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Inflammation is critical for the progression of hyperlipidemia. Although the exact mechanism through which inflammation affects hyperlipidemia is not very clear, evidence suggests that the tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT)LIGHT might regulate lipid metabolism. In this study we investigated the expression of LIGHT in patients with different stages of coronary disease. The expression of lipid metabolism-related enzymes and inflammation-related proteins were further explored in oxidized low-density lipoproteins (oxLDL)-induced THP-1 macrophages. We found that LIGHT is highly expressed and companied with severe inflammations in patients with coronary disease. LIGHT significantly enhanced inflammation response in oxLDL-induced THP-1 macrophages. We further demonstrated that LIGHT markedly decreased the levels of lipolytic genes and increased the expressions of lipogenic genes in oxLDL-induced THP-1 macrophages. In addition, our results showed that LIGHT exerts its pro-inflammatory and pro-lipogenesis roles through activating nuclear factor-kappa B (NF-κB) signaling pathway. Taken together our study has demonstrated that LIGHT NF-κB-dependently exacerbates inflammation response and promotes lipid accumulation, and provided a new potential target for treatment of hyperlipidemia-related disease.

Alcoholism is a psychiatric disorder that composes one of the principal causes of health disabilities in the world population. Furthermore, the available pharmacotherapy is limited. Therefore, this research was carried out to better understand the basis of the underlying neurobiological processes of this disorder and to discover potential therapeutic targets. Real-time PCR analysis was performed in the amygdala nuclei region of the brain of mice exposed to a chronic three-bottle free-choice model (water, 5 and 10% v/v ethanol). Based on individual ethanol intake, the mice were classified into three groups: "compulsive-like" (i.e., ethanol intake not affected by quinine adulteration), "ethanol-preferring" and "ethanol non-preferring". A fourth group had access only to tap water (control group). The candidate gene ACSS2 was genotyped in human alcoholics by real-time polymerase chain reaction using the markers rs6088638 and rs7266550. Seven genes were picked out (Acss2, Acss3, Acat1, Acsl1, Acaa2, Hadh, and Hadhb) and the mRNA level of the Acss2 gene was increased only in the "compulsive-like" group (p = 0.004). The allele frequency of rs6088638 for the gene ACSS2 was higher in the Alcoholic human group (p = 0.03), although sample size was very small. The gene ACSS2 is associated with alcoholism, suggesting that biochemical pathways where it participates may have a role in the biological mechanisms susceptible to the ethanol effects.

Beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) deficiency is an autosomal recessive disorder characterized by impaired metabolism of ketones and isoleucine. In this study, we report on the first two siblings with T2 deficiency from Libya. Both siblings presented with ketoacidosis, but the severity and outcomes were quite distinctive. T2 deficiency in patient 1, the younger sister, manifested as recurrent severe episodes of ketoacidosis during the first year of life. She unfortunately experienced neurodevelopmental complications, and died at 14 months old, after her 5th episode. In contrast, patient 2, the elder brother, experienced only one ketoacidotic episode at the age of 4 years. He recovered uneventfully and has continued to achieve age-appropriate development to date. Upon analysis, the siblings’ blood acylcarnitine profiles had shown increased levels of C5:1 and C5-OH carnitine. ACAT1 mutational analysis revealed patient 2 is homozygotic for a novel mutation–c.674C > A (p.Ala225Glu); this mutation was then confirmed by familial analysis. Transient expression analysis of c.674C > A mutant T2 cDNA revealed neither potassium ion-activated acetoacetyl-CoA thiolase activity, which represents T2 activity, nor mutant T2 protein. Therefore, this mutation is truly pathogenic. Interestingly, the incidence of T2 deficiency may be high among the Arab population. This disease should be considered in the differential diagnosis for unexplained ketoacidosis in children. Patients with T2 deficiency could have a favorable outcome if diagnosed and treated early.

MiR-21 is the most studied cancer-promoting oncomiR, which target numerous tumor suppressor genes associated with proliferation, apoptosis, and invasion. Here we have studied the synthesis of miR-21 and quantified the mRNA and protein levels for miR-21 potential target genes, i.e., Acat1, Armcx1, and Pten, in the livers of female Wistar rats after their treatment with either 1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) or benzo[a]pyrene (BP). The most important finding appears to be the significant decrease in the miR-21 level the day after treatment with DDT with subsequent rebound. These changes are accompanied by an increase and subsequent drop in the levels of mRNAs and proteins of the Acat1, Armcx1, and Pten genes. These observations indicate the involvement of miR-21 in the posttranscriptional regulation of the Acat1, Armcx1, and Pten genes in response to xenobiotics. We hypothesize that the toxic effects of xenobiotics may be indirect and may manifest by inducing epigenetic changes, particularly through the regulation of miRNAs and their target genes.

Previous studies have shown that miR-467b plays a central role in the progression of atherosclerosis via regulating LPL expression. However, the regulatory mechanism of miR-467b in regulateing the CE and FC formation is still unclear. Interestingly, computational analysis demonstrated that ACAT1 which converts intracellular FC into the storage form of CE, and ABCA1 which promotes cellular FC efflux may be target gene of miR-467b. Here, we examined whether miR-467b could target ACAT1 and ABCA1, thereby affecting the CE and FC formation in oxLDL-treatment RAW 264.7 cells. We found that miR-467b regulates the CE:FC ratio in oxLDL-treatment RAW 264.7 macrophages, and the luciferase activity of ACAT1 is regulated by the miR-467b, but the luciferase activity of ABCA1 has no effect. Furthermore, our data suggested that miR-467b highly regulates the endogenous levels of ACAT1 expression, thereby affecting the CE formation in oxLDL-treatment RAW 264.7 macrophages. Taken together, our findings demonstrate that ACAT1 is a target gene of miR-467b, and miR-467b regulated the CE and FC formation via directly target the ACAT1 3′UTR.

Amiodarone is known to induce hepatic injury in some recipients. We applied an untargeted metabolomics approach to identify endogenous metabolites with potential as biomarkers for amiodarone-induced liver injury. Oral amiodarone administration for 1 week in rats resulted in significant elevation of acylcarnitines and phospholipids in the liver. Hepatic short- and medium-chain acylcarnitines were dramatically increased in a dose-dependent manner, while the serum levels of these acylcarnitines did not change substantially. In addition, glucose levels were significantly increased in both the serum and liver. Gene expression profiling showed that the hepatic mRNA levels of Cpt1, Cpt2, and Acat1 were significantly suppressed, whereas those of Acot1, Acly, Acss2, and Acsl3 were increased. These results suggest that hepatic acylcarnitines and glucose levels might be increased due to disruption of mitochondrial function and suppression of glucose metabolism. Perturbation of energy metabolism might be associated with amiodarone-induced hepatotoxicity.

Endoplasmic reticulum (ER) stress induced by disruption of protein folding activates the unfolded protein response (UPR), which while generally pro-survival in effect can also induce cell death under severe ER stress. 24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the ER of neurons, plays an important role in maintaining brain cholesterol homeostasis but also shows neurotoxicity when subjected to esterification by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in the ER. In this study, we demonstrated that the accumulation of 24S-OHC esters in human neuroblastoma SH-SY5Y cells evoked the UPR with substantially no pro-survival adaptive response but with significant activation of pro-death UPR signaling via regulated IRE1-dependent decay (RIDD). We further found that accumulation of 24S-OHC esters caused disruption of ER membrane integrity and release of ER luminal proteins into cytosol. We also found that de novo synthesis of global proteins was robustly suppressed in 24S-OHC-treated cells. Collectively, these results show that ER dysfunction and the accompanying RIDD-mediated pro-death UPR signaling and global protein synthesis inhibition are responsible for 24S-OHC ester-induced unconventional cell death.

Beta-ketothiolase deficiency (BKTD) is an autosomal recessive disease caused by a defect of mitochondrial acetoacetyl-CoA thiolase. Beginning in 2014, we carried out newborn screening by tandem mass spectrometry (MS/MS) followed by next-generation sequencing (NGS) and identified two infants with BKTD among 203,750 newborns born in Jiangsu Province, China. Both infants showed the characteristic chemical abnormalities of BKTD. We used NGS to confirm variants in the ACAT1. Patient 1 had the compound heterozygous variants c.721dupA and c.928G > C. Patient 2 had compound heterozygosity for the c.238+1G > A and c.1163G > T variants. c.721dupA, c.928G > C and c.1163G > T were suspected to be likely pathogenic, whereas c.238+1G > A was determined to be pathogenic. None of the four variants have been reported in the literature. Patient 1 presented with onset of metabolic acidosis and neonatal hypoglycemia 8 days after birth, whereas patient 2 was detected through neonatal disease screening but had no clinical manifestations. These findings contribute to our understanding of the clinical characteristics and genetic basis of BKTD.

Metabolic alteration for adaptation of the local environment has been recognized as a hallmark of cancer. GNPAT dysregulation has been implicated in hepatocellular carcinoma (HCC). However, the precise posttranslational regulation of GNPAT is still undiscovered. Here we show that ACAT1 is upregulated in response to extra palmitic acid (PA). ACAT1 acetylates GNPAT at K128, which represses TRIM21-mediated GNPAT ubiquitination and degradation. Conversely, GNPAT deacetylation by SIRT4 antagonizes ACAT1's function. GNPAT represses TRIM21-mediated FASN degradation and promotes lipid metabolism. Furthermore, shRNA-mediated ACAT1 ablation and acetylation deficiency of GNPAT repress lipid metabolism and tumor progression in xenograft and DEN/CCl4-induced HCC. Otherwise, ACAT1 inhibitor combination with sorafenib enormously retards tumor formation in mice. Collectively, we demonstrate that stabilization of FASN by ACAT1-mediated GNPAT acetylation plays a critical role in hepatocarcinogenesis.

Acetoacetyl-CoA thiolase also known as acetyl-CoA acetyltransferase (ACAT) corresponds to two enzymes, one cytosolic (ACAT2) and one mitochondrial (ACAT1), which is thought to catalyse reversible formation of acetoacetyl-CoA from two molecules of acetyl-CoA during ketogenesis and ketolysis respectively. In addition to this activity, ACAT1 is also involved in isoleucine degradation pathway. Deficiency of ACAT1 is an inherited metabolic disorder, which results from a defect in mitochondrial acetoacetyl-CoA thiolase activity and is clinically characterized with patients presenting ketoacidosis. In this review I discuss the recent findings, which unexpectedly expand the known functions of ACAT1, indicating a role for ACAT1 well beyond its classical activity. Indeed ACAT1 has recently been shown to possess an acetyltransferase activity capable of specifically acetylating Pyruvate DeHydrogenase (PDH), an enzyme involved in producing acetyl-CoA. ACAT1-dependent acetylation of PDH was shown to negatively regulate this enzyme with a consequence in Warburg effect and tumor growth. Finally, the elevated ACAT1 enzyme activity in diverse human cancer cell lines was recently reported. These important novel findings on ACAT1's function and expression in cancer cell proliferation point to ACAT1 as a potential new anti-cancer target.

Background:Alzheimer´s disease (AD) is a chronic and progressive disease which impacts caregivers, families and societies physically, psychologically and economically. Currently available drugs can only improve cognitive symptoms, have no impact on progression and are not curative, so identifying and studying new drug targets is important. There are evidences which indicate disturbances in cholesterol homeostasis can be related with AD pathology, especially the compartmentation of intracellular cholesterol and cytoplasmic cholesterol esters formed by acyl-CoA: cholesterol acyltransferase 1 (ACAT1) can be implicated in the regulation of amyloid-beta (Aβ) peptide, involved in AD. Blocking ACAT1 activity, beneficial effects are obtained, so it has been suggested that ACAT1 can be a potential new therapeutic target. The present review discusses the role of cholesterol homeostasis in AD pathology, especially with ACAT inhibitors, and how they have been raised as a therapeutic approach. In addition, the genetic relationship of ACAT and AD is discussed.Conclusion:Although there are several lines of evidence from cell-based and animal studies that suggest that ACAT inhibition is an effective way of reducing cerebral Aβ, there is still an information gap in terms of mechanisms and concerns to cover before passing to the next level. Additionally, an area of interest that may be useful in understanding AD to subsequently propose new therapeutic approaches is pharmacogenetics; however, there is still a lot of missing information in this area.

Mitochondrial acetoacetyl‐CoA thiolase (T2, encoded by the ACAT1 gene) deficiency is an inherited disorder of ketone body and isoleucine metabolism. It typically manifests with episodic ketoacidosis. The presence of isoleucine‐derived metabolites is the key marker for biochemical diagnosis. To date, 105 ACAT1 variants have been reported in 149 T2‐deficient patients. The 56 disease‐associated missense ACAT1 variants have been mapped onto the crystal structure of T2. Almost all these missense variants concern residues that are completely or partially buried in the T2 structure. Such variants are expected to cause T2 deficiency by having lower in vivo T2 activity because of lower folding efficiency and/or stability. Expression and activity data of 30 disease‐associated missense ACAT1 variants have been measured by expressing them in human SV40‐transformed fibroblasts. Only two variants (p.Cys126Ser and p.Tyr219His) appear to have equal stability as wild‐type. For these variants, which are inactive, the side chains point into the active site. In patients with T2 deficiency, the genotype does not correlate with the clinical phenotype but exerts a considerable effect on the biochemical phenotype. This could be related to variable remaining residual T2 activity in vivo and has important clinical implications concerning disease management and newborn screening.

Introduction During post-natal development, the organism is subjected to 2 metabolic transitions. The weaning transition is particularly interesting as it is associated with a change in glucose metabolism. O-GlcNAcylation is an ubiquitous post-translational modification, described as a nutrient sensor. The GlcNAc moiety is added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Interestingly, cardiac O-GlcNAc levels decrease throughout post-natal development but its regulatory mechanism and impact on proteins have never been studied under this condition. Objective Study the regulatory mechanism involved in O-GlcNAc regulation and its potential impact on proteins implied in cardiac metabolism. Method Hearts from 28 day-olds (D28) Wistar rats fed with a standard or low-carbohydrate (low-CHO) diet (D28F: 50% fat, 30% protein, 3% carbohydrates) during the weaning period (D12 to D28) were studied. O-GlcNAc levels and regulatory enzymes expression were evaluated by western blot. The OGA activity was evaluated with a spectrophotometric assay. The impact of selected protein expression in response to O-GlcNAc levels variations (Thiamet-G, OGA inhibitor) was evaluated on cell culture (HEK and COS). Results No change was reported on O-GlcNAc levels between the diets (D28: 1.00 ± 0.06; D28F: 0.99 ± 0.08). Cardiac OGA expression was 5 times lower in D28F however OGA activity remained stable (D28: 1.00 ± 0.03; D28F: 0.83 ± 0.16 AU). Protein expression, acetyl-CoA transferase 1 (ACAT1) and acetyl-CoA synthetase 1 (ACSS1), was reduced both in HEK and COS cells in responses to O-GlcNAc stimulation with Thiamet-G. Conclusion While OGA expression is lower with the low-CHO diet, OGA activity and O-GlcNAc levels remain stable. This indicates a strong regulatory mechanism to control O-GlcNAc levels throughout post-natal development. This regulatory mechanism could help to modulate the expression of key metabolic enzymes such as ACAT1 or ACSS1.

We previously demonstrated that AKR vs. DBA/2 mouse bone marrow derived macrophages have higher levels of free cholesterol and lower levels of esterified cholesterol after cholesterol loading, and that AKR, but not DBA/2, macrophages induced C/EBP homologous protein (CHOP) expression after cholesterol loading. We earlier determined that the free and esterified cholesterol level effect is due to a truncation in the sterol O-acyltransferase 1 (Soat1) gene, encoding acetyl-coenzyme A acetyltransferase 1 (ACAT1). Here we examined the mechanism for the differential induction of CHOP by cholesterol loading. CHOP was induced in both strains after incubation with tunicamycin, indicating both strains have competent endoplasmic reticulum stress pathways. CHOP was induced when DBA/2 macrophages were cholesterol loaded in the presence of an ACAT inhibitor, indicating that the difference in free cholesterol levels were responsible for this strain effect. This finding was confirmed in macrophages derived from DBA/2 embryonic stem cells. Cholesterol loading of Soat1 gene edited cells, mimicking the AKR allele, led to increased free cholesterol levels and restored CHOP induction. The upstream pathway of free cholesterol induced endoplasmic reticulum stress was investigated; and, RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1 α protein kinase (IRE1α) pathways were required for maximal CHOP expression.

The process of vision is impossible without the photoreceptor cells, which have a unique structure and specific maintenance of cholesterol. Herein we report on the previously unrecognized cholesterol-related pathway in the retina discovered during follow-up characterizations of Cyp27a1(-/-)Cyp46a1(-/-) mice. These animals have retinal hypercholesterolemia and convert excess retinal cholesterol into cholesterol esters, normally present in the retina in very small amounts. We established that in the Cyp27a1(-/-)Cyp46a1(-/-) retina, cholesterol esters are generated by and accumulate in the photoreceptor outer segments (OS), which is the retinal layer with the lowest cholesterol content. Mouse OS were also found to express the cholesterol-esterifying enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT1), but not lecithin-cholesterol acyltransferase (LCAT), and to differ from humans in retinal expression of ACAT1. Nevertheless, cholesterol esters were discovered to be abundant in human OS. We suggest a mechanism for cholesterol ester accumulation in the OS and that activity impairment of ACAT1 in humans may underlie the development of subretinal drusenoid deposits, a hallmark of age-related macular degeneration, which is a common blinding disease. We generated Cyp27a1(-/-)Cyp46a1(-/-)Acat1(-/-) mice, characterized their retina by different imaging modalities, and confirmed that unesterified cholesterol does accumulate in their OS and that there is photoreceptor apoptosis and OS degeneration in this line. Our results provide insights into the retinal response to local hypercholesterolemia and the retinal significance of cholesterol esterification, which could be cell-specific and both beneficial and detrimental for retinal structure and function.

Alzheimer's disease (AD) is the most common cause of dementia with no cure at present. Cholesterol metabolism is closely associated with AD at several stages. ACAT1 converts free cholesterol to cholesteryl esters, and plays important roles in cellular cholesterol homeostasis. Recent studies show that in a mouse model, blocking ACAT1 provides multiple beneficial effects on AD. Here we review the current evidence that implicates ACAT1 as a therapeutic target for AD. We also discuss the potential usage of various ACAT inhibitors currently available to treat AD.

Earlier works identified the second generation (Z8R2) of a resistant Pekin duck line to duck hepatitis A virus genotype 3 (DHAV-3), which displays significantly strong resistance than that of the second generation (Z8S2) of a susceptible Pekin duck line. To understand the genetic mechanisms that determine the different resistance/susceptibility of Z8R2 and Z8S2 to DHAV-3, transcriptome analysis on livers of infected Pekin ducklings was performed to screen differentially expressed genes (DEGs). We found that DHAV-3 infection has a great effect on metabolism of Z8S2 at the transcription level. Using a newly created fourth generation of the resistant Pekin duck line (Z8R4) and an unselected Pekin duck flock (Z7) as models, hypoglycemia and dramatically increased aspartate aminotransferase and alanine aminotransferase were shown to be noticeable signs of fatal cases caused by DHAV-3 infection. These findings, together with expression analysis and verification of DEGs, support the view that DHAV-3 infection results in glucose metabolic abnormalities in susceptible individuals and that there are significant differences in expression patterns of glucose metabolism-related DEGs between susceptible and resistant individuals. Notably, cytokines displayed a negative correlation with glucose synthesis in terms of expression in susceptible individuals following DHAV-3 infection. Mechanism analyses suggests that cytokines will activate PI3K-AKT pathway and/or JAK-STAT pathway by up-regulated expression of JAK2, and thereby causes down-regulated expression of G6PC and/or ACAT1. Cytokines can also cause down-regulated expression of HPGDS by JAK2. The present work contributes to the understanding of pathogenesis of DHAV-3 infection and the resistance breeding project against DHAV-3.

Kilic-Yildirim G, Durmus-Aydogdu S, Ceylaner S, Sass JO. Beta-ketothiolase deficiency: An unusual cause of recurrent ketoacidosis. Turk J Pediatr 2017; 59: 471-474. Beta-ketothiolase deficiency (mitochondrial acetoacetyl-CoA thiolase, MAT or T2 deficiency) is a rare autosomal recessive disorder of isoleucine and ketone body metabolism due to acetyl-CoA acetyltransferase-1 (ACAT1) gene mutations. The disease is characterized by recurrent episodes of ketoasidosis which starts with vomiting and followed by dehydration and tachypnea. Here, we present a patient who was admitted to the hospital with severe acidosis and dehydration because of vomiting induced by protein rich nutrient and was diagnosed with MAT deficiency. 3-hydroxy-butyric acid, acetoacetic acid and 3-hydroxy-iso-valeric acid levels were significantly increased and tiglyglycine as trace amount in the urine organic acid analysis of the patient. Genetic analysis for ACAT-1 showed compound heterozygosity for the mutations c.949G > A (p.D317N) and c.951C > T (p.D317D), which both are known to cause exon 10 skipping and to be pathogenic missense mutations.

Beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase) deficiency is a genetic disorder characterized by impaired isoleucine catabolism and ketone body utilization that predisposes to episodic ketoacidosis. It results from biallelic pathogenic variants in the ACAT1 gene, encoding mitochondrial beta-ketothiolase. We report two cases of beta-ketothiolase deficiency presenting with acute ketoacidosis and “metabolic stroke.” The first patient presented at 28 months of age with metabolic acidosis and pallidal stroke in the setting of a febrile gastrointestinal illness. Although 2-methyl-3-hydroxybutyric acid and trace quantities of tiglylglycine were present in urine, a diagnosis of glutaric acidemia type I was initially suspected due to the presence of glutaric and 3-hydroxyglutaric acids. A diagnosis of beta-ketothiolase deficiency was ultimately made through whole exome sequencing which revealed compound heterozygous variants in ACAT1. Fibroblast studies for beta-ketothiolase enzyme activity were confirmatory. The second patient presented at 6 months of age with ketoacidosis, and was found to have elevations of urinary 2-methyl-3-hydroxybutyric acid, 2-methylacetoacetic acid, and tiglylglycine. Sequencing of ACAT1 demonstrated compound heterozygous presumed causative variants. The patient exhibited choreoathethosis 2 months after the acute metabolic decompensation. These cases highlight that, similar to a number of other organic acidemias and mitochondrial disorders, beta-ketothiolase deficiency can present with metabolic stroke. They also illustrate the variability in clinical presentation, imaging, and biochemical evaluation that make screening for and diagnosis of this rare disorder challenging, and further demonstrate the value of whole exome sequencing in the diagnosis of metabolic disorders.

2-methylacetoacetyl-coenzyme A thiolase (MAT) deficiency, also known as beta-ketothiolase deficiency, is an inborn error of ketone body utilization and isoleucine catabolism. It is caused by mutations in the ACAT1 gene and may present with metabolic ketoacidosis. In order to obtain a more comprehensive view on this disease, we have collected clinical and biochemical data as well as information on ACAT1 mutations of 32 patients from 12 metabolic centers in five countries. Patients were between 23 months and 27 years old, more than half of them were offspring of a consanguineous union. 63% of the study participants presented with a metabolic decompensation while most others were identified via newborn screening or family studies. In symptomatic patients, age at manifestation ranged between 5 months and 6.8 years. Only 7% developed a major mental disability while the vast majority was cognitively normal. More than one third of the identified mutations in ACAT1 are intronic mutations which are expected to disturb splicing. We identified several novel mutations but, in agreement with previous reports, no clear genotype-phenotype correlation could be found. Our study underlines that the prognosis in MAT deficiency is good and MAT deficient individuals may remain asymptomatic, if diagnosed early and preventive measures are applied.

Betaine is widely used in animal nutrition to promote growth, development and methyl donor during methionine metabolism through nutritional reprogramming via regulation of gene expression. Prenatal betaine exposure is reported to modulate hypothalamic cholesterol metabolism in chickens, yet it remains unknown whether feeding hens with betaine-supplemented diet may affect hypothalamic cholesterol metabolism in F1 offspring. In this study, hens were fed with basal or betaine-supplemented (0.5%) for 30days, and the eggs were collected for incubation. The hatchlings were raised under the same condition up to 56days of age. Betaine-treated group showed significantly (P

Background & Aims Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes the conversion of free cholesterol (FC) to cholesterol ester, which prevents excess accumulation of FC. We recently found that FC accumulation in hepatic stellate cells (HSCs) plays a role in progression of liver fibrosis, but the effect of ACAT1 on liver fibrosis has not been clarified. In this study, we aimed to define the role of ACAT1 in the pathogenesis of liver fibrosis. Methods ACAT1-deficient and wild-type mice, or Toll-like receptor 4 ( TLR4 ) −/− ACAT1 +/+ and TLR4 −/− ACAT1 −/− mice were subjected to bile duct ligation (BDL) for 3weeks or were given carbon tetrachloride (CCl 4 ) for 4weeks to induce liver fibrosis. Results ACAT1 was the major isozyme in mice and human primary HSCs, and ACAT2 was the major isozyme in mouse primary hepatocytes and Kupffer cells. ACAT1 deficiency significantly exaggerated liver fibrosis in the mouse models of liver fibrosis, without affecting the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation. ACAT1 deficiency significantly increased FC levels in HSCs, augmenting TLR4 protein and downregulating expression of transforming growth factor-β (TGFβ) pseudoreceptor Bambi (bone morphogenetic protein and activin membrane-bound inhibitor), leading to sensitization of HSCs to TGFβ activation. Exacerbation of liver fibrosis by ACAT1 deficiency was dependent on FC accumulation-induced enhancement of TLR4 signaling. Conclusions ACAT1 deficiency exaggerates liver fibrosis mainly through enhanced FC accumulation in HSCs. Regulation of ACAT1 activities in HSCs could be a target for treatment of liver fibrosis.

Macrophages in hyperlipidemic conditions accumulate cholesterol esters and develop into foamy transformed macrophages. During this transformation, macrophages demonstrate endoplasmic reticulum fragmentation and consequently produce acyl coenzyme A: cholesterol acyltransferase 1 (ACAT1)-positive late endosomes (ACAT1-LE). ACAT1-LE-positive macrophages effectively esterify modified or native low-density lipoprotein-derived free cholesterol, which results in efficient cholesterol esterification as well as atherosclerotic plaque formation. These macrophages show significant cholesterol ester formation even when free cholesterol egress from late endosomes is impaired, which indicates that free cholesterol is esterified at ACAT1-LE. Genetic blockade of cholesterol egress from late endosomes causes Niemann-Pick disease type C (NPC), an inherited lysosomal storage disease with progressive neurodegeneration. Induction of ACAT1-LE in macrophages with the NPC phenotype led to significant recovery of cholesterol esterification. In addition, in vivo ACAT1-LE induction significantly extended the lifespan of mice with the NPC phenotype. Thus, ACAT1-LE not only regulates intracellular cholesterol metabolism but also ameliorates NPC pathophysiology.

Scope The effects of sulforaphane (SFN) on the maturation of lipid droplets (LDs)-the storage units for free fatty acids and sterols as triacylglycerides (TAG) and cholesterol esters (CE)-are far from being understood, despite the fact that SFN is known to be beneficial for ameliorating lipid metabolism disorders. Methods and results High-fat-intake models are established in both HHL-5 hepatocytes and rodents. The numbers and sizes of LDs are decreased by SFN. The accumulation of lipid core components (TAG & CE) is reduced and the expression of their key synthetases, acyl-coenzyme A: diacylglycerol acyltransferases 2 (DGAT2) and acyl-coenzyme A: cholesterol acyltransferases 1 (ACAT1), is also inhibited. Moreover, SFN decreases LD-associated protein PLIN2 and PLIN5 expression, but not that of PLIN1 and PLIN3, both in vivo and in vitro. Furthermore, over-expression of peroxisome proliferator-activated receptor gamma (PPARγ) induces the accumulation of TAG and the up-regulation of PLIN2 and PLIN5, which are not reversed by SFN. These results suggest that PPARγ may be a target of SFN in lipid metabolism. Conclusion SFN disturbs LD maturation by inhibiting the formation of the neutral lipid core and decreases PLIN2 and PLIN5 via down-regulation of PPARγ.

MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by targeting mRNAs and control a wide range of biological functions. Recent studies have indicated that miRNAs can regulate lipid and cholesterol metabolism in mammals. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a key enzyme in cellular cholesterol metabolism. The accumulated cholesteryl esters are mainly synthesized by ACAT1 during the formation of foam cell, a hallmark of early atherosclerotic lesions. Here, we revealed that miR-9 could target the 3'-untranslated region of human ACAT1 mRNA, specifically reduce human ACAT1 or reporter firefly luciferase (Fluc) proteins but not their mRNAs in different human cell lines, and functionally decrease the formation of foam cells from THP-1-derived macrophages. Our findings suggest that miR-9 might be an important regulator in cellular cholesterol homeostasis and decrease the formation of foam cells in vivo by reducing ACAT1 proteins.

Both genetic inactivation and pharmacological inhibition of the cholesteryl ester synthetic enzyme acyl-CoA:cholesterol acyltransferase 1 (ACAT1) have shown benefit in mouse models of Alzheimer's disease (AD). In this study, we aimed to test the potential therapeutic applications of adeno-associated virus (AAV)-mediated Acat1 gene knockdown in AD mice. We constructed recombinant AAVs expressing artificial microRNA (miRNA) sequences, which targeted Acat1 for knockdown. We demonstrated that our AAVs could infect cultured mouse neurons and glia and effectively knockdown ACAT activity in vitro. We next delivered the AAVs to mouse brains neurosurgically, and demonstrated that Acat1-targeting AAVs could express viral proteins and effectively diminish ACAT activity in vivo, without inducing appreciable inflammation. We delivered the AAVs to the brains of 10-month-old AD mice and analyzed the effects on the AD phenotype at 12 months of age. Acat1-targeting AAV delivered to the brains of AD mice decreased the levels of brain amyloid-β and full-length human amyloid precursor protein (hAPP), to levels similar to complete genetic ablation of Acat1. This study provides support for the potential therapeutic use of Acat1 knockdown gene therapy in AD.

Perfluorooctanoic acid (PFOA, ‘C8’) and perfluoroctane sulphonate (PFOS) are environmentally stable compounds with industrial and consumer uses and long half-lives in humans. Concern has been raised over chronic exposure effects to human health, especially in relation to cholesterol metabolism. Here, we explore the association between exposure to PFOA and PFOS and the in vivo expression of genes involved in cholesterol metabolism. We studied 290 individuals exposed to background levels of PFOS and elevated concentrations of PFOA through drinking water. Using adjusted linear regression models, we found inverse associations between serum PFOA levels and the whole blood expression level of genes involved in cholesterol transport (NR1H2, NPC1 and ABCG1; p = 0.002, 0.026 and 0.014 respectively). A positive association was seen between PFOS and a transcript involved in cholesterol mobilisation (NCEH1; p = 0.018), and a negative relationship with a transcript involved in cholesterol transport (NR1H3; p = 0.044). When sexes were analysed separately, reductions in the levels of mRNAs involved in cholesterol transport were seen with PFOA in men (NPC1, ABCG1, and PPARA; p = 0.025, 0.024 and 0.012 respectively) and in women (NR1H2 expression; p = 0.019), whereas an increase in the levels of a cholesterol mobilisation transcript (NCEH1; p = 0.036) was noted in women alone. PFOS was positively associated with expression of genes involved in both cholesterol mobilisation and transport in women (NCEH1 and PPARA; p = 0.003 and 0.039 respectively), but no effects were evident in men. This is the first report of associations between the in vivo expression of genes involved in cholesterol metabolism and exposure to PFOA or PFOS, suggested that exposure to these compounds may promote a hypercholesterolaemic environment, with wider implications for human disease. Keywords: PFOA, PFOS, Cholesterol, Human, Toxicogenomics

Background β‐ketothiolase (T2, gene symbol ACAT1) deficiency is an autosomal recessive disorder, affecting isoleucine and ketone body metabolism. We encountered a patient (GK03) with T2 deficiency whose T2 mRNA level was A in the polypyrimidine stretch at the splice acceptor site of intron 9 of ACAT1. Initially, we regarded this variant as not being disease‐causing by a method of predicting the effect of splicing using in silico tools. However, based on other findings of exon 10 splicing, we eventually hypothesized that this mutation causes exon 10 skipping. Methods cDNA analysis was performed using GK03's fibroblasts treated with/without cycloheximide (CHX), since exon 10 skipping caused a frameshift and nonsense‐mediated mRNA decay (NMD). Minigene splicing experiment was done to confirm aberrant splicing. Results cDNA analysis using fibroblasts cultured with cycloheximide indeed showed the occurrence of exon 10 skipping. A minigene splicing experiment clearly showed that the c.941‐9T>A mutant resulted in transcripts with exon 10 skipping. There are few reports describing that single‐nucleotide substitutions in polypyrimidine stretches of splice acceptor sites cause aberrant splicing. Conclusion We showed that c.941‐9T>A induces aberrant splicing in the ACAT1 gene. Our ability to predict the effects of mutations on splicing using in silico tools is still limited. cDNA analysis and minigene splicing experiments remain useful alternatives to reveal splice defects.

Excess 2-hydroxy-(4-methylthio)butanoic acid (HMB) supplementation induces hyperhomocysteinemia, which contributes to hepatic cholesterol accumulation. However, it is unclear whether and how high levels of HMB break hepatic cholesterol homeostasis in nursery piglets. In this study, HMB oversupplementation suppressed food intake and decreased body weight in nursery piglets. Hyperhomocysteinemia and higher hepatic cholesterol accumulation were observed in HMB groups. Accordingly, HMB significantly increased the protein content of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and glycine N-methyltransferase (GNMT) but decreased that of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1). Significant downregulation of miR-150, miR-181d-5p, and miR-296-3p targeting the 3'-untranslated regions (UTRs) of GNMT and HMGCR was detected in the liver of HMB-treated piglets, and their functional validation was confirmed by dual-luciferase reporter assay. Furthermore, hypermethylation of miR-150 promoter was detected in association with suppressed miR-150 expression in the livers of HMB-treated piglets. This study indicated a new mechanism of hepatic cholesterol unhomeostasis by dietary methyl donor supplementation.

Mitochondrial acetoacetyl‑CoA thiolase (T2) (gene symbol: ACAT1) deficiency is an autosomal recessive disorder affecting isoleucine catabolism and ketone body utilization. In this study, mutational analysis of an Indian T2‑deficient patient revealed a homozygous mutation (c.121‑13T>A) located at the polypyrimidine tract of the splice acceptor site of intron 2, and exon 3 skipping was identified by cDNA analysis using cycloheximide. We made three mutant constructs (c.121‑13T>A, T>C, and T>G substitutions) followed by making a wild‑type minigene construct that included an ACAT1 segment from exon 2 to 4 for a splicing experiment. The minigene splicing experiment demonstrated that exon 3 skipping was induced not only by c.121‑13T>A mutation, but also by the other two substitutions. It was difficult to predict the effect of these mutations on splicing using in silico tools, as predictions of different tools were inconsistent with each other. The minigene splicing experiment remains the most reliable method to unravel splicing abnormalities.

Beta-ketothiolase deficiency (mitochondrial acetoacetyl-CoA thiolase (T2) deficiency) is an inherited disease of isoleucine catabolism and ketone body utilization caused by ACAT1 mutations. We identified ten Indian patients who manifested with ketoacidotic episodes of variable severity. The patients showed increased urinary excretion of isoleucine-catabolic intermediates: 2-methyl-3-hydroxybutyrate, 2-methylacetoacetate, and tiglylglycine. Six patients had a favorable outcome, one died, and three developed neurodevelopmental sequela. Mutational analysis revealed a common (p.Met193Arg) and four novel (p.Ile323Thr, p.Ala215Asn, c.1012_1015dup, and c.730+1G>A) ACAT1 mutations. Transient expression analyses of wild-type and mutant cDNA were performed at 30, 37, and 40°C. A p.Ile323Thr mutant T2 was detected with relative enzyme activity and protein amount of 20% and 25%, respectively, compared with wild type at 37°C; it was more prevalent at 30°C but ablated at 40°C. These findings showed that p.Ile323Thr had a significant residual T2 activity with temperature-sensitive instability. Neither residual enzymatic activity nor mutant T2 protein was identified in p.Met193Arg, p.Ala215Asn, and c.1012_1015dup mutations using supernatants; however, these mutant T2 proteins were detected in insoluble pellets by immunoblot analysis. Expression analyses confirmed pathogenicity of these mutations. T2 deficiency has a likely high incidence in India and p.Met193Arg may be a common mutation in the Indian population.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) 1 and 2 are both involved in intracellular cholesterol esterification. The expression of acyl-coenzyme A:cholesterol acyltransferase 1; (ACAT1) is ubiquitous, whereas, enterocytes and hepatocytes exclusively express acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2). Studies in mice lacking ACAT1 showed extensive deposition of unesterified cholesterol in skin and brain, and larger atherosclerotic lesions compared to controls. ACAT2 deficiency in mice protected against atherosclerosis, diet-induced hypercholesterolemia and cholesterol gallstone disease, and reduced hepatic lipid accumulation and decreased serum cholesterol levels. Two unspecific ACAT inhibitors have been tested in humans. In the face of the results in ACAT1 deficient mice, strongly arguing against an ACAT1 inhibition, it was not surprising that none of these inhibitors showed beneficial effects on the predefined primary endpoints of the studies. However, and still to be proven, ACAT2 inhibition might be a therapeutic option for the treatment and prevention of cardiovascular disease. In this review, we aim to outline some important aspects on the role of ACAT in cholesterol esterification and to reappraise ACAT2 as therapeutic target.

In this issue, Fan et al. (2016) show that oncogenic tyrosine kinases can promote glycolysis by phosphorylating and stabilizing the tetrameric form of mitochondrial acetyl-coA acetyltransferase 1 (ACAT1). The authors further identify a small molecule ACAT1 inhibitor that displays anti-cancer effects.