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ACC2 is expressed at high levels in human white adipose and has an isoform with a novel N-terminus [corrected].

Castle JC, Hara Y, Raymond CK, Garrett-Engele P, Ohwaki K, Kan Z, Kusunoki J, Johnson JM - PLoS ONE (2009)

Bottom Line: We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart.Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis.The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Rosetta Inpharmatics LLC, Seattle, Washington, United States of America. john_castle@merck.com

ABSTRACT
Acetyl-CoA carboxylases ACC1 and ACC2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA, regulating fatty-acid synthesis and oxidation, and are potential targets for treatment of metabolic syndrome. Expression of ACC1 in rodent lipogenic tissues and ACC2 in rodent oxidative tissues, coupled with the predicted localization of ACC2 to the mitochondrial membrane, have suggested separate functional roles for ACC1 in lipogenesis and ACC2 in fatty acid oxidation. We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart. Human adipose, along with human liver, expresses more ACC2 than ACC1. Using RT-PCR, real-time PCR, and immunoprecipitation we report a novel isoform of ACC2 (ACC2.v2) that is expressed at significant levels in human adipose. The protein generated by this isoform has enzymatic activity, is endogenously expressed in adipose, and lacks the N-terminal sequence. Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis. The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.

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Expression and activities of ACC2 proteins.(A) Expression of recombinant ACC2.v1 and ACC2.v2 proteins in FM3A cells. Recombinant ACC proteins in cell lysates from FM3A cells overexpressing ACC isoforms were detected by Western blotting. Arrows mark the locations of ACC2.V1, ACC1, and ACC2.v2, from top to bottom. (B) ACC activities of recombinant ACC2.v1 and ACC2.v2 protein. ACC activities in FM3A cell lysates were measured by an in vitro 14CO2 fixation assay, measuring the incorporation of [14C]bicarbonate into [14C]malonyl-CoA. (C) DNL activity in FM3A cells overexpressing recombinant ACCs. DNL activities were measured as incorporated radio activities of [14C]acetate into cells in the presence of Simvastatin, a HMG-CoA reductase inhibitor. The non-specific[14C]acetate incorporation was determined in the presence of 10 µM a specific dual ACC1 and ACC2 inhibitor, labeled ‘compound A,’ and subtracted from total DNL activity in each FM3A clone.
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pone-0004369-g005: Expression and activities of ACC2 proteins.(A) Expression of recombinant ACC2.v1 and ACC2.v2 proteins in FM3A cells. Recombinant ACC proteins in cell lysates from FM3A cells overexpressing ACC isoforms were detected by Western blotting. Arrows mark the locations of ACC2.V1, ACC1, and ACC2.v2, from top to bottom. (B) ACC activities of recombinant ACC2.v1 and ACC2.v2 protein. ACC activities in FM3A cell lysates were measured by an in vitro 14CO2 fixation assay, measuring the incorporation of [14C]bicarbonate into [14C]malonyl-CoA. (C) DNL activity in FM3A cells overexpressing recombinant ACCs. DNL activities were measured as incorporated radio activities of [14C]acetate into cells in the presence of Simvastatin, a HMG-CoA reductase inhibitor. The non-specific[14C]acetate incorporation was determined in the presence of 10 µM a specific dual ACC1 and ACC2 inhibitor, labeled ‘compound A,’ and subtracted from total DNL activity in each FM3A clone.

Mentions: To confirm the ACC2.v2 transcript isoform generates protein, we cloned ACC2.v2 and ACC2.v1 and overexpressed them in FM3A cells, a mammary carcinoma-derived cell line. Recombinant ACC2.v2 and ACC2.v1 were detected at expected molecular weights of ∼255 and ∼277 kDa, respectively and biotinylated in Western blotting (Figure 5A). We investigated ACC enzymological properties using the in vitro 14CO2 fixation assay using lysate from FM3A cells. Overexpression of either ACC2.v2 or ACC2.v1 increases ACC enzymatic activity >20-fold relative to vector alone (Figure 5B). Using purified recombinant human ACC1, ACC2.v1 and ACC2.v2, we found Km values for acetyl-CoA, CO2 and ATP among ACC subtypes and variants, with Km values for acetyl-CoA between 51 and 110 µM; for ATP between 44 and 110 µM; and for NaHCO3 between 2.9 and 6.5 mM (Table 1). Relative to ACC2.v1, ACC2.v2 showed 2 to 3-fold increases in Km values for ACC2.v2 with respect to ATP and NaHCO3. Finally, we assayed de novo lipogenesis (DNL) activity in FM3A cells overexpressing recombinant ACC clones using [14C]acetate incorporation as a marker for DNL (Figure 5C). After overexpression, incorporation was first measured in the presence of a non-specific ACC inhibitor, labeled ‘compound A,’ as a control, and then without inhibitor. Overexpression of ACC1 increased cellular DNL activity almost 4-fold relative to vector alone. Cells expressing either ACC2.v2 or ACC2.v1 showed an approximately 2-fold relative increase in DNL.


ACC2 is expressed at high levels in human white adipose and has an isoform with a novel N-terminus [corrected].

Castle JC, Hara Y, Raymond CK, Garrett-Engele P, Ohwaki K, Kan Z, Kusunoki J, Johnson JM - PLoS ONE (2009)

Expression and activities of ACC2 proteins.(A) Expression of recombinant ACC2.v1 and ACC2.v2 proteins in FM3A cells. Recombinant ACC proteins in cell lysates from FM3A cells overexpressing ACC isoforms were detected by Western blotting. Arrows mark the locations of ACC2.V1, ACC1, and ACC2.v2, from top to bottom. (B) ACC activities of recombinant ACC2.v1 and ACC2.v2 protein. ACC activities in FM3A cell lysates were measured by an in vitro 14CO2 fixation assay, measuring the incorporation of [14C]bicarbonate into [14C]malonyl-CoA. (C) DNL activity in FM3A cells overexpressing recombinant ACCs. DNL activities were measured as incorporated radio activities of [14C]acetate into cells in the presence of Simvastatin, a HMG-CoA reductase inhibitor. The non-specific[14C]acetate incorporation was determined in the presence of 10 µM a specific dual ACC1 and ACC2 inhibitor, labeled ‘compound A,’ and subtracted from total DNL activity in each FM3A clone.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2629817&req=5

pone-0004369-g005: Expression and activities of ACC2 proteins.(A) Expression of recombinant ACC2.v1 and ACC2.v2 proteins in FM3A cells. Recombinant ACC proteins in cell lysates from FM3A cells overexpressing ACC isoforms were detected by Western blotting. Arrows mark the locations of ACC2.V1, ACC1, and ACC2.v2, from top to bottom. (B) ACC activities of recombinant ACC2.v1 and ACC2.v2 protein. ACC activities in FM3A cell lysates were measured by an in vitro 14CO2 fixation assay, measuring the incorporation of [14C]bicarbonate into [14C]malonyl-CoA. (C) DNL activity in FM3A cells overexpressing recombinant ACCs. DNL activities were measured as incorporated radio activities of [14C]acetate into cells in the presence of Simvastatin, a HMG-CoA reductase inhibitor. The non-specific[14C]acetate incorporation was determined in the presence of 10 µM a specific dual ACC1 and ACC2 inhibitor, labeled ‘compound A,’ and subtracted from total DNL activity in each FM3A clone.
Mentions: To confirm the ACC2.v2 transcript isoform generates protein, we cloned ACC2.v2 and ACC2.v1 and overexpressed them in FM3A cells, a mammary carcinoma-derived cell line. Recombinant ACC2.v2 and ACC2.v1 were detected at expected molecular weights of ∼255 and ∼277 kDa, respectively and biotinylated in Western blotting (Figure 5A). We investigated ACC enzymological properties using the in vitro 14CO2 fixation assay using lysate from FM3A cells. Overexpression of either ACC2.v2 or ACC2.v1 increases ACC enzymatic activity >20-fold relative to vector alone (Figure 5B). Using purified recombinant human ACC1, ACC2.v1 and ACC2.v2, we found Km values for acetyl-CoA, CO2 and ATP among ACC subtypes and variants, with Km values for acetyl-CoA between 51 and 110 µM; for ATP between 44 and 110 µM; and for NaHCO3 between 2.9 and 6.5 mM (Table 1). Relative to ACC2.v1, ACC2.v2 showed 2 to 3-fold increases in Km values for ACC2.v2 with respect to ATP and NaHCO3. Finally, we assayed de novo lipogenesis (DNL) activity in FM3A cells overexpressing recombinant ACC clones using [14C]acetate incorporation as a marker for DNL (Figure 5C). After overexpression, incorporation was first measured in the presence of a non-specific ACC inhibitor, labeled ‘compound A,’ as a control, and then without inhibitor. Overexpression of ACC1 increased cellular DNL activity almost 4-fold relative to vector alone. Cells expressing either ACC2.v2 or ACC2.v1 showed an approximately 2-fold relative increase in DNL.

Bottom Line: We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart.Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis.The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Rosetta Inpharmatics LLC, Seattle, Washington, United States of America. john_castle@merck.com

ABSTRACT
Acetyl-CoA carboxylases ACC1 and ACC2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA, regulating fatty-acid synthesis and oxidation, and are potential targets for treatment of metabolic syndrome. Expression of ACC1 in rodent lipogenic tissues and ACC2 in rodent oxidative tissues, coupled with the predicted localization of ACC2 to the mitochondrial membrane, have suggested separate functional roles for ACC1 in lipogenesis and ACC2 in fatty acid oxidation. We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart. Human adipose, along with human liver, expresses more ACC2 than ACC1. Using RT-PCR, real-time PCR, and immunoprecipitation we report a novel isoform of ACC2 (ACC2.v2) that is expressed at significant levels in human adipose. The protein generated by this isoform has enzymatic activity, is endogenously expressed in adipose, and lacks the N-terminal sequence. Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis. The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.

Show MeSH
Related in: MedlinePlus