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PPAR? agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes.

ABSTRACT: BACKGROUND:Although the 3T3-L1 preadipocyte cell line represents an informative model for in vitro adipogenesis research, primary cultured cells are often needed to understand particular human or animal metabolic phenotypes. As demonstrated by in vitro cultured preadipocytes from large mammalian species, primary cultured cells require specific adipogenic differentiation conditions different to that of the 3T3-L1 cell line. These conditions are also species-specific and require optimization steps. However, efficient protocols to differentiate primary preadipocytes using alternative species to rodents are scarce. Sheep represent an amenable animal model for fetal biology and developmental origins of health and disease studies. In this work, we present with the first detailed procedure to efficiently differentiate primary fetal and adult ovine preadipocytes. METHODS:Fetal and adult ovine adipose and skin tissue harvest, preadipocyte and fibroblast isolation, proliferation, and standardization and optimization of a new adipogenic differentiation protocol. Use of commercial cell lines (3T3-L1 and NIH-3T3) for validation purposes. Oil red O stain and gene expression were used to validate adipogenic differentiation. ANOVA and Fisher's exact test were used to determine statistical significance. RESULTS:Our optimized adipogenic differentiation method included a prolonged adipogenic cocktail exposure time from 2 to 8 days, higher insulin concentration, and supplementation with the peroxisome proliferator-activated receptor gamma (PPAR?) agonist, rosiglitazone. This protocol was optimized for both, fetal and adult preadipocytes. CONCLUSIONS:Our protocol enables successful adipogenic differentiation of fetal and adult ovine preadipocytes. This work demonstrates that compared to the 3T3-L1 cell line, fetal ovine preadipocytes require a longer exposure to the differentiation cocktail, and the need for IMBX, dexamethasone, and/or the PPAR? agonist rosiglitazone through the terminal differentiation phase. They also require higher insulin concentration during differentiation to enhance lipid accumulation and similar to human primary preadipocytes, PPAR? agonist supplementation is also required for ovine adipogenic differentiation. This work highlights species-specific differences requirements for adipogenic differentiation and the need to develop standardized methods to investigate comparative adipocyte biology.

SUBMITTER: Pu Y

PROVIDER: S-EPMC5415806 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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PPARγ agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes.

Pu Yong Y Veiga-Lopez Almudena A

Cellular & molecular biology letters 20170323

<h4>Background</h4>Although the 3T3-L1 preadipocyte cell line represents an informative model for <i>in vitro</i> adipogenesis research, primary cultured cells are often needed to understand particular human or animal metabolic phenotypes. As demonstrated by <i>in vitro</i> cultured preadipocytes from large mammalian species, primary cultured cells require specific adipogenic differentiation conditions different to that of the 3T3-L1 cell line. These conditions are also species-specific and requ ...[more]

PMID: 28536637

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Project description:Obesity is a major global health issue that contributes to the occurrence of metabolic disorders. Based on this fact, understanding the underlying mechanisms and to uncover promising therapeutic approaches for obesity have attracted intense investigation. Brown adipose tissue (BAT) can help burns excess calories. Therefore, promoting White adipose tissue (WAT) browning and BAT activation is an attractive strategy for obesity treatment. MicroRNAs (miRNAs) are small, non-coding RNAs, which are involved in regulation of adipogenic processes and metabolic functions. Evidence is accumulating that miRNAs are important regulators for both brown adipocyte differentiation and white adipocyte browning. Here we report that the expression of miR-669a-5p increases during the adipogenic differentiation of 3T3-L1 and C3H10T1/2 adipocytes. miR-669a-5p supplementation promotes adipogenic differentiation and causes browning of 3T3-L1 and C3H10T1/2 cells. Moreover, the expression of miR-669a-5p is upregulated in iWAT of mice exposed to cold. These data demonstrate that miR-669a-5p plays a role in regulating adipocyte differentiation and fat browning.Abbreviations: Acadl: long-chain acyl-Coenzyme A dehydrogenase; Acadm: medium-chain acyl-Coenzyme A dehydrogenase; Acadvl: very long-chain acyl-Coenzyme A dehydrogenase, very long chain; Aco2: mitochondrial aconitase 2; BAT: brown adipose tissue; Bmper: BMP-binding endothelial regulator; Cpt1-b:carnitine palmitoyltransferase 1b; Cpt2: carnitine palmitoyltransferase 2; Crat: carnitine acetyltransferase; Cs: citrate synthase; C2MC: Chromosome 2 miRNA cluster; DMEM: Dulbecco's modified Eagle medium; eWAT: epididymal white adipose tissue; ETC: electron transport chain; FAO: fatty acid oxidation; Fabp4:fatty acid binding protein 4; FBS: fetal bovine serum; Hadha: hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha; Hadhb: hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta; HFD: high fat diet; Idh3a: isocitrate dehydrogenase 3 alpha; iWAT: inguinal subcutaneous white adipose tissue; Lpl: lipoprotein lipase; Mdh2: malate dehydrogenase 2; NBCS: NewBorn Calf Serum; mt-Nd1: mitochondrial NADH dehydrogenase 1; Ndufb8:ubiquinone oxidoreductase subunit B8; Nrf1: nuclear respiratory factor 1; Pgc1α: peroxisome proliferative activated receptor gamma coactivator 1 alpha; Pgc1b: peroxisome proliferative activated receptor, gamma, coactivator 1 beta; Pparγ: peroxisome proliferator activated receptor gamma; Prdm16: PR domain containing 16; Rgs4: regulator of G-protein signaling 4; Sdhb: succinate dehydrogenase complex, subunit B; Sdhc: succinate dehydrogenase complex, subunit C; Sdhd: succinate dehydrogenase complex, subunit D; Sh3d21: SH3 domain containing 21; Sfmbt2: Scm-like with four mbt domains 2; TG: triglyceride; TCA: tricarboxylic acid cycle; Tfam: transcription factor A, mitochondrial; TMRE: tetramethylrhodamine, methyl ester; Ucp1: uncoupling protein 1; Uqcrc2: ubiquinol cytochrome c reductase core protein 2; WAT: White adipose tissue.

Project description:MicroRNAs (miRNAs) are crucial regulatory molecules in lipid deposition and metabolism. However, the effect of miR-200b on the regulation of proliferation and adipogenesis of ovine preadipocytes is unknown in the sheep (Ovis aries). In this study, the expression profiles of miR-200b were investigated in the seven tissues of Tibetan ewes and differentiated preadipocytes. The effect of miR-200b, as well as its target genes p27 and KLF9, on the proliferation of ovine preadipocytes and adipogenesis was also investigated, using cell viability analysis, EdU staining, Oil Red O staining and reverse transcription-quantitative PCR (RT-qRCR). The miR-200b was expressed in all the tissues investigated, and it was highly expressed in lung, liver, subcutaneous adipose and spleen tissues. The expression of miR-200b continuously decreased when the differentiation of ovine preadipocytes initiated. The miR-200b mimic dramatically accelerated the proliferation but inhibited differentiation of ovine preadipocytes. The miR-200b inhibitor resulted in an opposite effect on the proliferation and differentiation of ovine preadipocytes. The dual luciferase reporter assay results showed that miR-200b mimic significantly decreased the luciferase activity of p27 and KLF9 in HEK293 cells transfected with wild-type dual luciferase reporter vectors. This suggests that p27 and KLF9 are the target genes of miR-200b. In over-expressed-p27 preadipocytes, the number of EdU-labeled preadipocytes and the expression levels of proliferation marker genes CDK2, CDK4, CCND1 and PCNA significantly decreased. In addition, the transfection of over-expressed-KLF9 vector into adipocytes remarkably increased the accumulation of lipid droplets and the expression levels of differentiation marker genes aP2, PPARγ, LPL and GLUT4. These results suggest that miR-200b accelerated the proliferation but inhibited the adipogenic differentiation of ovine preadipocytes by targeting p27 and KLF9, respectively.

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PPAR? agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes.

Publications

PPARγ agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes.

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