated with SAT adipocyte hypertrophy in FDRs. APCs from FDRs further demonstrated reduced capability to differentiate into adipocytes. Treatment with IGF2 protein decreased APC adipogenesis, while over-expression of hsa-miR-23a-5p, -193a-5p and -193b-5p enhanced adipogenesis by IGF2 targeting. Indeed, IGF2 increased the Wnt Family Member 10B gene expression in APCs. Down-regulation of the three miRNAs and IGF2 up-regulation were also observed in Peripheral Blood Leukocytes (PBLs) from FDRs. In conclusion, APCs from FDRs feature a specific miRNA/gene profile, which associates with SAT adipocyte hypertrophy and appears to contribute to impaired adipogenesis. PBL detection of this profile may help in identifying adipocyte hypertrophy in individuals at high risk of T2D.
2021-11-19 | GSE162133 | GEO
Project description:First-degree relatives (FDRs) of type 2 diabetics (T2D) feature dysfunction of subcutaneous adipose tissue (SAT) long before T2D onset. miRNAs have a role in adipocyte precursor cells (APC) differentiation and in adipocyte identity. Thus, impaired miRNA expression may contribute to SAT dysfunction in FDRs. In the present work, we have explored changes of miRNA expression associated with T2D family history which may affect gene expression in SAT APCs from FDRs. Small RNA-seq was performed in APCs from healthy FDRs and matched controls and omics data were validated by qPCR. Integrative analyses of APC miRNome and transcriptome from FDRs revealed down-regulated hsa-miR-23a-5p, -193a-5p and -193b-5p accompanied by up-regulated Insulin-like Growth Factor 2 (IGF2) gene which proved to be their direct target. The expression changes of these marks were associated with SAT adipocyte hypertrophy in FDRs. APCs from FDRs further demonstrated reduced capability to differentiate into adipocytes. Treatment with IGF2 protein decreased APC adipogenesis, while over-expression of hsa-miR-23a-5p, -193a-5p and -193b-5p enhanced adipogenesis by IGF2 targeting. Indeed, IGF2 increased the Wnt Family Member 10B gene expression in APCs. Down-regulation of the three miRNAs and IGF2 up-regulation were also observed in Peripheral Blood Leukocytes (PBLs) from FDRs. In conclusion, APCs from FDRs feature a specific miRNA/gene profile, which associates with SAT adipocyte hypertrophy and appears to contribute to impaired adipogenesis. PBL detection of this profile may help in identifying adipocyte hypertrophy in individuals at high risk of T2D.
2021-11-19 | GSE161989 | GEO
Project description:Akt is a serine/threonine protein kinase that is activated by a variety of growth factors or cytokines in a PI3-kinase dependent manner. Using a conditional transgenic system in which Akt signaling can be turned on or off in the adult heart, we have recently demonstrated that short-term Akt activation induces a ‘physiological’ form of cardiac hypertrophy with enhanced coronary angiogenesis and maintained contractility. Here we tested the hypothesis that induction of physiological hypertrophy by short-term Akt activation might improve contractile function in failing hearts. When Akt signaling was transiently activated in murine hearts with impaired contractility induced by pressure overload or adriamycin treatment, contractile dysfunction was attenuated in both cases. Importantly, improvement of contractility was observed before the development of cardiac hypertrophy, indicating that Akt improves contractile dysfunction independently of its growth-promoting effects. To gain mechanistic insights into Akt-mediated positive inotropic effects, transcriptional profiles in the heart were determined in a pressure overload-induced heart failure model. Biological network analysis of differentially expressed transcripts revealed significant alterations in the expression of genes associated with cell death, and these alterations were reversed by short-term Akt activation. Thus, short-term Akt activation improves contractile dysfunction in failing hearts. This beneficial effect of Akt on contractility is hypertrophy-independent and may be mediated in part by inhibition of cell death associated with heart failure. Keywords: transgenic mice, Akt1, cardiac hypertrophy after ascending aortic constriction and contractile dysfunction, DNA microarrays
2019-07-01 | GSE4728 | GEO
Project description:Agonist-induced cardiac hypertrophy in TG1306/1R transgenic mice with cardiac angiotensin II overproduction leads to a gradual transition from a compensatory hypertrophic state to heart failure. To gain insight into the molecular mechanisms that play a role in maintaining cardiac integrity in the diseased heart, we performed a comparative study of gene expression between wild-type (WT) and transgenic (TG) hearts using microarray analysis. TG1306/1R transgenics were separated into two phenotypic groups (hypertrophic and dilated) based on morphological parameters (cardiac weight index and histology) and either a moderate (hypertrophic) or a high (dilated) upregulation of molecular markers for hypertrophy and failure, and compared to age and sex-matched wild-types.
2008-10-25 | GSE13336 | GEO
Project description:Obesity is linked to the development of metabolic disorders. Expansion of white adipose tissue (WAT) from hypertrophy of pre-existing adipocytes and/or differentiation of precursors into new mature adipocytes contributes to obesity. We found that Nck2 expression is largely restricted to WAT, raising the hypothesis that it may play a unique function in that tissue. Using mice lacking Nck2, we found that Nck2 regulates adipocyte hypertrophy thus contributing to increased adiposity and progressive glucose intolerance, insulin resistance and hepatic steatosis. These findings were recapitulated in humans such that Nck2 expression in omental WAT was inversely correlated with the degree of obesity. Mechanistically, Nck2 deficiency promoted the induction of an adipocyte differentiation program and signaling by the PERK-eIF2α-ATF4 pathway in agreement with a role for the unfolded protein response in adipogenesis. These findings uncover Nck2 as a novel regulator of adipogenesis and that perturbation in its functionality contributes to adiposity-related metabolic disorders.
2016-07-01 | GSE63510 | GEO