Tetsuya Fukui

Expression of acetoacetyl-CoA synthetase, a novel cytosolic ketone body-utilizing enzyme, in human brain

Acetoacetyl-CoA synthetase (AACS, acetoacetate-CoA ligase, EC 6.2.1.16) is a ketone body-utilizing enzyme, the physiological role of which remains unclear yet in mammals, particularly has never been studied in human. In order to investigate the tissue distribution of AACS in human, cDNA encoding AACS was isolated from HepG2 cells. Amino acid sequence of human AACS deduced from the open reading frame showed high homology (89.3%) with that of rat AACS and much less homology (43.7%) with that of bacterial AACS. The expression level of the AACS mRNA was high in kidney, heart and brain, but low in liver, and the expression profile of AACS in the human brain was quite similar to that of 3-hydroxy-3-methylglutaryl-CoA reductase.

Transcriptional regulation of ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, by C/EBPα during adipocyte differentiation

Acetoacetyl-CoA synthetase (AACS), an essential enzyme for the synthesis of fatty acid and cholesterol from ketone bodies, was found to be highly expressed in mouse adipose tissue, and GC box and C/EBPs motif were crucial for AACS promoter activity in 3T3-L1 adipocytes. Moreover, we found that AACS promoter activity was controlled mainly by C/EBPalpha during adipogenesis.

Acetoacetyl-CoA synthetase, a ketone body-utilizing enzyme, is controlled by SREBP-2 and affects serum cholesterol levels.

Ketone bodies have been regarded as an energy source that is mainly produced in the liver, and exported to extrahepatic tissues. However, ketone bodies have also been suggested to be used during the lipogenesis by the ketone body-utilizing enzyme, acetoacetyl-CoA synthetase (AACS). To elucidate the physiological role of AACS in the liver, we investigated the mechanism of transcription of the AACS gene and performed knockdown experiments. We showed that SREBP-2 regulates the expression of AACS and that knockdown of AACS in vivo, by the hydrodynamics method, resulted in the reduction of total blood cholesterol. These results suggest that ketone body metabolism via AACS activity plays an important role in cholesterol homeostasis.

Enhancement of antioxidant activity of p-alkylaminophenols by alkyl chain elongation.

The initial finding that the p-methylaminophenol (6) exhibited antioxidant activity led us to investigate whether the length of alkyl chains linked to the aminophenol residue might affect antioxidative activity. Therefore, we synthesized p-butylaminophenol (5), p-hexylaminophenol (4), p-octylaminophenol (3), and p-methoxybenzylaminophenol (7). All p-alkylaminophenols quenched alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radicals, with 7 being the most potent DPPH radical scavenger. Lipid peroxidation by rat liver microsomes was reduced by p-alkylaminophenols in dose- and aminophenol alkyl chain length-dependent fashion (3>4>5>6), with 3 being the most potent lipid peroxidation inhibitor, at approximately 350-fold higher potency than 6. These results indicate that elongation of alkyl chains in p-alkylaminophenols may increase antioxidative activity, and that p-alkylaminophenols may potentially be useful in the development of antioxidants.

Combination of RET siRNA and irinotecan inhibited the growth of medullary thyroid carcinoma TT cells and xenografts via apoptosis

Medullary thyroid carcinoma (MTC) is a rare endocrine tumor that frequently metastasizes, and treatment with irinotecan (CPT‐11) is limited because of side effects. Mutations in the Rearranged during transfection (RET) proto‐oncogene are considered the causative event of MTC. The objective of this study was to examine whether small interfering RNA (siRNA) and its combined treatment with CPT‐11 could inhibit MTC cell growth in vitro and in vivo. The transfection of RET siRNA suppressed RET expression, reduced proliferation, and increased caspase‐3/7 activity via the down‐regulation of Bcl‐2 expression. Combined treatments with CPT‐11 or SN‐38 significantly increased caspase 3/7 activity compared with RET siRNA, CPT‐11 or SN‐38 treatment alone. Importantly, intratumoral injection of RET siRNA along with intravenous injection of CPT‐11 significantly inhibited the tumor growth of MTC xenografts via an increased apoptotic effect. These findings that RET siRNA enhanced sensitivity for CPT‐11 will provide a novel strategy for the treatment of MTC with RET mutation.

Effects of streptozotocin-induced diabetes on acetoacetyl-CoA synthetase activity in rats

In order to investigate the physiological role of acetoacetyl-CoA synthetase (acetoacetate-CoA ligase, EC 6.2.1.16), a cytosolic acetoacetate-activating enzyme, effects of streptozotocin (STZ)-induced diabetes on the enzyme activity was investigated in rats. At 72 hr of the STZ administration (80 mg/kg body weight, injected intravenously), hepatic enzyme specific activity decreased to 23% of its initial activity. However, the enzyme activities in non-hepatic tissues were not significantly affected by the STZ treatment. Feeding of rats with both 4% cholestyramine and 0.4% pravastatin for 3 days remarkably increased the hepatic acetoacetyl-CoA synthetase activity and decreased the plasma ketone bodies level in the diabetic rats. These results suggest that acetoacetyl-CoA synthetase has important roles in the regulation of ketone body utilization in rat liver and that these hypocholesterolemic agents have the ability to remedy the impaired utilization of ketone bodies under the diabetic condition.

Acetoacetyl-CoA synthetase is essential for normal neuronal development

Cholesterol and fatty acids are essential, abundant components of neuronal tissue. Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme for the synthesis of cholesterol and fatty acids and is highly expressed in the brain. In this study, we investigated the regulation of AACS during neurite outgrowth to clarify the physiological role of AACS in neurogenesis. Messenger RNA levels and the expression of AACS were increased during neurite outgrowth in Neuro-2a cells. The expression of HMG-CoA reductase, a key enzyme of cholesterol biosynthesis, was also increased. ChIP assays showed that the amount of SREBP-2, a key transcription factor of cholesterol synthesis, interacted with the AACS promoter was increased during neurite outgrowth, and knockdown of SREBP-2 down-regulated the mRNA levels of AACS in Neuro-2a cells. The expression of AACS in the brains of mouse embryos was dramatically increased between E16.5 and E18.5. Moreover, knockdown of AACS in primary neurons caused decreases in the expression of MAP-2 and NeuN, which are markers of neuronal differentiation, as well as synaptopodin, a marker of spine apparatus. These results suggest that AACS is regulated by SREBP-2 and involves in the normal development of neurons.

cDNA-derived amino acid sequence of acetoacetyl-CoA synthetase from rat liver1

In order to examine the primary structure of acetoacetyl‐CoA synthetase (acetoacetate‐CoA ligase, EC 6.2.1.16; AA‐CoA synthetase), the cDNA clone encoding this enzyme has been isolated from the cDNA library which was prepared from the liver of rat fed a diet supplemented with 4% cholestyramine and 0.4% pravastatin for 4 days. Nucleotide sequence analysis of cloned cDNA revealed that AA‐CoA synthetase of rat liver contains an open reading frame of 2019 nucleotides, and the deduced amino acid sequence (672 amino acid residues) bears 25.0 and 38.9% homologies with acetyl‐CoA synthetases of Saccharomyces cerevisiae and Archaeoglobus fulgidus, respectively.

Genetic Obesity Affects Neural Ketone Body Utilization in the Rat Brain

Obesity causes various physiological disorders between the central nervous system and peripheral tissues. Ketone bodies have a neuro‐protective role and are strongly affected by obesity‐related metabolic disorders. To clarify the effects of obesity on ketone body utilization in brain, we examined the mRNA localization of acetoacetyl‐CoA synthetase (AACS), which activates ketone bodies for the synthesis of fatty acid and cholesterol, in various brain regions of Zucker fatty rats by in situ hybridization. The AACS mRNA level was increased in the paraventricular thalamic nucleus (PVT) but not affected in the cerebrum and hippocampus in Zucker fatty rats. In contrast, the AACS mRNA level was reduced in the arcuate hypothalamic nucleus (Arc) and ventromedial hypothalamic nucleus (VMH) in the hypothalamus. Succinyl‐CoA:3‐oxoacid CoA‐transferase (SCOT) mRNA level was decreased only in the PVT but not affected in the Arc and VMH. These data raise the possibility that AACS is regulated by the leptin signaling pathway in the hypothalamus but not in the PVT. As AACS was expressed in neural‐like cells, ketone bodies are assumed to be utilized for the synthesis of lipidic substances and to cause metabolic disorders in the nervous system.

High-fat diet-induced obesity stimulates ketone body utilization in osteoclasts of the mouse bone.

Previous studies have shown that high-fat diet (HFD)-induced obesity increases the acetoacetyl-CoA synthetase (AACS) gene expression in lipogenic tissue. To investigate the effect of obesity on the AACS gene in other tissues, we examined the alteration of AACS mRNA levels in HFD-fed mice. In situ hybridization revealed that AACS was observed in several regions of the embryo, including the backbone region (especially in the somite), and in the epiphysis of the adult femur. AACS mRNA expression in the adult femur was higher in HFD-fed mice than in normal-diet fed mice, but this increase was not observed in high sucrose diet (HSD)-induced obese mice. In addition, HFD-specific increases were observed in the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and interleukin (IL)-6 genes. Moreover, we detected higher AACS mRNA expression in the differentiated osteoclast cells (RAW 264), and found that AACS mRNA expression was significantly up-regulated by IL-6 treatment only in osteoclasts. These results indicate the novel function of the ketone body in bone metabolism. Because the abnormal activation of osteoclasts by IL-6 induces bone resorption, our data suggest that AACS and ketone bodies are important factors in the relationship between obesity and osteoporosis.