Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A Time Course Analysis of the Effects of Prieurianin in the mouse preadipocytes 3T3-L1 cells

ABSTRACT: Gene expression profiling to examine the effets of prieurianin in a time-course study (0, 0.25, 0.5, 1, 2, 3, 5, 9, 12, 15, 24, 36, 48, 96, 144, 192, 240, and 288 hrs) on the differentiation of the 3T3-L1 preadipocytes into adipocytes. The sharp rise in the incidence of obesity in the last two decades has become one of the most serious public health risks worldwide. Currently approved therapies for obesity exhibit modest efficacy and limiting side effects. We show here that prieurianin, a limonoid, causes weight loss by reducing energy intake in morbidly obese mice and in mice on high-calorie diet. Prieurianin is also anti-adipogenic by inhibiting the proliferation and differentiation of preadipocytes into adipocytes, and induces either dedifferentiation or delipidation of mature adipocytes. Gene expression profiling shows that prieurianin suppresses the expression of genes involved in fat metabolism, and increases the expression of a transcription repressor, zinc finger protein 68 (Zfp68), which inhibits CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor γ (PPARγ), master transcriptional regulators of adipogenesis. The effects of prieurianin are reminiscent of several cytokines that regulate appetite and adipogenesis, and holds promise as an effective anti-obesity drug. Keywords: Time course, prieurianin, obesity, differentiation, adipogenesis, preadipocytes, adipocytes, Zfp68 L1 Cells were treated either with 0.5% DMSO or 2 µM of prieurianin for the following times: 0, 0.25, 0.5,1, 2, 3, 5, 9, 12, 15, 24, 36, 48, 96, 144, 192, 240, and 288 hrs, and then total RNA was purified at the indicated times using the RNeasy Mini kit according to the manufacturer’s instructions. Samples were labeled and hybridized to the Mouse OneArray (Phalanx Biotech Group, Palo Alto, CA) and analyzed for gene expression changes, compared to untreated control undergoing normal differentiation.

ORGANISM(S): Mus musculus

SUBMITTER: Khew-Voon Chin

PROVIDER: E-GEOD-15018 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To better understand the scale of gene expression changes that occur during the formation of mature adipocytes from preadipocytes, we compared and characterised the transcriptome profile of mesenchymal stromal cells derived from human adipose tissue, otherwise known as adipose-derived stromal cells (ASCs), undergoing adipocyte differentiation on day 1, 7, 14 and 21 (representing the early to late stage process of adipogenesis). Microarray technique was systematically employed to study gene expression in adipose-derived stromal cells during adipogenic differentiation over a 21 day period to identify genes that are important in driving adipogenesis in humans. We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived stromal cells (ASCs) induced to differentiate into adipocytes in vitro. Genes were differentially expressed on days 1, 7, 14 and 21 post-induction and numbered 128, 218, 253 and 240 respectively. Up-regulated genes were associated with neural and blood vessel development, leukocyte migration, and tumor growth, invasion and metastasis. They also shared common pathways with certain obesity-related pathophysiological conditions. Down-regulated genes were associated with osteogenesis and the immune response. KLF15, LMO3, FOXO1 and ZBTB16 transcription factors were up-regulated throughout the differentiation process. CEBPA, PPARG, ZNF117, MLXIPL, MMP3 and RORB were up-regulated only on days 14 - 21, which coincides with the maturation of adipocytes and could possibly serve as candidates for controlling fat accumulation and the size of mature adipocytes. We identified genes that were up-regulated only on day 1 - 7 and day 14 - 21 that could serve as potential early and late-stage differentiation markers. This study reveals potential markers for the different stages in ASC adipogenic differentiation which could serve as good candidates to combat obesity and further link the biology of fat cell formation to the co-morbidities of obesity.

Project description:Since the worldwide increase in obesity represents a growing challenge for the health care systems, new approaches are needed to treat obesity and associated disease effectively. The food intake is primarily stored in the adipose tissue and therefore this organ is in focus to develop new anti-obesity treatments. To provide a systematic analysis of genes that regulate adipose tissue biology and to establish a target-oriented compound screening, we performed a high throughput siRNA screen with primary (pre)adipocytes, using a druggable siRNA library targeting 7,784 human genes. Beside well known regulators of adipogenesis and neutral lipid storage (like PPAR?, RXR, Perilipin A) the screening revealed a large number of genes which were not previously described in the context of fatty tissue biology. An enrichment of genes was observed for axonemal dyneins. Five out of ten anxonemal dyneins were identified in our primary screen and retested positive with independent siRNAs and cell-donors. Quantitative RT-PCR- and immunoblot analysis revealed that axonemal dyneins are expressed in preadipocytes and maturing adipocytes. Using microarray analysis, we further characterize the remaining genes identified in our primary screen to determine their expression pattern during adipogenesis. In the course of fat cell differentiation, 149 among the 459 positive genes were regulated on the gene expression level. Assuming that an adipogenesis-specific expression pattern is another independent hind, that these genes are involved in neutral lipid storage and/or adipocyte differentiation, we retested this gene-pool with independent siRNAs and cell donors. Finally, to show that the genes identified are per se druggable we performed a proof of principle experiment using an antagonist for one randomly chosen gene. The results showed a very similar phenotype compared to knock-down experiments proofing the druggability. Thus, we identified new adipogenesis-associated genes and those involved in neutral lipid storage. Moreover, by using a druggable siRNA library the screen data provides a very attractive starting point, to identify anti-obesity compounds targeting the adipose tissue. For microarray analysis, RNA was isolated at 3 different points in time during adipogenesis (day 0, day 3 and day 7 after induction of differentiation). This experiment was carried out with cells obtained from three different donors. No replicates are included for each point in time.

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A Time Course Analysis of the Effects of Prieurianin in the mouse preadipocytes 3T3-L1 cells

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