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Metabolic flux analysis of mitochondrial uncoupling in 3T3-L1 adipocytes.

Si Y, Shi H, Lee K - PLoS ONE (2009)

Bottom Line: Chemical uncoupling significantly decreased TG content by ca. 35%.A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes.Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Tufts University, Medford, Massachusetts, USA.

ABSTRACT

Background: Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear.

Methodology/principal findings: This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects.

Conclusions/significance: The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism.

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Metabolic flux distribution (day 14 post-induction).Top to bottom, values refer to pRev control, UCP1, untreated control and FCCP, in order. Data shown are means±SD (n = 3 for pRev and UCP1; n = 6 for untreated control and FCCP). Statistical tests compared UCP1 against pRev and FCCP against untreated control. Significantly different fluxes are shown in bold blue font (p<0.05). Dashed and dotted lines denote electron and inter-compartmental transport steps. Mitochondrial metabolites are shown in grey shaded boxes.
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pone-0007000-g005: Metabolic flux distribution (day 14 post-induction).Top to bottom, values refer to pRev control, UCP1, untreated control and FCCP, in order. Data shown are means±SD (n = 3 for pRev and UCP1; n = 6 for untreated control and FCCP). Statistical tests compared UCP1 against pRev and FCCP against untreated control. Significantly different fluxes are shown in bold blue font (p<0.05). Dashed and dotted lines denote electron and inter-compartmental transport steps. Mitochondrial metabolites are shown in grey shaded boxes.

Mentions: We applied MFA to integrate the metabolite measurements and quantify the broader impact of mitochondrial uncoupling on adipocyte intermediary metabolism. The flux analysis used metabolite data collected between days 12 and 14 post-induction, because the newly differentiated adipocyte were expected to exhibit the mature phenotype by this time [27]. Figure 5 summarizes the estimated fluxes through the major pathways. The complete flux data are shown in Table S2. Overall, there were 18 reactions significantly affected by both UCP1 expression and FCCP treatment. These reactions participated in the following pathways: glycolysis, PPP, lipid metabolism and amino acid metabolism.


Metabolic flux analysis of mitochondrial uncoupling in 3T3-L1 adipocytes.

Si Y, Shi H, Lee K - PLoS ONE (2009)

Metabolic flux distribution (day 14 post-induction).Top to bottom, values refer to pRev control, UCP1, untreated control and FCCP, in order. Data shown are means±SD (n = 3 for pRev and UCP1; n = 6 for untreated control and FCCP). Statistical tests compared UCP1 against pRev and FCCP against untreated control. Significantly different fluxes are shown in bold blue font (p<0.05). Dashed and dotted lines denote electron and inter-compartmental transport steps. Mitochondrial metabolites are shown in grey shaded boxes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2734990&req=5

pone-0007000-g005: Metabolic flux distribution (day 14 post-induction).Top to bottom, values refer to pRev control, UCP1, untreated control and FCCP, in order. Data shown are means±SD (n = 3 for pRev and UCP1; n = 6 for untreated control and FCCP). Statistical tests compared UCP1 against pRev and FCCP against untreated control. Significantly different fluxes are shown in bold blue font (p<0.05). Dashed and dotted lines denote electron and inter-compartmental transport steps. Mitochondrial metabolites are shown in grey shaded boxes.
Mentions: We applied MFA to integrate the metabolite measurements and quantify the broader impact of mitochondrial uncoupling on adipocyte intermediary metabolism. The flux analysis used metabolite data collected between days 12 and 14 post-induction, because the newly differentiated adipocyte were expected to exhibit the mature phenotype by this time [27]. Figure 5 summarizes the estimated fluxes through the major pathways. The complete flux data are shown in Table S2. Overall, there were 18 reactions significantly affected by both UCP1 expression and FCCP treatment. These reactions participated in the following pathways: glycolysis, PPP, lipid metabolism and amino acid metabolism.

Bottom Line: Chemical uncoupling significantly decreased TG content by ca. 35%.A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes.Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Tufts University, Medford, Massachusetts, USA.

ABSTRACT

Background: Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear.

Methodology/principal findings: This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects.

Conclusions/significance: The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism.

Show MeSH
Related in: MedlinePlus