Acot9: A Novel Target for NAFLD by Shuttling Mitochondrial Short-Chain Fatty Acids?

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease globally. It can progress to the more severe nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). The liver is a major site for lipid metabolism, a process that is strictly regulated by a series of steps, including fatty acid uptake; de novo lipogenesis (DNL); triglyceride (TG) synthesis, esterification, and storage; and fatty acid β-oxidation and very low-density lipoprotein (VLDL) secretion. An imbalance between the lipid input (intake and biosynthesis of TGs) and output (export and catabolism of TGs) can result in NAFLD and further progress to NASH.⁽¹⁾

Free fatty acids (FFAs) play an important role in the development of NAFLD. FFAs are converted to fatty acid-coenzyme A, or acyl CoA, which regulates protein acylation, synthesis of signaling lipids and membrane phospholipids, and energy production and storage. Acyl CoA synthetase (Acs) regulates the formation of acyl CoA by ligating an FFA to CoA. Conversely, acyl CoA is hydrolyzed and deactivated into FFAs and coenzyme A (CoASH) by members of the acyl CoA thioesterase (Acot) gene family.⁽²⁾ Acots belong to a group of thioester hydrolases, which cleave the thioester bond between a sulfur group and a carboxyl group. Acots are divided into two structurally distinct types: type I (Acots 1-6), which have a molecular mass of approximately 40 kDa and contain an α/β-hydrolase domain near the C-terminus, and type II (Acots 7-13), which are approximately 110 kDa and share a distinct “hotdog” domain.⁽³⁾ Despite these structural differences, both type I and type II enzymes catalyze the same reactions, indicating that they are analogous but not homologous. Both types of Acots have distinct subcellular localization and substrate specificities as well as tissue expression patterns.^{(2, 4)} Acot1 is cytosolic, whereas Acot2 is located in the mitochondria. Both are primarily responsible for long-chain acyl CoA metabolism. Mouse Acots 3-6 and human Acot4 are located at peroxisomes and exhibit substrate specificities for long- and medium-chain acyl CoA as well as short-chain dicarboxylic acyl CoA esters. Acot9 is reported to hydrolyze a number of long- and short-chain acyl CoA as well as short-chain methyl-branched CoA esters, suggesting Acot9 regulates both mitochondrial lipid and amino acid metabolism.⁽⁵⁾ Acot11, also known as thioesterase superfamily member 1 (Them1), is enriched in brown adipose tissue (BAT) and induced by high-fat diet (HFD) in the mouse liver.⁽⁶⁾ Acot13 (Them2) is highly expressed in metabolic tissues, including adipose, muscle, and liver, and is localized to mitochondria.⁽⁷⁾ Both Acot11 and Acot13 prefer to hydrolyze long-chain acyl CoA and play critical roles in hepatic steatosis and glucose metabolism.^{(6, 7)} Increasing evidence suggests that Acots are critical regulators of lipid and glucose metabolism, which may contribute to the pathogenesis of NAFLD.

To better characterize the metabolic functions of Acots in NAFLD, Steensels et al. used an elegant unbiased RNA interference (RNAi) screening of various Acot genes for lipid accumulation in Caenorhabditis elegans.⁽⁸⁾ They found that knockdown of Acot9 led to the highest level of lipid accumulation. Intriguingly, they found that levels of Acot9 increased in the livers of mice and obese humans with NAFLD, suggesting a maladaptive response of Acot9 in NAFLD. Following HFD feeding, global Acot9 knockout (KO) mice had decreased body weight gain, fat mass, and hepatic steatosis and glucose production compared with wild-type (WT) mice. Global deletion of Acot9 did not affect glucose tolerance or insulin sensitivity as well as plasma levels of TG, non-esterified fatty acids, cholesterol, and phospholipids. Similar trends were observed in liver-specific Acot9 (L-Acot9) KO mice using AAV- thyroid hormone-binding globulin (TBG)-Cre except that L-Acot9 KO mice had the same body weight gain as the matched WT mice. These results indicate that hepatic Acot9 primarily regulates lipid and glucose metabolism. Indeed, when Acot9 was re-expressed in the global Acot9 KO mice, protection against HFD-induced steatosis and glucose production was abolished. Because Acot9 is highly expressed in many tissues, including brown and white adipose, kidney, lung, and brain, future work is needed to further dissect the role of Acot9 in these tissues and its contribution to body weight gain. The authors found increased energy expenditure (EE) in global Acot9 KO mice, which further supports the notion that Acot9 in other tissues may contribute to body weight gain.

How does loss of hepatic Acot9 protect against steatosis and glucose production? The authors found no difference in hepatic FFA uptake, β-oxidation, and VLDL secretion between WT and Acot9 KO mice. In order to elucidate additional mechanisms of how Acot9 regulates hepatic lipid and glucose metabolism, the authors performed a cellular fractionation assay along with detergent digestion. They found that Acot9 localized specifically to the mitochondrial inner membrane. Using a thioesterase activity assay, they confirmed that Acot9 catalyzes the short- but not long-chain acyl CoA within the mitochondrial inner membrane. It should be noted that a previous study reported that Acot9 can simultaneously hydrolyze both short- and long-chain acyl CoA.⁽⁵⁾ One explanation for this inconsistency may be that the different tissue mitochondrial Acot9 was assessed in these two studies (liver vs. BAT and kidney tissue). It is also likely that other unidentified cofactors in different tissues may alter the substrate specificities of Acot9.

Mitochondria are critical for fatty acid β-oxidation, the citric acid or tricarboxylic acid (TCA) cycle, and ketogenesis, which are important in supporting lipogenesis and gluconeogenesis. When the TCA cycle runs, intermediates from the cycle are transported from mitochondria into the cytosol where they serve as raw materials for lipid and glycogen synthesis. Steensels et al.⁽⁸⁾ performed metabolomic analysis and found that loss of hepatic Acot9 decreased TCA cycle–prominent metabolites, such as citrate, cis-aconitate, and 2-oxoglutarate, and also decreased amino acids that are interchangeable with the TCA cycle. Subsequent results from primary hepatocytes and mouse liver DNL experiments revealed that Acot9 promoted the bioavailability of acetyl CoA for DNL in the setting of overnutrition. Therefore, increased hepatic Acot9 in obese NALFD patients may represent a maladaptation that provides mitochondrial TCA metabolites from the short-chain acyl CoA as substrates for DNL and gluconeogenesis.

Steatosis is a common early feature of NAFLD that can progress to more severe NASH. Steensels et al.⁽⁸⁾ next investigated the role of Acot9 in diet-induced NASH. They found that although Acot9 KO mice had decreased hepatic steatosis, inflammation, and fibrosis, the histological NASH score did not improve between WT and Acot9 KO mice. Moreover, the levels of hepatic Acot9 did not differ between obese NAFLD and NASH patients. The authors concluded that Acot9 may be important for development of early steatosis but not for progression to NASH; more studies are needed to further examine the role of Acot9 in the progression of NAFLD to NASH. Using more rigorous NASH mouse models, such as extended feeding with NASH diets, or using NAFLD-associated HCC models may help provide more insights for Acot9 in NASH- and NAFLD-associated HCC.

In summary, the work of Steensels et al.⁽⁸⁾ broadens our understanding of the role of Acot9 in regulating lipid and glucose metabolism under conditions of overnutrition (FIG. 1). Loss of Acot9 decreases hepatic glucose production (HGP) and steatosis, which may also halt the progression of NAFLD to NASH. Therefore, Acot9 may be a potential therapeutic target for NAFLD. Despite this, several crucial questions still remain. How do HFD and obesity induce hepatic Acot9? Would pharmacological inhibition of Acot9 improve NAFLD? It is imperative to identify small molecule inhibitors for Acot9 and other Acot family proteins for potential treatment of NAFLD. Acot9 primarily hydrolyzes short-chain acyl CoA, whereas other Acots favor long-chain acyl CoA (such as Them1 and Them2), and therefore it is likely that simultaneous inhibition of several Acots may offer additive beneficial effects for treating NALFD.

FIG. 1.

Proposed cellular events in Acot9-induced DNL and HGP. Inside hepatocytes, Acs promotes FFA activation by esterification to CoA to form acyl CoA, which is transported into mitochondria by CPT1. Short-chain acyl CoA is deactivated by Acot9 to generate short-chain FFA and acetyl CoA, which increases TCA cycle flux to promote HGP and DNL, resulting in the development of NAFLD/NASH. Abbreviation: CPT1, carnitine palmitoyltransferase 1.

Abbreviations:

Acot

acyl coenzyme A thioesterase

Acs

acyl coenzyme A synthetase

DNL

de novo lipogenesis

HCC

hepatocellular carcinoma

HFD

high fat diet

HGP

hepatic glucose production

NAFLD

Non-alcoholic liver disease

NASH

Nonalcoholic Steatohepatitis

TCA

tricarboxylic acid

VLDL

very-low-density lipoprotein

WT

wildtype