acyl-CoA:cholesterol Acyltransferase Research Articles

Fatty acids abrogate the growth-suppressive effects induced by inhibition of cholesterol flux in pancreatic cancer cells

BackgroundDespite therapeutic advances, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. Metabolic reprogramming is increasingly recognized as a key contributor to tumor progression and therapy resistance in PDAC. One of the main metabolic changes essential for tumor growth is altered cholesterol flux. Targeting cholesterol flux appears an attractive therapeutic approach, however, the complex regulation of cholesterol balance in PDAC cells remains poorly understood.MethodsThe lipid content in human pancreatic duct epithelial (HPDE) cells and human PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) was determined. Cells exposed to eight different inhibitors targeting different regulators of lipid flux, in the presence or absence of oleic acid (OA) stimulation were assessed for changes in viability, proliferation, migration, and invasion. Intracellular content and distribution of cholesterol was assessed. Lastly, proteome profiling of PANC-1 exposed to the sterol O-acyltransferase 1 (SOAT1) inhibitor avasimibe, in presence or absence of OA, was performed.ResultsPDAC cells contain more free cholesterol but less cholesteryl esters and lipid droplets than HPDE cells. Exposure to different lipid flux inhibitors increased cell death and suppressed proliferation, with different efficiency in the tested PDAC cell lines. Avasimibe had the strongest ability to suppress proliferation across the three PDAC cell lines. All inhibitors showing cell suppressive effect disturbed intracellular cholesterol flux and increased cholesterol aggregation. OA improved overall cholesterol balance, reduced free cholesterol aggregation, and reversed cell death induced by the inhibitors. Treatment with avasimibe changed the cellular proteome substantially, mainly for proteins related to biosynthesis and metabolism of lipids and fatty acids, apoptosis, and cell adhesion. Most of these changes were restored by OA.ConclusionsThe study reveals that disturbing the cholesterol flux by inhibiting the actions of its key regulators can yield growth suppressive effects on PDAC cells. The presence of fatty acids restores intracellular cholesterol balance and abrogates the alternations induced by cholesterol flux inhibitors. Taken together, targeting cholesterol flux might be an attractive strategy to develop new therapeutics against PDAC. However, the impact of fatty acids in the tumor microenvironment must be taken into consideration.

Reversible translocation of acyl-CoA:cholesterol acyltransferase (ACAT) between the endoplasmic reticulum and vesicular structures.

The enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is normally localized in the endoplasmic reticulum (ER) where it can esterify cholesterol for storage in lipid droplets and/or the formation of lipoproteins. Here, we report that ACAT can translocate from the ER into vesicular structures in response to different ACAT inhibitors. The translocation was fast (within minutes), reversible and occurred in different cell types. Interestingly, oleic acid was able to fasten the re-translocation from vesicles back into the reticular ER network. The process of ACAT translocation could also be induced by cyclodextrins, cholesterol, lanosterol (but not 4-cholestene-3 one), 25-hydroxycholesterol, and by certain stress stimuli such as hyperosmolarity (sucrose treatment), temperature change, or high-density cultivation. In vitro esterification showed that ACAT remains fully active after it has been translocated to vesicles in response to hyperosmotic sucrose treatment of the cells. The translocation process was not accompanied by changes in the electrophoretic mobility of ACAT, even after chemical crosslinking. Interestingly, the protein synthesis inhibitor cycloheximide showed a stimulating effect on ACAT activity and prevented the translocation of ACAT from the ER into vesicles.

Cholesterol Metabolism in Pancreatic Cancer.

Pancreatic cancer's substantial impact on cancer-related mortality, responsible for 8% of cancer deaths and ranking fourth in the US, persists despite advancements, with a five-year relative survival rate of only 11%. Forecasts predict a 70% surge in new cases and a 72% increase in global pancreatic cancer-related deaths by 2040. This review explores the intrinsic metabolic reprogramming of pancreatic cancer, focusing on the mevalonate pathway, including cholesterol biosynthesis, transportation, targeting strategies, and clinical studies. The mevalonate pathway, central to cellular metabolism, significantly shapes pancreatic cancer progression. Acetyl coenzyme A (Acetyl-CoA) serves a dual role in fatty acid and cholesterol biosynthesis, fueling acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) development. Enzymes, including acetoacetyl-CoA thiolase, 3-hydroxy-3methylglutaryl-CoA (HMG-CoA) synthase, and HMG-CoA reductase, are key enzymes in pancreatic cancer. Inhibiting HMG-CoA reductase, e.g., by using statins, shows promise in delaying PanIN progression and impeding pancreatic cancer. Dysregulation of cholesterol modification, uptake, and transport significantly impacts tumor progression, with Sterol O-acyltransferase 1 (SOAT1) driving cholesterol ester (CE) accumulation and disrupted low-density lipoprotein receptor (LDLR) expression contributing to cancer recurrence. Apolipoprotein E (ApoE) expression in tumor stroma influences immune suppression. Clinical trials targeting cholesterol metabolism, including statins and SOAT1 inhibitors, exhibit potential anti-tumor effects, and combination therapies enhance efficacy. This review provides insights into cholesterol metabolism's convergence with pancreatic cancer, shedding light on therapeutic avenues and ongoing clinical investigations.

Characterization of thraustochytrid-specific sterol O-acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186.

Since the global market for sterols and vitamin D are grown with a high compound annual growth rate, a sustainable source of these compounds is required to keep up with the increasing demand. Thraustochytrid is a marine oleaginous microorganism that can synthesize several sterols, which are stored as SE in lipid droplets. DGAT2C is an unconventional SE synthase specific to thraustochytrids. Although the primary structure of DGAT2C shows high similarities with that of DGAT, DGAT2C utilizes sterol as an acceptor substrate instead of diacylglycerol. In this study, we examined more detailed enzymatic properties, intracellular localization, and structure-activity relationship of DGAT2C. Furthermore, we successfully developed a method to increase sterol and provitamin D3 productivity of thraustochytrid by more than threefold in the process of elucidating the function of the DGAT2C-specific N-terminal region. Our findings could lead to sustainable sterol and vitamin D production using thraustochytrid.

Sparassis latifolia Polysaccharide Attenuates Cholesterol in Rats Maintained on a High-Fat, High-Cholesterol Diet

Sparassis latifolia polysaccharide (SLP) has broad application prospects as a raw material of functional food. This study aimed to investigate the effects of SLP on liver cholesterol metabolism and its mechanism. Sprague–Dawley rats were fed a high-fat, high-cholesterol diet, and various assessments were conducted including hematoxylin and eosin staining, measurement of liver and metabolic indices, and antioxidant analysis. In addition, fluorescence quantitative reverse transcription-polymerase chain reaction was performed to analyze mRNA expression, and gas chromatography-mass spectrometry was employed for screening differential metabolites. The results demonstrated that SLP reduced the abnormal increase in the liver index caused by high fat and cholesterol and improved liver tissue morphology, metabolism level, and antioxidant-related enzyme activities. The polysaccharide regulated the liver sterol metabolism pathway, decreased the mRNA expression levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR) and sterol O-acyltransferase 1 (SOAT1), upregulated the mRNA expression levels of cholesterol 7α hydroxylase (CYP7α-1) and ABCA1, and increased the contents of propionic acid and butyric acid in the liver. These findings indicated a novel pathway through which SLP prevented hepatic lipid deposition and ameliorated sterol metabolism via regulating the HMG-CoAR-CYP7α-1 axis.

The metabolic adaptation of bile acids and cholesterol after biliary atresia in lamprey via transcriptome-based analysis

Lamprey underwent biliary atresia (BA) at its metamorphosis stage. In contrast to patients with BA who develop progressive disease, lamprey can grow and develop normally, suggesting that lamprey has several adaptations for BA. Here we show that adaptive changes in bile acid and cholesterol metabolism are produced after lamprey BA. Among 1102 differentially expressed genes (DGEs) after BA in lamprey, many are enriched in gene ontology (GO) terms and pathways related to steroid metabolism. We find that among the DGEs related to bile acids and cholesterol metabolism, the expression of cytochrome P450 family 7 subfamily A member 1 (CYP7A1), sodium-dependent taurine cotransport polypeptide (NTCP) are significantly downregulated, whereas nuclear receptor farnesoid X receptor (FXR), multidrug resistance-associated protein 3 (MRP3), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), sterol O-acyltransferase 1 (SOAT1), and ATP binding cassette subfamily A member 1 (ABCA1) are remarkably upregulated. The changes in expression level are also validated by RT-qPCR. Furthermore, the level of high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) in juvenile serum is higher compared to larvae. Taken together, the findings collectively indicate that after BA, lamprey may maintain bile acids and cholesterol homeostasis in liver tissue by inhibiting bile acids synthesis and uptake, promoting its efflux back to circulation, and enhancing cholesterol esterification for storage as lipid droplets and its egress to form nascent HDL (nHDL). Understanding the possible molecular mechanisms of lamprey metabolic adaptation sheds new light on the understanding of the development and treatment of diseases caused by abnormal bile acid and cholesterol metabolism in humans.

Regulation of cholesterol metabolism during high fatty acid–induced lipid deposition in calf hepatocytes

Cholesterol in the circulation is partly driven by changes in feed intake, but aspects of cholesterol metabolism during development of fatty liver are not well known. The objective of this study was to investigate mechanisms of cholesterol metabolism in calf hepatocytes challenged with high concentrations of fatty acids (FA). To address mechanistic insights regarding cholesterol metabolism, liver samples were collected from healthy control dairy cows (n = 6; 7-13 d in milk) and cows with fatty liver (n = 6; 7-11 d in milk). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old) were challenged with or without a mix of 1.2 mM FA to induce metabolic stress. In addition, hepatocytes were processed with 10 µmol/L of the cholesterol synthesis inhibitor simvastatin or 6 µmol/L of the cholesterol intracellular transport inhibitor U18666A with or without the 1.2 mM FA mix. To evaluate the role of cholesterol addition, hepatocytes were treated with 0.147 mg/mL methyl-β-cyclodextrin (MβCD + FA) or 0.147 mg/mL MβCD with or without 10 and 100 µmol/L cholesterol before incubation with FA (CHO10 + FA and CHO100 + FA). In vivo data from liver biopsies were analyzed by 2-tailed unpaired Student's t-test. Data from in vitro calf hepatocytes were analyzed by one-way ANOVA. Compared with healthy cows, blood plasma total cholesterol and plasma low-density lipoprotein cholesterol content in cows with fatty liver was markedly lower, whereas the hepatic total cholesterol content did not differ. In contrast, compared with healthy controls, the triacylglycerol content in the liver and the content of FA, β-hydroxybutyrate, and aspartate aminotransferase in the plasma of cows with fatty liver were greater. The results revealed that both fatty liver in vivo and challenge of calf hepatocytes with 1.2 mM FA in vitro led to greater mRNA and protein abundance of sterol regulatory element binding transcription factor 1 (SREBF1) and fatty acid synthase (FASN). In contrast, mRNA and protein abundance of sterol regulatory element binding transcription factor 2 (SREBF2), acyl coenzyme A-cholesterol acyltransferase, and ATP-binding cassette subfamily A member 1 (ABCA1) were lower. Compared with the FA group, the cholesterol synthesis inhibitor simvastatin led to greater protein abundance of microsomal triglyceride transfer protein and mRNA abundance of SREBF2, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), ACAT2, and lower ABCA1 and FASN protein abundance. In contrast, compared with the FA group, the cholesterol intracellular transport inhibitor U18666A + FA led to greater total cholesterol concentration and greater protein and mRNA abundance of FASN. Compared with the MβCD + FA group, the addition of 10 µmol/L cholesterol led to greater concentration of cholesteryl ester and excretion of apolipoprotein B100, and greater protein and mRNA abundance of ABCA1 and microsomal triglyceride transfer protein, and lower concentration of malondialdehyde. Overall, a reduction in cholesterol synthesis promoted FA metabolism in hepatocytes likely to relieve the oxidative stress caused by the high FA load. The data suggest that maintenance of normal cholesterol synthesis promotes very low-density lipoprotein excretion and can reduce lipid accumulation and oxidative stress in dairy cows that experience fatty liver.

Efektivitas Ekstrak Daun Annona Muricata Terhadap Penurunan Kolesterol Total Pada Lansia Dengan Hiperkolesterolemia Di Panti Werdha Mojopahit Mojokerto

The elderly experience a decrease in function, especially in the function of blood circulation. Blood circulation diseases and hormonal metabolism disorders experienced by the elderly include hypercholesterolemia. Hypercholesterolemia is a disorder of fat metabolism that triggers an increase in total blood cholesterol levels, namely> 200 mg/dl. The study aimed to determine the effectiveness of A. muricata leaf extract in decreasing total cholesterol levels in the elderly with hypercholesterolemia. Quantitative research method, using a quasi-experimental type with a group pretest-posttest design, with a sample size of 20 elderly. The sampling technique used purposive sampling, was carried out for a week consuming three times a day. The paired t-test showed a value of 8.73 and a p-value of 0.001. This study's results indicated a significant difference between the average cholesterol levels in the pre-test and post-test administration of Annona Muricata leaf extract. Compounds contained in soursop leaves, namely flavonoids, by reducing the activity of HMG-koa reductase (enzyme β-Hydroxy β-methylglutaryl-CoA) can reduce the activity of ACAT (enzyme acyl-Coa cholesterol acyltransferase), and reduce the absorption activity of cholesterol in the liver. Digestive tract. Saponin compounds in soursop leave lower cholesterol by binding to cholesterol in the intestinal lumen, thereby preventing the reabsorption of cholesterol. Saponins can bind with bile acids, which in turn will reduce the enterohepatic circulation of bile acids and cause an increase in the process of excretion of cholesterol.

Targeting sterol-O-acyltransferase 1 to disrupt cholesterol metabolism for cancer therapy.

Cholesterol esterification is often dysregulated in cancer. Sterol O-acyl-transferase 1 (SOAT1) plays an important role in maintaining cellular cholesterol homeostasis by catalyzing the formation of cholesterol esters from cholesterol and long-chain fatty acids in cells. Many studies have implicated that SOAT1 plays a vital role in cancer initiation and progression and is an attractive target for novel anticancer therapy. In this review, we provide an overview of the mechanism and regulation of SOAT1 in cancer and summarize the updates of anticancer therapy targeting SOAT1.

DNA methyltransferase 1 deficiency improves macrophage motility and wound healing by ameliorating cholesterol accumulation

Healing of the cutaneous wound requires macrophage recruitment at the sites of injury, where chemotactic migration of macrophages toward the wound is regulated by local inflammation. Recent studies suggest a positive contribution of DNA methyltransferase 1 (Dnmt1) to macrophage pro-informatory responses; however, its role in regulating macrophage motility remains unknown. In this study, myeloid-specific depletion of Dnmt1 in mice promoted cutaneous wound healing and de-suppressed the lipopolysaccharides (LPS)-inhibited macrophage motility. Dnmt1 inhibition in macrophages eliminated the LPS-stimulated changes in cellular mechanical properties in terms of elasticity and viscoelasticity. LPS increased the cellular accumulation of cholesterol in a Dnmt1-depedent manner; cholesterol content determined cellular stiffness and motility. Lipidomic analysis indicated that Dnmt1 inhibition altered the cellular lipid homeostasis, probably through down-regulating the expression of cluster of differentiation 36 CD36 (facilitating lipid influx) and up-regulating the expression of ATP-binding cassette transporter ABCA1 (mediating lipid efflux) and sterol O-acyltransferase 1 SOAT1 (also named ACAT1, catalyzing the esterification of cholesterol). Our study revealed a Dnmt1-dependent epigenetic mechanism in the control of macrophage mechanical properties and the related chemotactic motility, indicating Dnmt1 as both a marker of diseases and a potential target of therapeutic intervention for wound healing.

Sphingolipid desaturase DEGS1 is essential for mitochondria-associated membrane integrity.

Sphingolipids function as membrane constituents and signaling molecules, with crucial roles in human diseases, from neurodevelopmental disorders to cancer, best exemplified in the inborn errors of sphingolipid metabolism in lysosomes. The dihydroceramide desaturase Δ4-dihydroceramide desaturase 1 (DEGS1) acts in the last step of a sector of the sphingolipid pathway, de novo ceramide biosynthesis. Defects in DEGS1 cause the recently described hypomyelinating leukodystrophy-18 (HLD18) (OMIM #618404). Here, we reveal that DEGS1 is a mitochondria-associated endoplasmic reticulum membrane-resident (MAM-resident) enzyme, refining previous reports locating DEGS1 at the endoplasmic reticulum only. Using patient fibroblasts, multiomics, and enzymatic assays, we show that DEGS1 deficiency disrupts the main core functions of the MAM: (a) mitochondrial dynamics, with a hyperfused mitochondrial network associated with decreased activation of dynamin-related protein 1; (b) cholesterol metabolism, with impaired sterol O-acyltransferase activity and decreased cholesteryl esters; (c) phospholipid metabolism, with increased phosphatidic acid and phosphatidylserine and decreased phosphatidylethanolamine; and (d) biogenesis of lipid droplets, with increased size and numbers. Moreover, we detected increased mitochondrial superoxide species production in fibroblasts and mitochondrial respiration impairment in patient muscle biopsy tissues. Our findings shed light on the pathophysiology of HLD18 and broaden our understanding of the role of sphingolipid metabolism in MAM function.

Inhibition of ACAT as a Therapeutic Target for Alzheimer's Disease Is Independent of ApoE4 Lipidation.

APOE4, encoding apolipoprotein E4 (apoE4), is the greatest genetic risk factor for Alzheimer's disease (AD), compared to the common APOE3. While the mechanism(s) underlying APOE4-induced AD risk remains unclear, increasing the lipidation of apoE4 is an important therapeutic target as apoE4-lipoproteins are poorly lipidated compared to apoE3-lipoproteins. ACAT (acyl-CoA: cholesterol-acyltransferase) catalyzes the formation of intracellular cholesteryl-ester droplets, reducing the intracellular free cholesterol (FC) pool. Thus, inhibiting ACAT increases the FC pool and facilitates lipid secretion to extracellular apoE-containing lipoproteins. Previous studies using commercial ACAT inhibitors, including avasimibe (AVAS), as well as ACAT-knock out (KO) mice, exhibit reduced AD-like pathology and amyloid precursor protein (APP) processing in familial AD (FAD)-transgenic (Tg) mice. However, the effects of AVAS with human apoE4 remain unknown. In vitro, AVASinduced apoE efflux at concentrations of AVASmeasured in the brains of treated mice. AVAS treatment of male E4FAD-Tg mice (5xFAD+/-APOE4+/+) at6-8months had no effect on plasma cholesterol levels or distribution, the original mechanism forAVAS treatment of CVD. In the CNS, AVAS reduced intracellular lipid droplets, indirectly demonstrating target engagement. Surrogate efficacy was demonstrated by an increase in Morris water maze measures of memory and postsynaptic protein levels. Amyloid-beta peptide (Aβ) solubility/deposition and neuroinflammation were reduced, critical components of APOE4-modulated pathology. However, there was no increase in apoE4 levels or apoE4 lipidation, while amyloidogenic and non-amyloidogenic processing of APP were significantly reduced. This suggests that the AVAS-induced reduction in Aβ via reduced APP processing was sufficient to reduce AD pathology, as apoE4-lipoproteins remained poorly lipidated.

An ACAT inhibitor suppresses SARS-CoV-2 replication and boosts antiviral T cell activity.

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314.

Effect of the DGAT1 K232A mutation and breed on milk traits in cattle populations of Ethiopia

Non-conservative K232A substitution in the diacylglycerol acyl-CoA acyltransferase 1 (DGAT1) gene has been reported to explain variation in milk traits in cattle. The objective of this study was to estimate allele and genotype frequencies and to assess associations between K232A variants and milk yield and composition in cattle populations of Ethiopia. Blood samples for genomic DNA extraction and milk samples for analysis of milk components were collected from 92 randomly selected cattle of five Ethiopian breeds. Gene-specific primers were used to amplify 278 bp of the exon 8 region of DGAT1. Allele and genotype frequencies were calculated using Power Marker, and the GLM function of SAS software was used to assess the associations of detected genetic variation with milk traits. Boran * Holstein Friesian (HF) crosses produced a higher daily milk yield than the other breeds (p< 0.05), whereas the Boran and Begait breeds produced milk with higher fat and protein contents (p< 0.05), and Horro produced milk with a higher content of lactose, than the other breeds studied (p< 0.05). Alleles K and A and genotypes AA, KA, and KK were detected. The frequency of K232 ranged from 0.50 in Boran * HF crosses to 0.97 in the Horro population. The frequency of the KK and KA genotypes in the zebu population ranged from 0.50 to 0.94 and from 0.03 to 0.50, respectively. The AA genotype was associated with higher milk yield in Boran * HF crosses, whereas the KA genotype was associated with higher milk yield in the zebu populations (p< 0.05). The fat and lactose contents of milk produced with the KA genotype were lower than those of milk produced with the KK genotype in all genetic groups (p< 0.05). Substitution of one copy of the K allele led to a significant (p< 0.05) increase in fat content, of up to 0.81%, a decrease in daily milk yield of up to 3 L, and a decrease in lactose content of 0.58% in the sampled populations. The association study confirmed that the DGAT1 K232A marker had significant effects on daily milk yield, milk fat and lactose contents in the investigated cattle. These results suggested that the DGAT1 K232A marker may be utilized to accelerate future molecular breeding of dairy cattle, tropical zebu, their crossbreeds, after validation in a larger population.

Luteolin Mitigates Diabetic Dyslipidemia in Rats by Modulating ACAT-2, PPARα, SREBP-2 Proteins, and Oxidative Stress.

Diabetic dyslipidemia is a crucial link between type-2 diabetes mellitus (T2DM) and atherosclerotic cardiovascular diseases (ASCVD). Natural biologically active substances have been advocated as complementary remedies for ASCVD and T2DM. Luteolin, a flavonoid, exhibits antioxidant, hypolipidemic, and antiatherogenic effects. Hence, we aimed to determine influence of luteolin on lipid homeostasis and hepatic damage in rats with T2DM induced by high-fat-diet (HFD) and streptozotocin (STZ). After being fed HFD for 10days, male Wistar rats received 40mg/kg STZ intraperitoneal injection on 11th day. Seventy-two hours later, hyperglycemic rats (fasting glucose > 200mg/dL) were randomized into groups, and oral hydroxy-propyl-cellulose, atorvastatin (5mg/kg), or luteolin (50mg/kg or 100mg/kg) administered daily, while continuing HFD for 28days. Luteolin significantly ameliorated dyslipidemia levels and concomitantly improved atherogenic index of plasma in a dose-dependent manner. Increased levels of malondialdehyde and diminished levels of superoxide dismutase, catalase, and glutathione in HFD-STZ-diabetic rats were significantly regulated by luteolin. Luteolin significantly intensified PPARα expression while decreasing expression of acyl-coenzyme A:cholesterol acyltransferase-2 (ACAT-2) and sterol regulatory element binding protein-2 (SREBP-2) proteins. Moreover, luteolin effectively alleviated hepatic impairment in HFD-STZ-diabetic rats to near-normal control levels. The findings of the present study expound mechanisms by which luteolin mitigated diabetic dyslipidemia and alleviated hepatic impairment in HFD-STZ-diabetic rats by amelioration of oxidative stress, modulation of PPARα expression, and downregulation of ACAT-2 and SREBP-2. In conclusion, our results imply that luteolin may be efficacious in management of dyslipidemia in T2DM, and future research may be essential to substantiate our findings.

Evaluation of sterol‑o‑acyl transferase 1 and cholesterol ester levels in plasma, peritoneal fluid and tumor tissue of patients with endometrial cancer: A pilot study.

Endometrial cancer (EC) is the most prevalent gynecological malignancy. Abnormal accumulation of sterol-O-acyl transferase 1 (SOAT1) and SOAT1-mediated cholesterol ester (CE) contributes to cancer progression in various malignancies, including ovarian cancer. Therefore, it was hypothesized that similar molecular changes may occur in EC. The present study aimed to evaluate the diagnostic and/or prognostic potential of SOAT1 and CE in EC by: i) Determining SOAT1 and CE levels in plasma, peritoneal fluid and endometrial tissue from patients with EC and control subjects; ii) performing receiver operating characteristic curve analysis to determine diagnostic performance; iii) comparing SOAT1 and CE expression to that of the tumor proliferation marker Ki67; and iv) assessing the association between SOAT1 expression and survival. Enzyme-linked immunosorbent assay was used to determine the levels of SOAT1 protein in tissue, plasma and peritoneal fluid. The mRNA and protein expression levels of SOAT1 and Ki67 in tissues were detected by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. CE levels were determined colorimetrically in plasma and peritoneal fluid. SOAT1-associated survival data from the cBioPortal cancer genomics database were used to assess prognostic relevance. The results revealed that SOAT1 and CE levels were significantly elevated in tumor tissue and peritoneal fluid samples collected from the EC group. By contrast, the plasma levels of SOAT1 and CE in the EC and control groups were similar. Significant positive associations between CE and SOAT1, SOAT1/CE and Ki67, and SOAT1/CE and poor overall survival in patients with EC suggested that SOAT1/CE may be associated with malignancy, aggressiveness and poor prognosis. In conclusion, SOAT1 and CE may serve as potential biomarkers for prognosis and target-specific treatment of EC.

SOAT1 missense variant in two cats with sebaceous gland dysplasia

Spontaneously arisen hereditary diseases in domestic animals provide an excellent opportunity to study the physiological functions of the altered genes. We investigated two 4-month-old sibling domestic short haired kittens with dry dark debris around the eyes, nose, and ears, dark crusting on the legs and a thin poor hair coat. Skin biopsies revealed abnormal sebaceous gland morphology with lack of normal sebocyte arrangement and differentiation. Hair follicles had a distorted silhouette, interpreted as a change secondary to the observed sebaceous gland dysplasia. Whole genome sequencing on both affected kittens and 65 genetically diverse feline genomes was performed. Filtering for variants that were present in both kittens but absent from the control genomes revealed a homozygous missense variant in SOAT1, encoding sterol O-acyltransferase 1. The protein is localized in the endoplasmic reticulum and catalyzes the formation of cholesteryl esters, an essential component of sebum and meibum. The identified SOAT1:c.1531G > A variant is predicted to change a highly conserved glycine residue within the last transmembrane domain of SOAT1, p.Gly511Arg. In mice, variants in Soat1 or complete knockout of the gene lead to the “hair interior defect” (hid) or abnormal Meibomian glands, respectively. SOAT1:c.1531G > A represents a plausible candidate variant for the observed sebaceous gland dysplasia in both kittens of this study. The variant was not present in 10 additional cats with a similar clinical and histopathological phenotype suggesting genetic heterogeneity. SOAT1 variants should be considered as potential cause in hereditary sebaceous gland dysplasias of humans and domestic animals.

Abstract 2690: In vivo CRISPR screen identifies SOAT1 as a chemotherapy chemosensitizing target for non-small cell lung cancer (NSCLC)

Abstract Purpose: To identify druggable gene targets that would sensitize NSCLCs to available chemotherapy agents. Experimental Procedures: We developed a CRISPR-Cas9 lentivirus library of 12,474 sgRNAs targeting 660 FDA proved ‘druggable’ putative proteins (18 guides/gene) and investigated their potential to chemosensitize (“drop out” in the presence of taxane) the NSCLC lung adenocarcinoma (LUAD) line NCI-H2009 (TP53 and KRAS mutant) in parallel studies in vitro (tissue culture) & in vivo (xenografts). Key elements of our experiments were: the use low doses of paclitaxel (IC10 values for in vitro and in vivo doses that barely affected tumor growth) compared to control treatment; treatment schedules in vitro and in vivo that mirrored those used in patients; multiple biologic replicates for each transfection and drug selection; and large representation (2,000 cells/sgRNA) for each guide. At the end time point (1 month) we harvested multiple replicates of the taxane and control treatments and sequenced each to identify each guide that “remained” or “dropped out.” Data Summary: We were looking for guides that selectively dropped out in the setting of low dose taxane exposure comparing taxane to control treatment and in vitro to in vivo results. Of importance, we found little overlap between guides that dropped out during taxane exposure in tissue culture vs those that dropped out in xenografts. From the top 10 gene “sensitizers” that selectively dropped out only with taxane treatment and selectively dropped out in xenografts compared to 2D mass tissue culture, we focused on SOAT1 (Sterol O-Acyltransferase 1). SOAT1 is a key enzyme for lipid metabolism which mediates conversion of intracellular free cholesterol to cholesteryl esters which are then stored as lipid droplets. SOAT1 is a potential cancer therapeutic target with a clinically available inhibitor, avasimibe. H2009 cells with SOAT1 hemizygously removed (CRISPR) grew well in vitro and in vivo in the absence of chemotherapy treatment, but were dramatically sensitized to taxanes compared to parental H2009 cells. While avasimibe treatment, at concentrations achievable in patients, sensitized NSCLC to taxanes, this sensitization was not as dramatic as hemizygous removal by CRISPR. Of equal importance, SOAT1 H2009 hemizygous knockout (KO) cells were also dramatically sensitized to etoposide, pemetrexed, and gemcitabine, other chemotherapy agents used in NSCLC treatment. Mechanistically, RNAseq analysis identified G2/M cell cycle related gene sets as enriched in the H2009 SOAT1 KO xenografts compared to SOAT1 wildtype xenografts with paclitaxel treatment. Conclusions: Our results using CRISPR screening in vivo, identified SOAT1 as a critical therapeutic chemosensitizing target requiring only 50% inhibition of activity for sensitizing NSCLC to several chemotherapy agents routinely used in the clinic. Citation Format: Long Shan Li, Kenneth Huffman, Huiyu Li, Michael Peyton, Hyunsil Park, Kimberley Avila, Luc Girard, Mathew Augustine, Joshua T. Mendell, John D. Minna. In vivo CRISPR screen identifies SOAT1 as a chemotherapy chemosensitizing target for non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2690.

Abstract 5880: The role of SOAT1 on CD8+T cells-mediated immune response in ovarian cancer

Abstract Introduction: Sterol-O acyltransferase 1 (SOAT1) functions by converting cholesterol and acyl-CoA into cholesterol ester and coA-SH. High SOAT1 expression with its mediated tumorigenesis has been shown in multiple cancers. Studies showed that SOAT1 inhibition by its inhibitors augmented the outcome of immunotherapies, in terms of the immune checkpoint blockades, cancer vaccines, and CAR-T cells, by boosting the function of T cells. However, in order to have a better understanding of the role played by SOAT1 in the tumor microenvironment of ovarian cancer (OC) and optimize strategies of combination immunotherapy, it’s necessary to detect the role of SOAT1 in OC cells on T cell-mediated immune response. Methods: SOAT1 knockdown was manipulated by siRNAs transfection. SOAT1 was inhibited by avasimibe and OC cells were treated in different doses of avasimibe. qPCR was performed to detect gene expressions in ovarian cancer cells. T cells, isolated and activated from healthy donors, were cocultured with SOAT1-silenced and avasimibe pre-treated OC cells and their tumor-conditioned medium (TCM). Flow cytometry analysis was used to measure cytotoxic secretory molecules of human T cells, like IFN-r, TNF-a, and Granzyme B. Results: The downregulation of intracellular cytotoxic cytokines was shown in the T cells cocultured with SOAT1-silenced and avasimibe-pretreated OC cells or treated by their TCM, thereby impairing the cytotoxic capacity of T cells, compared to the corresponding control group. qPCR results showed that several immunosuppressive cytokines, like IL-6, IL-8, TGF- were upregulated in the SOAT1-silenced or avasimibe-treated OC cells. Conclusion: SOAT1 inhibition in ovarian tumor cells impairs CD8+ T cell cytotoxic function, to some extent, via the upregulation of immunosuppressive-related cytokines. Citation Format: Jiangnan He, Michelle K.Y. Siu, Runying Long, Ruiqian Zhang, Mingo M.H. Yung, Hextan Y. S. Ngan, Karen K.L. Chan. The role of SOAT1 on CD8+T cells-mediated immune response in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5880.

Microalgal glycerol-3-phosphate acyltransferase role in galactolipids and high-value storage lipid biosynthesis.

Glycerolipids are the most abundant lipids in microalgae, and glycerol-3-phosphate:acyl-CoA acyltransferase (GPAT) plays an important role in their biosynthesis. However, the biochemical and biological functions of algal GPAT remain poorly characterized. Here, we characterized the endoplasmic reticulum (ER)-associated GPAT of the model unicellular green alga Chlamydomonas reinhardtii (CrGPATer). Enzymatic assays indicated that CrGPATer is an sn-1 acyltransferase using a variety of acyl-CoAs as the acyl donor. Subcellular localization revealed that CrGPATer was associated with ER membranes and lipid droplets. We constructed overexpression (OE) and knockdown (KD) transgenic C. reinhardtii lines to investigate the in vivo function of CrGPATer. Lipidomic analysis indicated that CrGPATer OE enhanced the cellular content of galactolipids, especially monogalactosyldiacylglycerol, under nitrogen deficiency stress. Correspondingly, CrGPATer KD lines contained lower contents of galactolipids than the control. Feeding experiments with labeled phosphatidic acid revealed that the intermediate of the eukaryotic Kennedy pathway could be used for galactolipid biosynthesis in the chloroplasts. These results provided multiple lines of evidence that the eukaryotic Kennedy pathway mediated by CrGPATer may be involved in galactolipid biosynthesis in C. reinhardtii. OE of CrGPATer significantly increased the content of triacylglycerol and the yield of biomass. Moreover, the content and yield of 1, 3-olein-2-palmitin, a high-value lipid that can be used as an alternative for human milk fat in infant formula, were significantly enhanced in the OE transgenic lines. Taken together, this study provided insights into the biochemical and biological functions of CrGPATer and its potential as a genetic engineering target in functional lipid manufacturing.