4EBP1 is a chief downstream factor of mTORC1, and PPARγ is a key lipogenesis-related transcription factor. mTORC1 and PPARγ are associated with lipid metabolism. However, it is unknown which effector protein connects mTORC1 and PPARγ. This study investigated the interaction between 4EBP1 with PPARγ as part of the underlying mechanism by which insulin-induced lipid synthesis and secretion are regulated by mTORC1 in primary bovine mammary epithelial cells (pBMECs). Rapamycin, a specific inhibitor of mTORC1, downregulated 4EBP1 phosphorylation and the expression of PPARγ and the following lipogenic genes: lipin 1, DGAT1, ACC, and FAS. Rapamycin also decreased the levels of intracellular triacylglycerol (TAG); 10 types of fatty acid; and the accumulation of TAG, palmitic acid (PA), and stearic acid (SA) in the cell culture medium. Inactivation of mTORC1 by shRaptor or shRheb attenuated the synthesis and secretion of TAG and PA. In contrast, activation of mTORC1 by Rheb overexpression promoted 4EBP1 phosphorylation and PPARγ expression and upregulated the mRNA and protein levels of lipin 1, DGAT1, ACC, and FAS, whereas the levels of intracellular and extracellular TAG, PA, and SA also rose. Further, 4EBP1 interacted directly with PPARγ. Inactivation of mTORC1 by shRaptor prevented the nuclear location of PPARγ. These results demonstrate that mTORC1 regulates lipid synthesis and secretion by inducing the expression of lipin 1, DGAT1, ACC, and FAS, which is likely mediated by the 4EBP1/PPARγ axis. This finding constitutes a novel mechanism by which lipid synthesis and secretion are regulated in pBMECs.
The mechanistic target of rapamycin complex 2 (mTORC2) primarily functions as an effector of insulin/PI3K signaling to regulate cell proliferation and is associated with cell metabolism. However, the function of mTORC2 in lipid metabolism is not well understood. In the present study, mTORC2 was inactivated by the ATP-competitive mTOR inhibitor AZD8055 or shRNA targeting RICTOR in primary bovine mammary epithelial cells (pBMECs). MTT assay was performed to examine the effect of AZD8055 on cell proliferation. ELISA assay and GC-MS analysis were used to determine the content of lipid. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that cell proliferation, mTORC2 activation, and lipid secretion were inhibited by AZD8055. RICTOR was knocked down and mTORC2 activation was specifically attenuated by the shRNA. Compared to control cells, the expression of the transcription factor gene PPARG and the lipogenic genes LPIN1, DGAT1, ACACA, and FASN was downregulated in RICTOR silencing cells. As a result, the content of intracellular triacylglycerol (TAG), palmitic acid (PA), docosahexaenoic acid (DHA), and other 16 types of fatty acid was decreased in the treated cells; the accumulation of TAG, PA, and DHA in cell culture medium was also reduced. Overall, mTORC2 plays a critical role in regulating lipogenic gene expression, lipid synthesis, and secretion in pBMECs, and this process probably is through PPARγ. This finding provides a model by which lipogenesis is regulated in pBMECs.
Background. Several large-scale studies suggest that Bacille Calmette–Guerin (BCG) vaccination in early childhood may reduce the risk of atopic diseases, but the findings remain controversial. Here, we aimed to investigate the potential correlation between early childhood BCG vaccination and the risk of developing atopic diseases. Methods. Eligible studies published on PubMed, EMBASE, and Cochrane CENTRAL were systematically sourced from 1950 to July 2021. Studies with over 100 participants and focusing on the association between BCG vaccine and atopic diseases including eczema, asthma, and rhinitis were included. Preliminary assessment of methods, interventions, outcomes, and study quality was performed by two independent investigators. Odds ratio (OR) with 95% confidence interval (CI) was calculated. Random effects of the meta-analysis were performed to define pooled estimates of the effects. Results. Twenty studies with a total of 222,928 participants were selected. The quantitative analysis revealed that administering BCG vaccine in early childhood reduced the risk of developing asthma significantly (OR 0.77, 95% CI 0.63 to 0.93), indicating a protective efficacy of 23% against asthma development among vaccinated children. However, early administration of BCG vaccine did not significantly reduce the risk of developing eczema (OR 0.94, 95% CI 0.76 to 1.16) and rhinitis (OR 0.99, 95% CI 0.81 to 1.21). Further analysis revealed that the effect of BCG vaccination on asthma prevalence was significant especially in developed countries (OR 0.73, 95% CI 0.58 to 0.92). Conclusion. BCG vaccination in early childhood is associated with reduced risk of atopic disease, especially in developed countries.
BackgroundMammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that is a central regulator of cell growth and metabolism. CCI-779 is a specific inhibitor of the mTORC1 signaling pathway.ResultsWe performed comparative transcriptome profiling on Inner Mongolia Cashmere goat fetal fibroblasts (GFbs) that were treated with CCI-779 and untreated cells. A total of 365 differentially expressed genes (DEGs) appeared between untreated and CCI-779-treated GFbs, with an FDR ≤0.001 and fold-change ≥2. These 365 DEGs were associated with mTOR signaling; 144 were upregulated in CCI-779-treated cells, and 221 were downregulated.Additionally, 300 genes were annotated with 43 Gene Ontology (GO) terms, and 293 genes were annotated with 194 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Three RNA polymerase II and polymerase III subunits, 3 transcription factors, and 5 kinases in mTOR signaling were differentially expressed in CCI-779-treated GFbs. Further 6 DEGs were related to amino acid metabolism, 11 mediated lipid metabolism, 11 participated in carbohydrate metabolism, and 5 were involved in single-nucleotide metabolism. Based on our quantitative transcriptomic analysis, 40 significant DEGs with important function related to metabolism, RNA polymerase, transcription factors and mTOR signaling were selected for qPCR analysis, and the quantitative results between the two analysis methods were concordant. The qPCR data confirmed the differential expression in the RNA-Seq experiments. To validate the translational significance of the findings in certain differentially expressed genes, S6K1 and VEGF were detected by western blot, and these two proteins showed a differential expression between non-treated and treated with CCI-779 groups, which were consistent with mRNA abundance. The data showed a preliminary significance of the findings in the protein levels.ConclusionsCCI-779 induces transcriptomic changes, and mTOR signaling might have significant function in the activation of RNA polymerase and certain transcription factors and in the metabolism of amino acids, lipids, carbohydrates, and single nucleotides in GFbs. These data filled the vacancy in the systematical profiling of mTOR signaling on Cashmere goat fetal fibroblasts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3151-y) contains supplementary material, which is available to authorized users.
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