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Metabolic and tissue-specific regulation of acyl-CoA metabolism.

Ellis JM, Bowman CE, Wolfgang MJ - PLoS ONE (2015)

Bottom Line: We find patterns of coordinated regulation within and between these gene families as well as distinct regulation occurring in a tissue- and physiologically-dependent manner.Due to observed changes in long-chain ACOT mRNA and protein abundance in liver and adipose tissue, we determined the consequence of increasing cytosolic long-chain thioesterase activity on fatty acid metabolism in these tissues by generating transgenic mice overexpressing a hyperactive mutant of Acot7 in the liver or adipose tissue.Together, these data suggest distinct modes of regulation of the ACS and ACOT enzymes and that these enzymes act in a coordinated fashion to control fatty acid metabolism in a tissue-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Center for Metabolism and Obesity Research, Baltimore, Maryland 21205 United States of America.

ABSTRACT
Acyl-CoA formation initiates cellular fatty acid metabolism. Acyl-CoAs are generated by the ligation of a fatty acid to Coenzyme A mediated by a large family of acyl-CoA synthetases (ACS). Conversely, acyl-CoAs can be hydrolyzed by a family of acyl-CoA thioesterases (ACOT). Here, we have determined the transcriptional regulation of all ACS and ACOT enzymes across tissues and in response to metabolic perturbations. We find patterns of coordinated regulation within and between these gene families as well as distinct regulation occurring in a tissue- and physiologically-dependent manner. Due to observed changes in long-chain ACOT mRNA and protein abundance in liver and adipose tissue, we determined the consequence of increasing cytosolic long-chain thioesterase activity on fatty acid metabolism in these tissues by generating transgenic mice overexpressing a hyperactive mutant of Acot7 in the liver or adipose tissue. Doubling cytosolic acyl-CoA thioesterase activity failed to protect mice from diet-induced obesity, fatty liver or insulin resistance, however, overexpression of Acot7 in adipocytes rendered mice cold intolerant. Together, these data suggest distinct modes of regulation of the ACS and ACOT enzymes and that these enzymes act in a coordinated fashion to control fatty acid metabolism in a tissue-dependent manner.

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Development of a transgenic mouse model with a conditional tissue-specific and cytoplasmically targeted long-chain acyl-CoA thioesterase.(A) Transgenic construct schematic and representative western blot confirming Acot7HA-FLAG overexpression in liver. (B) Thioesterase activity for oleoyl-CoA in liver lysate from control and Acot7HA-Liv transgenic mice, n = 5–7. Overnight fasted control and Acot7HA-Liv liver slice rates of (C) 14C-oleate oxidation, (D) 14C-oleate incorporation into complex lipids, and (E) 3H-acetate incorporation into lipids, n = 5–7. Data represent mean ± SEM, * represent p≤0.05 by Student’s t-test.
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pone.0116587.g007: Development of a transgenic mouse model with a conditional tissue-specific and cytoplasmically targeted long-chain acyl-CoA thioesterase.(A) Transgenic construct schematic and representative western blot confirming Acot7HA-FLAG overexpression in liver. (B) Thioesterase activity for oleoyl-CoA in liver lysate from control and Acot7HA-Liv transgenic mice, n = 5–7. Overnight fasted control and Acot7HA-Liv liver slice rates of (C) 14C-oleate oxidation, (D) 14C-oleate incorporation into complex lipids, and (E) 3H-acetate incorporation into lipids, n = 5–7. Data represent mean ± SEM, * represent p≤0.05 by Student’s t-test.

Mentions: To further understand the functional consequence and tissue-specific role of cytosolic thioesterase-induced fatty acid deactivation, we designed a transgenic mouse model to conditionally over-express an active and cytoplasmic long-chain acyl-CoA thioesterase. We chose Acot7 as a model thioesterase because its enzymatic and regulatory properties have been previously characterized including a crystal structure [4,5,29–31]. Acot7 is a typical hotdog fold type II thioesterase that encodes two thioesterase domains per polypeptide, one of which is inactive yet binds substrate resulting in a “half-of-sites inhibition” regulation of the enzyme [29]. Engineering the inactive site to an active thioesterase doubles the enzyme activity [29]. We capitalized on this discovery by mutating the mouse Acot7 E39D and T198N residues thereby generating a hyperactive Acot7 expressing two active thioesterase domains. This hyperactive Acot7 (Acot7HA) was cloned downstream of a floxed red fluorescent protein, mCherry, in a manner that allowed Cre recombinase-dependent excision of the mCherry to cause a shift in the translational reading frame allowing expression of Acot7HA (Fig. 7A). The Acot7HA vector was used to generate transgenic mice which were subsequently crossed with albumin-driven Cre expressing mice to yield liver-specific Acot7HA expressing mice (Acot7HA-Liv). We confirmed Cre-dependent Acot7-HA expression in liver resulting in robust protein expression and ~2-fold greater cytosolic long-chain acyl-CoA thioesterase activity (Fig. 7A,B). To determine how doubling cytosolic long-chain acyl-CoA thioesterase activity altered fatty acid metabolic flux, we traced radiolabeled substrates in liver slices from control and Acot7HA-Liv mice. Surprisingly, increased cytosolic thioesterase activity under these conditions did not reduce the rate of fatty acid metabolic flux into either catabolic or anabolic products (Fig. 7C-E).


Metabolic and tissue-specific regulation of acyl-CoA metabolism.

Ellis JM, Bowman CE, Wolfgang MJ - PLoS ONE (2015)

Development of a transgenic mouse model with a conditional tissue-specific and cytoplasmically targeted long-chain acyl-CoA thioesterase.(A) Transgenic construct schematic and representative western blot confirming Acot7HA-FLAG overexpression in liver. (B) Thioesterase activity for oleoyl-CoA in liver lysate from control and Acot7HA-Liv transgenic mice, n = 5–7. Overnight fasted control and Acot7HA-Liv liver slice rates of (C) 14C-oleate oxidation, (D) 14C-oleate incorporation into complex lipids, and (E) 3H-acetate incorporation into lipids, n = 5–7. Data represent mean ± SEM, * represent p≤0.05 by Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356623&req=5

pone.0116587.g007: Development of a transgenic mouse model with a conditional tissue-specific and cytoplasmically targeted long-chain acyl-CoA thioesterase.(A) Transgenic construct schematic and representative western blot confirming Acot7HA-FLAG overexpression in liver. (B) Thioesterase activity for oleoyl-CoA in liver lysate from control and Acot7HA-Liv transgenic mice, n = 5–7. Overnight fasted control and Acot7HA-Liv liver slice rates of (C) 14C-oleate oxidation, (D) 14C-oleate incorporation into complex lipids, and (E) 3H-acetate incorporation into lipids, n = 5–7. Data represent mean ± SEM, * represent p≤0.05 by Student’s t-test.
Mentions: To further understand the functional consequence and tissue-specific role of cytosolic thioesterase-induced fatty acid deactivation, we designed a transgenic mouse model to conditionally over-express an active and cytoplasmic long-chain acyl-CoA thioesterase. We chose Acot7 as a model thioesterase because its enzymatic and regulatory properties have been previously characterized including a crystal structure [4,5,29–31]. Acot7 is a typical hotdog fold type II thioesterase that encodes two thioesterase domains per polypeptide, one of which is inactive yet binds substrate resulting in a “half-of-sites inhibition” regulation of the enzyme [29]. Engineering the inactive site to an active thioesterase doubles the enzyme activity [29]. We capitalized on this discovery by mutating the mouse Acot7 E39D and T198N residues thereby generating a hyperactive Acot7 expressing two active thioesterase domains. This hyperactive Acot7 (Acot7HA) was cloned downstream of a floxed red fluorescent protein, mCherry, in a manner that allowed Cre recombinase-dependent excision of the mCherry to cause a shift in the translational reading frame allowing expression of Acot7HA (Fig. 7A). The Acot7HA vector was used to generate transgenic mice which were subsequently crossed with albumin-driven Cre expressing mice to yield liver-specific Acot7HA expressing mice (Acot7HA-Liv). We confirmed Cre-dependent Acot7-HA expression in liver resulting in robust protein expression and ~2-fold greater cytosolic long-chain acyl-CoA thioesterase activity (Fig. 7A,B). To determine how doubling cytosolic long-chain acyl-CoA thioesterase activity altered fatty acid metabolic flux, we traced radiolabeled substrates in liver slices from control and Acot7HA-Liv mice. Surprisingly, increased cytosolic thioesterase activity under these conditions did not reduce the rate of fatty acid metabolic flux into either catabolic or anabolic products (Fig. 7C-E).

Bottom Line: We find patterns of coordinated regulation within and between these gene families as well as distinct regulation occurring in a tissue- and physiologically-dependent manner.Due to observed changes in long-chain ACOT mRNA and protein abundance in liver and adipose tissue, we determined the consequence of increasing cytosolic long-chain thioesterase activity on fatty acid metabolism in these tissues by generating transgenic mice overexpressing a hyperactive mutant of Acot7 in the liver or adipose tissue.Together, these data suggest distinct modes of regulation of the ACS and ACOT enzymes and that these enzymes act in a coordinated fashion to control fatty acid metabolism in a tissue-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Center for Metabolism and Obesity Research, Baltimore, Maryland 21205 United States of America.

ABSTRACT
Acyl-CoA formation initiates cellular fatty acid metabolism. Acyl-CoAs are generated by the ligation of a fatty acid to Coenzyme A mediated by a large family of acyl-CoA synthetases (ACS). Conversely, acyl-CoAs can be hydrolyzed by a family of acyl-CoA thioesterases (ACOT). Here, we have determined the transcriptional regulation of all ACS and ACOT enzymes across tissues and in response to metabolic perturbations. We find patterns of coordinated regulation within and between these gene families as well as distinct regulation occurring in a tissue- and physiologically-dependent manner. Due to observed changes in long-chain ACOT mRNA and protein abundance in liver and adipose tissue, we determined the consequence of increasing cytosolic long-chain thioesterase activity on fatty acid metabolism in these tissues by generating transgenic mice overexpressing a hyperactive mutant of Acot7 in the liver or adipose tissue. Doubling cytosolic acyl-CoA thioesterase activity failed to protect mice from diet-induced obesity, fatty liver or insulin resistance, however, overexpression of Acot7 in adipocytes rendered mice cold intolerant. Together, these data suggest distinct modes of regulation of the ACS and ACOT enzymes and that these enzymes act in a coordinated fashion to control fatty acid metabolism in a tissue-dependent manner.

Show MeSH
Related in: MedlinePlus