Molecular Regulation Of The Cardiacenriched Acetyl-Coa Carboxylase Isoform (Accp): A Novel Target For Therapeutic Interventions In Cardiovascular Disease

Abstract

Metabolic remodeling is thought to be an important contributor towards the

development of various cardiac pathophysiologic conditions. Therefore, studies

attempting to delineate undenying mechanisms driving cardiac metabolic

remodeling represent an important initiative toward the development of novel

therapeutic interventions. To further investigate the role of metabolic substrate

switches in the heart, we focused on a pivotal, rate-limiting step of cardiac fatty

acid metabolism i.e. an upstream modulator of long-chain fatty acid importation

into the mitochondrion. In the heart, long-chain fatty acids are transported into

the mitochondrion by the rate-limiting enzyme, carnitine palmitoyl transferase 1

(CPT1). CPT1 is potently inhibited by malonyl-CoA, the product of the acetylCoA

carboxylation reaction that is catalyzed by acetyl-CoA carboxylase (ACC).

Recent studies have demonstrated that metabolic fuels such as fatty acids and

glucose can function as signaling ligands, directing transcriptional regulation of

numerous metabolic genes. However, transcriptional mechanisms directing the

gene expression of the cardiac isoform of acetyl-CoA carboxylase (ACC[3) are

less well understood. Previously, four E-box (CANNTG) sequence motifs were

identified on the human ACC[3 promoter. Since E-boxes act as binding sites for

upstream stimulatory factors (US F), putative glucose-responsive transcriptional

modulators, we hypothesized that ACC[3 is induced by USF1 in a glucosedependent

manner.

To investigate this, we began by acutely fasting and subsequently refeeding

Balb/C mice with a carbohydrate-enriched diet. Here, high carbohydrate feeding

resulted in elevated systemic glucose levels associated with increased cardiac

ACC[3 gene and protein expression. To further explore these interesting findings,

we tranSiently cotransfected neonatal card iom yocytes , H9C2 myoblasts, CV-1

fibroblasts and HepG2 hepatocytes with the full-length and deletion constructs of

the human ACC[3 gene promoter together with a putative activator and repressor expression vector, respectively: a) USF1 (glucose-responsive transcription

factor) - the rationale that it should elevate ACCI3 gene promoter activity in

accordance with the glucose-fatty acid cycle, and b) nuclear respiratory factor 1

(NRF1) - the hypothesis being that this mitochondrial biogenesis and l3-oxidation

enhancing modulator would be expected to attenuate ACCI3 promoter activity in

order to increase fatty acid oxidation capacity.

To assess whether USF1 plays a role in ACCI3 gene induction in response to

increased glucose supply, we performed cotransfection stUdies together with a

USF1 expression vector under low or high glucose exposure. We found that

USF1 overexpression markedly elevates ACCI3 promoter activity in neonatal

cardiomyocytes and CV-1 'fibroblasts under low glucose culturing conditions.

Moreover, high glucose levels Significantly increased USF1-mediated ACCI3

promoter activation compared to the low glucose exposure in CV-1 fibroblasts.

These data suggest that glucose-responsive USF1 transactivation of the human

ACCI3 gene promoter occurs in a cell-type specific manner i.e. unlike the CV-1

fibroblasts the USF1 response in neonatal cardiac myocytes appears to be

glucose-independent. We next performed transfection assays in the presence of

2-deoxyglucose, a glucose analog that is taken up into the cell and not further

metabolized. In agreement with our earlier findings, 2-deoxyglucose did not

inhibit the USF1-mediated transactivation of the human ACCI3 gene promoter

activity in neonatal cardiomyocytes. In contrast, 2-deoxyglucose administration

robustly inhibited USF1-mediated human ACCI3 promoter activity in CV-1

fibroblasts. We next tested whether phosphatase 2A (PP2A), a downstream

target of the pentose phosphate metabolite xylulose-5-phosphate. plays a role in

the USF1-mediated transcriptional activation of human ACCI3 promoter. Here,

PP2A inhibition attenuated USF1-mediated transactivation of the human ACCI3

gene promoter in CV-1 fibroblasts. Surprisingly, PP2A inhibition also reduced

USF1-mediated transactivation of the human ACCI3 gene promoter in neonatal

cardiomyocytes. Furthermore. endogenous USF1 transcriptional activity was

also reduced following exposure to okadaic add.Since four E-box sequence elements were previously identified on the human ACCI) gene promoter, we next performed cotransfection studies with a USF1 overexpression vector to determine sequence elements responsible for the USF1-mediated transactivation of the ACCI) gene promoter. Employing deletion

constructs, we found that the shortest ACCI) promoter deletion construct was

able to induce ACCI) promoter activity in both cell lines. However, the induction

was reduced compared to the full-length construct indicating that the observed

USF1 induction may be mediated via E-box 4 interacting with other upstream

regions of the promoter predominantly through E-box 4 located close to the

transcription start site. In summary, these data show that glucose-mediated

USF1 induction of the human ACCI) gene promoter occurs in a cell-specific

manner. In CV-1 fibroblasts, USF1 transactivates the human ACCI) promoter in

a glucose-dependent manner, in part via dephosphorylation of USF1 by PP2A.

On the other hand, USF1-mediated induction of ACCI) also occurs through

dephosphorylation of USF1 by PP2A, although unlike CV-1 fibroblasts, glucose

metabolites do not appear to mediate this process.

We also found that NRF1 overexpression markedly attenuated ACCI) promoter

activity in neonatal cardiomyocytes and cardiac-derived H9C2 myoblasts.

However, NRF1 overexpression induced ACCI) promoter activity in both CV-1

fibroblasts and HepG2 hepatocytes. Interestingly. we found that USF1-mediated

induction of ACCI) promoter activity was markedly attenuated in all cell lines

tested. We therefore propose that NRF1 may interfere with USF1 binding of the

ACCI) gene promoter binding, probably via E-box4 located close to the

transcription start site. In summary, we suggest that under energy sparing

conditions, the transcriptional modulator NRF1 not only induces genes required

for mitochondrial biogenesis, but can also negatively regulate ACCI) gene

expression. This in turn should result in reduced malonyl-CoA levels and

elevated mitochondrial fatty acid oxidation In conclusion, we have identified a novel transactivator (USF1) and repressor (NRF1) of the human ACCI3 gene promoter in the heart. Our data may eventually result in the development of novel therapeutic interventions since we

have identified unique modulators regulating ACCI3 gene transcription, and by implication malonyl-CoA levels and mitochondrial fatty acid l3-oxidation.