Induced polymerization of mammalian acetyl-CoA carboxylase by MIG12 provides a tertiary level of regulation of fatty acid synthesis

Abstract

Acetyl-CoA carboxylase (ACC), the first committed enzyme in fatty acid (FA) synthesis, is regulated by phosphorylation/dephosphorylation, transcription, and an unusual mechanism of protein polymerization. Polymerization of ACC increases enzymatic activity and is induced in vitro by supraphysiological concentrations of citrate (>5 mM). Here, we show that MIG12, a 22 kDa cytosolic protein of previously unknown function, binds to ACC and lowers the threshold for citrate activation into the physiological range (<1 mM). In vitro, recombinant MIG12 induced polymerization of ACC (as determined by nondenaturing gels, FPLC, and electron microscopy) and increased ACC activity by >50-fold in the presence of 1 mM citrate. In vivo, overexpression of MIG12 in liver induced ACC polymerization, increased FA synthesis, and produced triglyceride accumulation and fatty liver. Thus, in addition to its regulation by phosphorylation and transcription, ACC is regulated at a tertiary level by MIG12, which facilitates ACC polymerization and enhances enzymatic activity.lipogenesis | SREBPs | steatosis F atty acids (FAs) are the essential components of phospholipids and provide the most important energy depot for the body in the form of triglycerides (TG). Newly synthesized FAs also serve as signaling molecules and modulators of transcription factor activity (1-3). Excess accumulation of FAs in tissues contributes to the pathogenesis of many common diseases, including insulin resistance, nonalcoholic fatty liver disease, diabetes, and cancer (4, 5). Given the central role of FAs in these diseases, the enzymes of FA biosynthesis are potential therapeutic targets.The first committed step in FA biosynthesis is carried out by acetyl-CoA carboxylase (ACC). ACC1 was isolated and characterized by Wakil et al. (6) and functions to carboxylate acetyl-CoA to form malonyl-CoA. Subsequently, a second ACC isoform, ACC2, was identified in mammals that is encoded by a separate gene but carries out the same enzymatic reaction (7,8).ACCs are members of a larger family of carboxylases that require biotin and ATP and use bicarbonate as a carbon donor. The malonyl-CoA produced by ACC can be used by FA synthase (FAS) for the sequential 2-carbon elongation reactions that generate palmitic acid (C16:0) in the cytosol. While both ACC1 and ACC2 produce malonyl-CoA, ACC1 is predominantly cytosolic and generates malonyl-CoA that is used by FAS to synthesize palmitic acid. ACC2 is associated with the mitochondrial membrane (9) and produces malonyl-CoA that serves to allosterically inhibit carnitine palmitoyl transferase I, the protein responsible for transport of long chain FAs into mitochondria for β-oxidation (10).Regulation of ACC1 and ACC2 occurs at multiple levels. ACC activity is acutely regulated by phosphorylation/dephosphorylation. AMP-activated protein kinase phosphorylates ACC and inhibits enzyme activity (11). In the fed state, excess glucose must be converted to FAs for energy storage in the form of TGs. Increased insulin signaling in the fed state res...

Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium

“…SP1 and SP3 have been found to bind to the promoters of Acly, Acaca, and Fasn, with SP3 stimulating transcription. MIG12 increases the activity of ACACA1 by increasing its polymerization but did not increase protein translation in the liver (19). While we readily detect the ChREBP protein by Western blot in WTLs, we found neither ChREBP protein nor its mRNA in MEC from late-pregnant and lactating mice (M. C. Rudolph and S. M. Anderson, unpublished data), suggesting that it does not regulate lipogenic genes in the lactating alveolar cell.…”

“…MIG12 is expressed only at low levels in the mammary alveolar cell of mice (P. Ramanathan and M. C. Neville, unpublished observation) and cows (15); so it does not seem to be a candidate for regulation of de novo lactogenesis. The role of THRSP is unclear, but Kim et al (19) found no evidence that it altered polymerization of ACACA1.…”

“…Factors other than SREBF that have been implicated in the transcriptional control of lipogenic genes include upstream stimulatory factors (USF) (12), the carbohydrate response element binding protein (ChREBP) (44), SP1 and SP3 (7,10), and MIG12 (19). USF and ChREBP bind to E-boxes, which are known to be present in the promoter regions of Acaca.…”

Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium

“…Conversely, Spot14-null hepatic de novo fatty acid synthesis was increased 2-fold under lipogenic stimuli, suggesting Spot14 is an inhibitor of this process in the liver ( 40 ). Coexpression of Spot14 with Spot14-R was determined to be inhibitory, as Spot14 heterodimerizes with Spot14-R to prevent Spot14-R association with ACC and decrease activation of the hepatic de novo fatty acid synthesis pathway ( 43 ( 29,41 ). In mammary cancer cells, antisense Spot14 knock down decreased in acetonitrile to initiate derivatization and incubated at room temperature for 30 min ( 50 ).…”

Molecular cloning and expression of the porcine S14R gene in Escherichia coli

“…Signal peptide analysis showed that the MID1IP1 protein is a non-secretory protein. MID1IP1 is a cytoplasmic protein, which has been confirmed in humans and mice (Berit et al, 2004;Kim et al, 2010). To further investigate the detailed structure of porcine MID1IP1, SABLE on-line analysis software was employed to determine secondary structure.…”