Myeloid-specific Acat1 ablation attenuates inflammatory responses in macrophages, improves insulin sensitivity, and suppresses diet-induced obesity.

Macrophages are phagocytes that play important roles in health and diseases. Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) converts cellular cholesterol to cholesteryl esters and is expressed in many cell types. Unlike global Acat1 knockout (KO), myeloid-specific Acat1 KO ( Acat1-) does not cause overt abnormalities in mice. Here, we performed analyses in age- and sex-matched Acat1-M/-M and wild-type mice on chow or Western diet and discovered that Acat1-M/-M mice exhibit resistance to Western diet-induced obesity. On both chow and Western diets, Acat1-M/-M mice display decreased adipocyte size and increased insulin sensitivity. When fed with Western diet, Acat1-M/-M mice contain fewer infiltrating macrophages in white adipose tissue (WAT), with significantly diminished inflammatory phenotype. Without Acat1, the Ly6Chi monocytes express reduced levels of integrin-ß1, which plays a key role in the interaction between monocytes and the inflamed endothelium. Adoptive transfer experiment showed that the appearance of leukocytes from Acat1-M/-M mice to the inflamed WAT of wild-type mice is significantly diminished. Under Western diet, Acat1-M/-M causes suppression of multiple proinflammatory genes in WAT. Cell culture experiments show that in RAW 264.7 macrophages, inhibiting ACAT1 with a small-molecule ACAT1-specific inhibitor reduces inflammatory responses to lipopolysaccharide. We conclude that under Western diet, blocking ACAT1 in macrophages attenuates inflammation in WAT. Other results show that Acat1-M/-M does not compromise antiviral immune response. Our work reveals that blocking ACAT1 suppresses diet-induced obesity in part by slowing down monocyte infiltration to WAT as well as by reducing the inflammatory responses of adipose tissue macrophages.

Abrogating cholesterol esterification suppresses growth and metastasis of pancreatic cancer.

Cancer cells are known to execute reprogramed metabolism of glucose, amino acids and lipids. Here, we report a significant role of cholesterol metabolism in cancer metastasis. By using label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, mediated by acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme. Expression of ACAT-1 showed a correlation with poor patient survival. Abrogation of cholesterol esterification, either by an ACAT-1 inhibitor or by shRNA knockdown, significantly suppressed tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Mechanically, ACAT-1 inhibition increased intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum stress and caused apoptosis. Collectively, our results demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification.

Acyl-coenzyme A:cholesterol acyltransferase 1 blockage enhances autophagy in the neurons of triple transgenic Alzheimer's disease mouse and reduces human P301L-tau content at the presymptomatic stage.

Patients with Alzheimer's disease (AD) display amyloidopathy and tauopathy. In mouse models of AD, pharmacological inhibition using small molecule enzyme inhibitors or genetic inactivation of acyl-coenzyme A (Acyl-CoA):cholesterol acyltransferase 1 (ACAT1) diminished amyloidopathy and restored cognitive deficits. In microglia, ACAT1 blockage increases autophagosome formation and stimulates amyloid ß peptide1-42 degradation. Here, we hypothesize that in neurons ACAT1 blockage augments autophagy and increases autophagy-mediated degradation of P301L-tau protein. We tested this possibility in murine neuroblastoma cells ectopically expressing human tau and in primary neurons isolated from triple transgenic AD mice that express mutant forms of amyloid precursor protein, presenilin-1, and human tau. The results show that ACAT1 blockage increases autophagosome formation and decreases P301L-tau protein content without affecting endogenous mouse tau protein content. In vivo, lacking Acat1 decreases P301L-tau protein content in the brains of young triple transgenic AD mice but not in those of old mice, where extensive hyperphosphorylations and aggregation of P301L-tau take place. These results suggest that, in addition to ameliorating amyloidopathy in both young and old AD mice, ACAT1 blockage may benefit AD by reducing tauopathy at early stage.

Role of ACAT1-positive late endosomes in macrophages: cholesterol metabolism and therapeutic applications for Niemann-Pick disease type C.

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.

The roles of salusins in atherosclerosis and related cardiovascular diseases.

Human salusin-α and -ß are related peptides of 28 and 20 amino acids, respectively, produced from the same precursor, prosalusin. Salusin-ß exerts more potent mitogenic effects on human vascular smooth muscle cells (VSMCs) and fibroblasts than salusin-α. Human macrophage foam cell formation is significantly stimulated by salusin-ß, but suppressed by salusin-α. Chronic salusin-ß infusion into apolipoprotein E-knockout mice enhances atherosclerotic lesions, paralleling increases in foam cell formation and upregulation of scavenger receptors and of acyl-CoA:cholesterol acyltransferase-1 (ACAT1) in macrophages. In contrast, chronic salusin-α infusion reduces atherosclerotic lesions accompanied by significant suppression of foam cell formation owing to ACAT1 downregulation. Salusin-ß is expressed in proliferative neointimal lesions of porcine coronary arteries after stenting. Salusin-α and -ß immunoreactivity has been detected in human coronary atherosclerotic plaques, with dominance of salusin-ß in macrophage foam cells, VSMCs, and fibroblasts. Serum salusin-α levels are markedly decreased in patients with angiographically proven coronary artery disease compared with patients with mild hypertension and healthy volunteers. Furthermore, among patients with acute coronary syndrome, serum salusin-α levels are decreased in accordance with the severity of coronary atherosclerotic lesions. These findings suggest that salusin-ß may contribute to the pathogenesis of atherosclerosis. Decreased levels of salusin-α in circulating blood and vascular tissue are closely linked with human atherosclerosis. Therefore, salusin-α could be a candidate biomarker for atherosclerosis and may be therapeutically useful for prevention of atherosclerotic cardiovascular diseases.

ACAT inhibition and amyloid beta reduction.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder. Accumulation and deposition of the beta-amyloid (Abeta) peptide generated from its larger amyloid precursor protein (APP) is one of the pathophysiological hallmarks of AD. Intracellular cholesterol was shown to regulate Abeta production. Recent genetic and biochemical studies indicate that not only the amount, but also the distribution of intracellular cholesterol is critical to regulate Abeta generation. Acyl-coenzyme A: cholesterol acyl-transferase (ACAT) is a family of enzymes that regulates the cellular distribution of cholesterol by converting membrane cholesterol into hydrophobic cholesteryl esters for cholesterol storage and transport. Using pharmacological inhibitors and transgenic animal models, we and others have identified ACAT1 as a potential therapeutic target to lower Abeta generation and accumulation. Here we discuss data focusing on ACAT inhibition as an effective strategy for the prevention and treatment of AD.

Pivotal advance: macrophages become resistant to cholesterol-induced death after phagocytosis of apoptotic cells.

One of the most important functions of macrophages is the phagocytosis of apoptotic cells (ACs). ACs deliver large amounts membrane-derived cholesterol to phagocytes, which, if not handled properly, can be cytotoxic. In atherosclerosis, where the ACs are cholesterol-loaded, this situation is exaggerated, because the ACs deliver both endogenous membrane cholesterol and stored lipoprotein-derived cholesterol. To examine how phagocytes handle this very large amount of cholesterol, we incubated macrophage phagocytes with cholesterol-loaded ACs. Our results show that the phagocytes call into play a number of cellular responses to protect them from cholesterol-induced cytotoxicity. First, through efficient trafficking of the internalized AC-derived cholesterol to acyl-CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum, phagocytes efficiently esterify the cholesterol and thus prevent its toxic effects. However, the phagocytes show no signs of cytotoxicity even when ACAT is rendered dysfunctional, as might occur in advanced atherosclerotic lesions. Under these conditions, the phagocytes remain viable through massive efflux of AC-derived cholesterol. Remarkably, these phagocytes still show a survival response even when high cholesterol levels are maintained in the post-phagocytosis period by subsequent incubation with atherogenic lipoproteins, as also may occur in atheromata. In this case, death in phagocytes is prevented by activation of survival pathways involving PI-3 kinase/Akt and NF-kappaB. Thus, macrophages that have ingested ACs successfully employ three survival mechanisms -- cholesterol esterification, massive cholesterol efflux, and cell-survival signaling. These findings have implications for macrophage physiology in both AC clearance and atherosclerotic plaque progression.

Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-x gene function.

Gene targeting in mice was used to investigate the unknown function of Scp2, encoding sterol carrier protein-2 (SCP2; a peroxisomal lipid carrier) and sterol carrier protein-x (SCPx; a fusion protein between SCP2 and a peroxisomal thiolase). Complete deficiency of SCP2 and SCPx was associated with marked alterations in gene expression, peroxisome proliferation, hypolipidemia, impaired body weight control, and neuropathy. Along with these abnormalities, catabolism of methyl-branched fatty acyl CoAs was impaired. The defect became evident from up to 10-fold accumulation of the tetramethyl-branched fatty acid phytanic acid in Scp2(-/-) mice. Further characterization supported that the gene disruption led to inefficient import of phytanoyl-CoA into peroxisomes and to defective thiolytic cleavage of 3-ketopristanoyl-CoA. These results corresponded to high-affinity binding of phytanoyl-CoA to the recombinant rat SCP2 protein, as well as high 3-ketopristanoyl-CoA thiolase activity of the recombinant rat SCPx protein.

Enzymes of ketone body utilization in human tissues: protein and messenger RNA levels of succinyl-coenzyme A (CoA):3-ketoacid CoA transferase and mitochondrial and cytosolic acetoacetyl-CoA thiolases.

We describe the distribution in human tissues of three enzymes of ketone body utilization: succinyl-CoA:3-ketoacid CoA transferase (SCOT), mitochondrial acetoacetyl-CoA thiolase (T2), and cytosolic acetoacetyl-CoA thiolase (CT). Hereditary deficiency of each of these enzymes has been associated with ketoacidosis. Physiologically the two mitochondrial enzymes have different roles: SCOT mediates energy production from ketone bodies (ketolysis), whereas T2 functions both in ketogenesis and ketolysis. In contrast, CT is implicated in cytosolic cholesterol synthesis. We investigated the tissue distribution of these enzymes in humans by quantitative immunoblots and by Northern blots. In most tissues, polypeptide and mRNA levels were proportional. CT and T2 proteins were detected in all tissues examined. CT levels were highest in liver, were 4-fold lower in adrenal glands, kidney, brain, and lung, and were lowest in skeletal and heart muscles. T2 was most abundant in liver but substantial amounts were present in kidney, heart, adrenal glands, and skeletal muscle. SCOT was detected in all tissues except liver: myocardium > brain, kidney and adrenal glands. The relative amounts of T2 and SCOT were similar in all tissues except for liver (T2 > > SCOT) and brain (SCOT > T2). The observed distribution of SCOT, T2, and CT is consistent with current views of their physiologic roles.

Utilization of ketone bodies by the rat liver, brain and heart in chronic alcohol intoxication.

The time-course of ketone body concentrations, the activities of enzymes of their utilization as well as the activities of acetyl-CoA synthetase and ATP-citrate lyase were studied in the liver, brain and heart of rats receiving ethanol for 40 days (3 g/kg, intragastrally). Ethanol increased the concentration of 3-hydroxybutyrate 3 hr following the last ethanol treatment in the blood and tissues investigated and that of acetoacetate in the liver with raised acetoacetyl-CoA synthetase activity in all three tissues. The activities of acetyl-CoA-generating enzymes were, however, increased only in the liver and heart. Chronic alcohol intoxication diminished the activities of ketone body utilizing enzymes (3-hydroxybutyrate dehydrogenase and 3-oxo acid-CoA transferase) in the heart but not in the brain. The data obtained indicate both disturbed ketone body utilization and increased importance of acetate produced from ethanol as an energy source in the heart during long-term ethanol treatment.

Isolation and characterization of yeast mutants blocked in mevalonic acid formation.

Yeast mutants defective in beta-hydroxy-beta-methylglutaryl-CoA synthase and acetoacetyl-CoA thiolase have been isolated. Mutants impaired in acetoacetyl-CoA thiolase range into two linked complementation units, erg 10 A and erg 10 B. Mutants deficient in beta-hydroxy-beta-methylglutaryl-CoA synthase belong to two unlinked complementation groups, erg 11 and erg 13. In strictly anaerobic growth conditions, mutants impaired in beta-hydroxy-beta-methylglutaryl-CoA synthase require mevalonic acid in addition to sterol and oleic acid, pointing out the role of mevalonic acid in other physiological function than ergosterol precursor. Growth of mutants impaired in acetoacetyl-CoA thiolase cannot be recovered by mevalonic acid supplementation, suggesting a role of acetoacetyl-CoA or thiolase not linked to sterol pathway.

The charge heterogeneity of the mitochondrial acetyl-CoA acetyltransferase from rat liver.

The charge heterogeneity of the mitochondrial acetyl-CoA acetyltransferase (EC 2.3.1.9) referred to transferases A and B, seems to be preformed in vivo and can be demonstrated in vitro by a spontaneous transformation of transferase A into transferase B. These two enzymes, although remarkably similar in amino acid composition, show structural dissimilarities. This becomes evident from their tryptic maps, which are substantially different. Isoelectric focusing in the presence of urea confirms the charge heterogeneity of the mitochondrial acetyl-CoA acetyltransferase by indicating different patterns of protein bands for transferases A and B. Transferase B lacks the protein band with an isoelectric point of 7.2 which is normally shown with transferase A. However, this 7.2-pI protein band can be demonstrated after a [32P]CoASH treatment of transferase B in the presence of urea by protein staining and by autoradiography. This change in the isoelectric focusing band pattern after interaction of the subunits with CoASH in interpreted as being the result of an apparently covalent secondary modification of the protein side chains. The CoASH-modification may be the only molecular basis of the acetyl-CoA acetyltransferase charge heterogeneity, a veiw, which is further substantiated by the CoASH-mediated transformation of transferase B into transferase A. An additional heterogeneity of the enzyme as caused by a limited proteolysis seems unlikely but cannot be definitely excluded.