Dataset Information

The Fatty Acid ?-Oxidation Pathway is Activated by Leucine Deprivation in HepG2 Cells: A Comparative Proteomics Study.

ABSTRACT: Leucine (Leu) is a multifunctional essential amino acid that plays crucial role in various cellular processes. However, the integral effect of Leu on the hepatic proteome remains largely unknown. Here, we for the first time applied an isobaric tags for relative and absolute quantification (iTRAQ)-based comparative proteomics strategy to investigate the proteome alteration induced by Leu deprivation in human hepatocellular carcinoma (HepG2) cells. A total of 4,111 proteins were quantified; 43 proteins were further identified as differentially expressed proteins between the normal and Leu deprivation groups. Bioinformatics analysis showed that the differentially expressed proteins were involved in various metabolic processes, including amino acid and lipid metabolism, as well as degradation of ethanol. Interestingly, several proteins involved in the fatty acid ?-oxidation pathway, including ACSL1, ACADS, and ACOX1, were up-regulated by Leu deprivation. In addition, Leu deprivation led to the reduction of cellular triglycerides in HepG2 cells. These results reveal that the fatty acid ?-oxidation pathway is activated by Leu deprivation in HepG2 cells, and provide new insights into the regulatory function of Leu in multiple cellular processes, especially fatty acid metabolism.

SUBMITTER: Yan G

PROVIDER: S-EPMC5432498 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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The Fatty Acid β-Oxidation Pathway is Activated by Leucine Deprivation in HepG2 Cells: A Comparative Proteomics Study.

Yan Guokai G Li Xiuzhi X Peng Ying Y Long Baisheng B Fan Qiwen Q Wang Zhichang Z Shi Min M Xie Chunlin C Zhao Li L Yan Xianghua X

Scientific reports 20170515 1

Leucine (Leu) is a multifunctional essential amino acid that plays crucial role in various cellular processes. However, the integral effect of Leu on the hepatic proteome remains largely unknown. Here, we for the first time applied an isobaric tags for relative and absolute quantification (iTRAQ)-based comparative proteomics strategy to investigate the proteome alteration induced by Leu deprivation in human hepatocellular carcinoma (HepG2) cells. A total of 4,111 proteins were quantified; 43 pro ...[more]

PMID: 28507299

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Project description:Salusin‑β and adiponectin receptor 1 (adipoR1) serve important roles in the development of certain cardiovascular diseases and lipid metabolism. However, to the best of our knowledge, the relationship between salusin‑β and adipoR1, and their underlying mechanisms of action, currently remain unclear. In the present study, lentiviral vectors designed to overexpress salusin‑β or knock down salusin‑β expression were used in 293T and HepG2 cells. Semi‑quantitative PCR was performed to investigate the relationship between salusin‑β and adipoR1 mRNA expression in 293T cells. Western blotting was used to assess the protein expression levels of adipoR1, adenosine monophosphate‑activated protein kinase (AMPK), acetyl‑CoA carboxylase (ACC) and carnitine palmitoyl transferase 1A (CPT‑1A) in transfected HepG2 cells. Simultaneously, HepG2 cells were treated with an adipoR1 inhibitor (thapsigargin) or agonist (AdipoRon) and the resultant changes in the expression levels of the aforementioned proteins were observed. Oil Red O staining and measurements of cellular triglyceride levels were performed to assess the extent of lipid accumulation in HepG2 cells. The results demonstrated that salusin‑β overexpression downregulated adipoR1 expression and inhibited the phosphorylation of AMPK and ACC, which led to decreased CPT‑1A protein expression. By contrast, salusin‑β knockdown increased adipoR1 expression and promoted the phosphorylation of AMPK and ACC, which conversely enhanced CPT‑1A protein expression. Treatment with adipoR1 agonist, AdipoRon, reversed the effects of salusin‑β overexpression. In addition, salusin‑β overexpression enhanced intracellular lipid accumulation in HepG2 cells induced by free fatty acid treatment. These findings highlighted the potential regulatory role of salusin‑β in adipoR1‑mediated signaling pathways. To conclude, the present study provided insights into the regulation of fatty acid metabolism by the liver. In particular, salusin‑β may serve as a potential target for the therapeutic intervention of metabolic disorders of lipids.

Project description:BackgroundMedium-chain fatty acids are molecules with applications in different industries and with growing demand. However, the current methods for their extraction are not environmentally sustainable. The reverse β-oxidation pathway is an energy-efficient pathway that produces medium-chain fatty acids in microorganisms, and its use in Saccharomyces cerevisiae, a broadly used industrial microorganism, is desired. However, the application of this pathway in this organism has so far either led to low titers or to the predominant production of short-chain fatty acids.ResultsWe genetically engineered Saccharomyces cerevisiae to produce the medium-chain fatty acids hexanoic and octanoic acid using novel variants of the reverse β-oxidation pathway. We first knocked out glycerolphosphate dehydrogenase GPD2 in an alcohol dehydrogenases knock-out strain (△adh1-5) to increase the NADH availability for the pathway, which significantly increased the production of butyric acid (78 mg/L) and hexanoic acid (2 mg/L) when the pathway was expressed from a plasmid with BktB as thiolase. Then, we tested different enzymes for the subsequent pathway reactions: the 3-hydroxyacyl-CoA dehydrogenase PaaH1 increased hexanoic acid production to 33 mg/L, and the expression of enoyl-CoA hydratases Crt2 or Ech was critical to producing octanoic acid, reaching titers of 40 mg/L in both cases. In all cases, Ter from Treponema denticola was the preferred trans-enoyl-CoA reductase. The titers of hexanoic acid and octanoic acid were further increased to almost 75 mg/L and 60 mg/L, respectively, when the pathway expression cassette was integrated into the genome and the fermentation was performed in a highly buffered YPD medium. We also co-expressed a butyryl-CoA pathway variant to increase the butyryl-CoA pool and support the chain extension. However, this mainly increased the titers of butyric acid and only slightly increased that of hexanoic acid. Finally, we also tested the deletion of two potential medium-chain acyl-CoA depleting reactions catalyzed by the thioesterase Tes1 and the medium-chain fatty acyl CoA synthase Faa2. However, their deletion did not affect the production titers.ConclusionsBy engineering the NADH metabolism and testing different reverse β-oxidation pathway variants, we extended the product spectrum and obtained the highest titers of octanoic acid and hexanoic acid reported in S. cerevisiae. Product toxicity and enzyme specificity must be addressed for the industrial application of the pathway in this organism.

Dataset Information

The Fatty Acid ?-Oxidation Pathway is Activated by Leucine Deprivation in HepG2 Cells: A Comparative Proteomics Study.

Publications

The Fatty Acid β-Oxidation Pathway is Activated by Leucine Deprivation in HepG2 Cells: A Comparative Proteomics Study.

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