Molecular cloning of ratacss3and characterization of mammalian propionyl-CoA synthetase in the liver mitochondrial matrix

Abstract

Among the three acyl-CoA synthetase short-chain family members (ACSS), ACSS3 is poorly characterized. To characterize ACSS3, we performed molecular cloning and protein expression of rat acss3 and determined its intracellular localization, tissue distribution, and substrate specificity. Transient expression of rat ACSS3 in HeLa cells resulted in a 10-fold increase of acetyl-CoA synthetase activity compared with that in control cells. The acss3 transcripts are expressed in a wide range of tissues, with the highest levels observed in liver tissue followed by kidney tissue. Subcellular fractionation using liver tissue showed that ACSS3 is localized into the mitochondrial matrix. Among the short-chain fatty acids examined, recombinant ACSS3, purified from Escherichia coli cells transformed with the plasmid containing rat acss3, preferentially utilized propionate with a KM value of 0.19 mM. Knockdown of acss3 in HepG2 cells resulted in a significant decrease of ACSS3 expression level and propionyl-CoA synthetase activity in cell lysates. Levels of ACSS3 in the liver and the activity of propionyl-CoA synthetase in the mitochondria were significantly increased by fasting. These results suggested that ACSS3 is a liver mitochondrial matrix enzyme with high affinity to propionic acid, and its expression level is upregulated under ketogenic conditions.

The effects of dietary supplementation with inulin and inulin‐propionate ester on hepatic steatosis in adults with non‐alcoholic fatty liver disease

“…Previous work has also showed that chronic intragastric acetate infusion in rats promotes postprandial hyperinsulinaemia and increases liver triglyceride content . The conversion of SCFAs into metabolic intermediates is initially determined by the acyl‐CoA synthetase short‐chain family members (ACSS) . Human hepatocytes express the cytosolic isoform ACSS2 , which has high specificity for acetate and increases the availability of acetyl‐CoA for lipid synthesis .…”

“…The conversion of SCFAs into metabolic intermediates is initially determined by the acyl‐CoA synthetase short‐chain family members (ACSS) . Human hepatocytes express the cytosolic isoform ACSS2 , which has high specificity for acetate and increases the availability of acetyl‐CoA for lipid synthesis . Evidence highlights that, together with higher insulin levels, humans with NAFLD have an elevated expression of hepatic genes that favour fat accumulation, with increased expression of acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FASN), which are key enzymes in hepatic DNL .…”

“…Studies using rat hepatocytes have also highlighted that propionate inhibits lipid synthesis when acetate is a major source of acetyl‐CoA . Recent evidence has showed a third ACSS isoform, ACSS3 , for which propionate is the preferred substrate over acetate, and which is highly expressed in the mitochondrial matrix of hepatocytes . ACSS3 converts propionate to propionyl‐CoA allowing it to enter mitochondrial respiration through succinate and the TCA cycle .…”

“…16 ACSS3 converts propionate to propionyl-CoA allowing it to enter mitochondrial respiration through succinate and the TCA cycle. 16 Elevating hepatic propionate metabolism would therefore increase competition with acetate for conversion into their CoA adducts at tissue level, which may reduce cytosolic acetyl-CoA availability for DNL. This potential mechanism is supported by a recent observation that exposing HepG2 cells to elevated ratios of propionate : acetate increases the formation of heptadecanoic acid derived from propionyl-CoA, which inhibits the synthesis of palmitate from acetyl-CoA.…”

Increasing N-acetylaspartate in the Brain during Postnatal Myelination Does Not Cause the CNS Pathologies of Canavan Disease

“…The enzymes involved in converting free acetate to acetyl-CoA are known as ACSS and three forms are currently known (family members 1–3) (Yoshimura et al, 2017). These enzymes expend ATP to generate acetyl-CoA from acetate and coenzyme A. ACSS1 and ACSS3 are mitochondrial matrix enzymes involved in the oxidation of acetate and propionate respectively for energy derivation, whereas Acss2 is a nuclear-cytoplasmic enzyme involved in lipid synthesis and protein acetylation reactions.…”