Free Cholesterol Research Articles

Microbiome and lipidomic analysis reveal the interplay between skin bacteria and lipids in a cohort study.

Human skin acts as a protective barrier between the body and the external environment. Skin microbiome and intercellular lipids in the stratum corneum (SC) are essential for maintaining skin barrier function. However, the interplay between skin bacteria and the lipids is not fully understood. In this study, we characterized the skin microbiome and SC lipid profiles from the forearm and face in a cohort of 57 healthy participants. 16S rRNA gene sequencing showed the skin microbial composition is significantly different between body locations and genders. Female forearm samples have the highest microbial diversity. The relative abundance of Staphylococcus hominis, Micrococcus luteus, Corynebacterium tuberculostearicum, Finegoldia magna, and Moraxellaceae sp. are significantly higher in the forearm than the face. The predictive functional analysis of 16S rRNA gene sequencing by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) and ANCOM-BC showed different bacterial metabolic pathway profiles between body locations or genders, and identified 271 differential pathways, including arginine and polyamine biosynthesis, chorismate biosynthesis pathways, which are more abundant in the female forearm, and sulfur oxidation pathway, which is more abundant in the male face. The SC lipid profiles differ between the body locations as well. Total free fatty acids (FFA), cholesterol sulfate and sphingosine are more abundant in the face. Dihydro-/6-hydroxy/phyto-ceramides are more abundant in the forearm. The correlation analysis of 16S rRNA gene sequencing and lipids revealed novel interplay between the bacteria and skin lipids. Shannon entropy and S. hominis negatively correlated with FFA, cholesterol sulfate and sphingosine; while positively correlated with dihydro-/6-hydroxy/phyto-ceramides. The correlation of predictive pathway profiles and lipids identified pathways involved in amino acids metabolism, carbohydrates degradation, aromatic compounds metabolism and fatty acid degradation metabolism are positively correlated with dihydro-/6-hydroxy/phyto-ceramides and negatively correlated with FFA, cholesterol sulfate and sphingosine. This study provides insights on the potential correlation between skin microbiome and lipids.

The Role of Fatty Acid Synthase in the Vascular Smooth Muscle Cell to Foam Cell Transition.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

Mendelian Randomization of Blood Metabolites Suggests Circulating Glutamine Protects Against Late-Onset Alzheimer's Disease.

Late-onset Alzheimer's disease (LOAD) represents a growing health burden. Previous studies suggest that blood metabolite levels influence risk of LOAD. We used a genetics-based study design which may overcome limitations of other epidemiological studies to assess the influence of metabolite levels on LOAD risk. We applied Mendelian randomization (MR) to evaluate bi-directional causal effects using summary statistics from the largest genome-wide association studies (GWAS) of 249 blood metabolites (n = 115,082) and GWAS of LOAD (ncase = 21,982, ncontrol = 41,944). MR analysis of metabolites as exposures revealed a negative association of genetically-predicted glutamine levels with LOAD (Odds Ratio (OR) = 0.83, 95% CI = 0.73, 0.92) that was consistent in multiple sensitivity analyses. We also identified a positive association of genetically-predicted free cholesterol levels in small LDL (OR = 1.79, 95% CI = 1.36, 2.22) on LOAD. Using genetically-predicted LOAD as the exposure, we identified associations with phospholipids to total lipids ratio in large LDL (OR = 0.96, 95% CI = 0.94, 0.98), but not with glutamine, suggesting that the relationship between glutamine and LOAD is unidirectional. Our findings support previous evidence that higher circulating levels of glutamine may be a target for protection against LOAD.

Relevance of plasma lipoproteins and small metabolites in assessment of nutritional status among patients with severe injuries

BackgroundThis study aimed to identify plasma lipoproteins and small metabolites associated with high risk of malnutrition during intensive care unit (ICU) stay in patients with severe injuries. MethodsThis observational prospective exploratory study was conducted at two level-1 trauma centers in the Netherlands. Adult patients (aged ≥18 years) who were admitted to the ICU for more than 48 h between July 2018 and April 2022 owing to severe injuries (polytrauma, as defined by Injury Severity Scores of ≥16) caused by blunt trauma were eligible for inclusion. Partial least squares discriminant analysis was used to analyze the relationship of 112 lipoprotein-related components and 23 small metabolites with the risk of malnutrition (modified Nutrition Risk in Critically Ill score). Malnutrition was diagnosed based on Subjective Global Assessment scores. The relationship of lipoprotein properties and small metabolite concentrations with malnutrition (during ICU admission) was evaluated using mixed effects logistic regression. ResultsOverall, 51 patients were included. Lower (very) low-density lipoprotein ([V]LDL) (free) cholesterol and phospholipid levels, low particle number, and higher levels of LDL triglycerides were associated with a higher risk of malnutrition (variable importance in projection [VIP] value >1.5). Low levels of most (V)LDL and intermediate-density lipoprotein subfractions and high levels of high-density lipoprotein Apo-A1 were associated with the diagnosis of malnutrition (VIP value >1.5). Increased levels of dimethyl sulfone, trimethylamine N-oxide, creatinine, N, N-dimethylglycine, and pyruvic acid and decreased levels of creatine, methionine, and acetoacetic acid were also indicative of malnutrition (VIP value >1.5). Overall, 14 lipoproteins and 1 small metabolite were significantly associated with a high risk of malnutrition during ICU admission (P <0.05); however, the association did not persist after correcting the false discovery rate (P=0.35 for all). ConclusionIncreased triglyceride in several lipoprotein subfractions and decreased levels of other lipoprotein subfraction lipids and several small metabolites (involved in the homocysteine cycle, ketone body formation, and muscle metabolism) may be indicative of malnutrition risk. Following validation in larger cohorts, these indicators may guide institution of preventive nutritional measures in patients admitted to the ICU with severe injuries.

Can Triglycerides-HDL-C Ratio, HOMA-IR, ApoB, Non-HDL Cholesterol, and Free Cholesterol be Laboratory-Associated Flags of Female Coronary Atherosclerosis Regardless of Being Diabetic?

Aim The study aimed to evaluate biomarkers, ratios, and indexes as flags of coronary obstructions in women with established coronary atherosclerotic disease regardless of diabetes. Methods A cross-sectional study was conducted on 42 confirmed atherosclerotic coronary female patients, stratified into diabetic or not and scheduled for angioplasty or coronary graft bypass surgery. Blood samples were collected immediately before coronary intervention for laboratory determinations, such as glycemia, HbA1c, insulin, HDL-C, PON-1, free cholesterol, LDL-C, Apo A-1, Apo B, and TG. Results In diabetic patients, insulin was positively correlated with triglycerides (p &lt; 0.0108; r = 0.2009), apo B (p &lt; 0.0006; r = 0.3737), non-HDL cholesterol (p &lt; 0.0084; r = 0.2156), and free cholesterol (p &lt; 0.0084; r = 0.3251). Applying a linear regression model, insulin from diabetic patients showed an association with glycemia, triglycerides, and HOMA-IR (p &lt; 0.001, R2 = 0.9868), but in non-diabetics, the association was only found between insulin and HOMA-IR (p = 0.002, R2 = 0.9031). On the other hand, using triglycerides as a dependent variable, its association has been found in both groups, but only with HOMA-IR (diabetics: p = 0.006, R2 = 0.2504; non-diabetics: p = 0.014, R2 = 0.4697). Also, the TG/HDL-C ratio was higher than 2.5 in 90% of diabetics and 83.33% of non-diabetic patients. Conclusion The high prevalence of females with a TG/HDL-C ratio above 2.5, the association among insulin, HOMA-IR, and TG/HDL-C, and correlations with apoB, non-HDL-C, and free cholesterol, should be evaluated as flags of female precocious coronary atherosclerosis.

Abstract 4442: Associations between pre-diagnostic plasma metabolites and biliary tract cancer risk in the prospective UK Biobank cohort

Abstract Background Annually, approximately 200,000 people are diagnosed with biliary tract cancer (BTC). Most present with metastatic disease which encompasses a 5-year survival rate of &amp;lt;5%. Etiology is poorly understood though some studies suggest metabolic dysregulation may contribute to development. Previous evaluations of metabolites and BTC risk have primarily been limited to case-control studies, few metabolites, or post-diagnostic blood samples. Methods We evaluated 248,285 adult participants with metabolite data in the UK Biobank, a prospective cohort study. Participants were excluded (6.2%) primarily due to a baseline history of cancer. To reduce potential influences of reverse causation, those who developed cancer or died within one year after baseline blood collection were also removed. Metabolites were trimmed to two standard deviations, log-transformed (ln[x+0.5]), z-scored, and evenly divided into tertiles. Cox proportional hazards regression was implemented to evaluate associations of BTC risk per 1-standard deviation (SD) and across tertiles with hazard ratios (HR) and 95% confidence intervals (95% CI). Models were adjusted for age, sex, education, income, fasting time, and statin use. A p-value of 0.001 was considered statistically significant after multiple comparison adjustment (0.05 divided by 50, the number of independent tests). Results The analyzed cohort includes 232,781 UKB participants with a median age of 58, a median follow-up time of 11.8 years, and 268 first primary incident BTC cases. Multiple metabolites were significantly associated with BTC risk using continuous variables (per 1-SD increment). High triglyceride to total lipid ratios were associated with higher BTC risk, and the strongest association was for intermediate-density lipoproteins (the HR (95% CI) was 1.33 (1.15-1.54), p=0.0001). On the other hand, high cholesterol to total lipid ratios were associated with lower BTC risk, and the strongest association was for the free cholesterol to total lipids in small very-low-density lipoproteins (VLDL) (0.76 (0.66-0.88), p =0.0002). High glutamine was associated with lower BTC risk (0.68 (0.69-0.89), p =0.0003) as was the phospholipid to total lipid ratio in small VLDL (0.75 (0.65-0.87), p =0.0002). Analysis by tertile identified additional significant associations, including a higher polyunsaturated fatty acid to total fatty acid ratio being associated with lower BTC risk (the HR (95% CI) comparing the highest to lowest tertile, 0.50 (0.35-0.71), p-trend=0.0001). Notably, within polyunsaturated fatty acids, a higher omega-6 to omega-3 ratio trended toward higher risk (1.32 (0.94-1.85), p-trend=0.11). Conclusion These findings indicate circulating metabolites may be biomarkers for BTC and suggest that triglycerides, cholesterols, fatty acids, phospholipids, and glutamine may be involved in BTC etiology. Citation Format: Valerie Gunchick, Guochong Gia, Wei Zheng. Associations between pre-diagnostic plasma metabolites and biliary tract cancer risk in the prospective UK Biobank cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4442.

Aging and brain free cholesterol concentration on amyloid-β peptide accumulation in guinea pigs.

Alzheimer's disease is a complex multifactorial neurodegenerative disorder wherein age is a major risk factor. The appropriateness of the Hartley guinea pig (GP), which displays high sequence homologies of its amyloid-β (Aβ40 and Aβ42) peptides, Mdr1 and APP(amyloid precursor protein) and similarity in lipid handling to humans, was appraised among 9-40weeks old guinea pigs. Protein expression levels of P-gp (Abcb1) and Cyp46a1 (24(S)-hydroxylase) for Aβ40, and Aβ42 efflux and cholesterol metabolism, respectively, were decreased with age, whereas those for Lrp1 (low-density lipoprotein receptor related protein 1), Rage (receptor for advanced glycation endproducts) for Aβ efflux and influx, respectively, and Abca1 (the ATP binding cassette subfamily A member 1) for cholesterol efflux, were unchanged among the ages examined. There was a strong, negative correlation of the brain Aβ peptide concentrations and Abca1 protein expression levels with free cholesterol. The correlation of Aβ peptide concentrations with Cyp46a1 was, however, not significant, and concentrations of the 24(S)-hydroxycholesterol metabolite revealed a decreasing trend from 20weeks old toward 40weeks old guinea pigs. The composite data suggest a role for free cholesterol on brain Aβ accumulation. The decreases in P-gp and Lrp1 protein levels should further exacerbate the accumulation of Aβ peptides in guinea pig brain.

A two-sample Mendelian randomization study explores metabolic profiling of different glycemic traits

We assessed the causal relation of four glycemic traits and type 2 diabetes liability with 167 metabolites using Mendelian randomization with various sensitivity analyses and a reverse Mendelian randomization analysis. We extracted instruments for fasting glucose, 2-h glucose, fasting insulin, and glycated hemoglobin from the Meta-Analyses of Glucose and Insulin-related traits Consortium (n = 200,622), and those for type 2 diabetes liability from a meta-analysis of multiple cohorts (148,726 cases, 965,732 controls) in Europeans. Outcome data were from summary statistics of 167 metabolites from the UK Biobank (n = 115,078). Fasting glucose and 2-h glucose were not associated with any metabolite. Higher glycated hemoglobin was associated with higher free cholesterol in small low-density lipoprotein. Type 2 diabetes liability and fasting insulin were inversely associated with apolipoprotein A1, total cholines, lipoprotein subfractions in high-density-lipoprotein and intermediate-density lipoproteins, and positively associated with aromatic amino acids. These findings indicate hyperglycemia-independent patterns and highlight the role of insulin in type 2 diabetes development. Further studies should evaluate these glycemic traits in type 2 diabetes diagnosis and clinical management.

Millets based alternative sustainable cost-effective culture media for microbial growth

Millets are a rich source of starch, proteins, lipids, and other nutrients. This study aimed to assess whether millets can be used to formulate sustainable and economically viable culture media, thus potentially replacing the expensive traditional media used for growing microorganisms. Millet samples from Maharashtra, Rajasthan, and Tamil Nadu were assayed for their protein and lipid content. Sorghum vulgare (Jowar), Pennisetum glaucum (Bajra), and Eleusine coracana (Ragi) demonstrated high protein content ranging from 0.63–0.78 mg/ml. Using Thin Layer Chromatography, lipids extracted with hexane were fractionated into several bands and compared against standard fatty acids and cholesterol. Panicum miliaceum (Proso) and Setaria italica (Kang) showed the maximum levels of free fatty acids and cholesterol. Based on the protein, lipid, and nutrient content, millets were used in different compositions to formulate media for growing bacteria and fungi. A mixture of Ragi and Bajra, which serves as calcium and phosphate sources respectively, exhibited zones of phosphate solubilization, thus confirming its potential as an alternative to Pikovskaya medium, which is used to cultivate phosphate- solubilizing bacteria. A mixture of Varai and Rajgira, which serves as carbohydrate and protein sources respectively, showed luxurious growth of fungi, thus confirming its potential as an alternative to Sabouraud’s Agar medium. Phosphate-solubilising bacteria are utilized in biofertilizer formation, thereby contributing to increased agricultural productivity. Besides proving a sustainable, environmentally friendly, and cost-effective alternative, the use of millets for media preparation can boost the agriculture sector and the economy of farmers as well.

Hawthorn leaf flavonoids alleviate the deterioration of atherosclerosis by inhibiting SCAP-SREBP2-LDLR pathway through sPLA2-ⅡA signaling in macrophages in mice

Ethnopharmacological relevanceHawthorn leaves are a combination of the dried leaves of the Rosaceae plants, i.e., Crataegus pinnatifida Bge. or Crataegus pinnatifida Bge. var. major N. E. Br., is primarily cultivated in East Asia, North America, and Europe. hawthorn leaf flavonoids (HLF) are the main part of extraction. The HLF have demonstrated potential in preventing hypertension, inflammation, hyperlipidemia, and atherosclerosis. However, the potential pharmacological mechanism behind its anti-atherosclerotic effect has yet to be explored. Aim of the studyThe in vivo and in vitro effects of HLF on lipid-mediated foam cell formation were investigated, with a specific focus on the levels of secreted phospholipase A2 type IIA (sPLA2-II A) in macrophage cells. Materials and methodsThe primary constituents of HLF were analyzed using ultra-high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. In vivo, HLF, at concentrations of 5 mg/kg, 20 mg/kg, and 40 mg/kg, were administered to apolipoprotein E knockout mice (ApoE−/−) fed by high-fat diet (HFD) for 16 weeks. Aorta and serum samples were collected to identify lesion areas and lipids through mass spectrometry analysis to dissect the pathological process. RAW264.7 cells were incubated with oxidized low-density lipoprotein (ox-LDL) alone, or ox-LDL combined with different doses of HLF (100, 50, and 25 μg/ml), or ox-LDL plus 24-h sPLA2-IIA inhibitors, for cell biology analysis. Lipids and inflammatory cytokines were detected using biochemical analyzers and ELISA, while plaque size and collagen content of plaque were assessed by HE and the Masson staining of the aorta. The lipid deposition in macrophages was observed by Oil Red O staining. The expression of sPLA2-IIA and SCAP-SREBP2-LDLR was determined by RT-qPCR and Western blot analysis. ResultsThe chemical profile of HLF was studied using UPLC-Q-TOF-MS/MS, allowing the tentative identification of 20 compounds, comprising 1 phenolic acid, 9 flavonols and 10 flavones, including isovitexin, vitexin-4″-O-glucoside, quercetin-3-O-robibioside, rutin, vitexin-2″-O-rhamnoside, quercetin, etc. HLF decreased total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and non-high-density lipoprotein cholesterol (non-HDL-C) levels in ApoE−/− mice (P < 0.05), reduced ox-LDL uptake, inhibited level of inflammatory factors, such as IL-6, IL-8, TNF-α, and IL-1ꞵ (P < 0.001), and alleviated aortic plaques with a thicker fibrous cap. HLF effectively attenuated foam cell formation in ox-LDL-treated RAW264.7 macrophages, and reduced levels of intracellular TC, free cholesterol (FC), cholesteryl ester (CE), IL-6, TNF-α, and IL-1β (P < 0.001). In both in vivo and in vitro experiments, HLF significantly downregulated the expression of sPLA2-IIA, SCAP, SREBP2, LDLR, HMGCR, and LOX-1 (P < 0.05). Furthermore, sPLA2-IIA inhibitor effectively mitigated inflammatory release in RAW264.7 macrophages and regulated SCAP-SREBP2-LDLR signaling pathway by inhibiting sPLA2-IIA secretion (P < 0.05). ConclusionHLF exerted a protective effect against atherosclerosis through inhibiting sPLA2-IIA to diminish SCAP-SREBP2-LDLR signaling pathway, to reduce LDL uptake caused foam cell formation, and to slow down the progression of atherosclerosis in mice.

NMR-Based Analysis of Plasma Lipoprotein Subclass and Lipid Composition Demonstrate the Different Dietary Effects in ApoE-Deficient Mice.

Plasma lipid levels are commonly measured using traditional methods such as triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and cholesterol (CH). However, the use of newer technologies, such as nuclear magnetic resonance (NMR) with post-analysis platforms, has made it easier to assess lipoprotein profiles in research. In this study involving ApoE-deficient mice that were fed high-fat diets, significant changes were observed in TG, CH, free cholesterol (FC), and phospholipid (PL) levels within the LDL fraction. The varied proportions of TG in wild-type mice and CH, FC, and PL in ApoE-/- mice were strikingly different in very low-density lipoproteins (VLDL), LDL, intermediate-density lipoprotein (IDL), and HDL. This comprehensive analysis expands our understanding of lipoprotein subfractions and the impacts of the APOE protein and high-fat diet in mouse models. The new testing method allows for a complete assessment of plasma lipids and their correlation with genetic background and diet in mice.

PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST INDIAN PAPER WASP ROPALIDIA MARGINATA VENOM TOXINS AND THEIR EFFICACY IN THE REVERSAL OF TOXIC EFFECTS

Objective: In this study, albino mice were injected with a sub-lethal dosage of purified wasp Ropalidia Marginata venom toxins to assess the effectiveness of polyclonal anti-venom antibodies. Methods: To neutralize the toxic effects, polyclonal antibodies were generated by immunizing albino mice. The antibody underwent partial purification using ammonium sulphate treatment and octanoic acid precipitation. To detect the presence of antibodies in the antiserum, an immunodouble diffusion test was conducted using Ouchterlony's method (1962). This involved allowing both antigens and antibodies to diffuse radially towards each other from their respective wells. When they reached an equivalence zone, a precipitation complex of antigen and antibody became visible as a concentric band, indicating the development of the combination. To quantitatively determine the amount of antibodies in the antiserum, the equivalency zone approach was used. Results: Experimental mice were injected with a combination containing 400, 800, and 1200 µg of pure antibody, which had been treated serum biomolecules, including metabolic enzymes, completely reversed in the experimental with 40% of the LD50 of wasp venom the elevated serum parameters were glucose, pyruvic acid, lipid, protein and free amino acid, reached to normal (100%) in the treated with 40% of LD50 of the venom and polyclonal treated after 6 h of administration. Anti-serum treatment also successfully normalized the alteration in serum enzyme just after 4h. Similarly, anti-serum treatment also successfully normalized the alteration in serum enzyme just after 4h treated with 40% of LD50 of the venom. Serum ACP content was obtained as 125.35% after 40% of LD50 venom injection, which was get normalized up to 102.81% after 4 h of the anti-venom treatment. Serum ALP content of 114.8% elevation was reversed back to 102.40% after anti-venom treatment. The GPT level significantly reversed up to 102.5%, while it was 130% in the venom-treated mice. A complete reversal was obtained in GPT level, which was obtained as 104.54% in the venom-treated animal. Similarly, LDH which was elevated up to 112.45 % in venom-injected mice was successfully reversed up to 100.25% after anti-venom treatment. Similarly, Ache concentration was fully recovered after anti-venom treatment 6 h, all animals (group B-E) that had received 40% of the LD50 of venom treated with pure antiserum. Conclusion: The venom-injected group showed a complete restoration of serum protein, free amino acid, uric acid, cholesterol, pyruvic acid, total lipid, and glucose level in experimental mice.

Recent Progresses on Pathophysiology, Diagnosis, Therapeutic Modalities, and Management of Non-alcoholic Fatty Liver Disorder

Abstract: Non-alcoholic fatty liver disease (NAFLD) is currently the utmost common chronic liver disorder that happens through all age groups and is identified to occur in 14%-30% of the general population, demonstrating a critical and grossing clinical issue because of the growing incidence of obesity and overweight. From the histological aspect, it looks like alcoholic liver damage, but it happens in patients who avoid remarkable alcohol usage. NAFLD comprises a broad spectrum, ranging from benign hepatocellular steatosis to inflammatory nonalcoholic steatohepatitis (NASH), different levels of fibrosis, and cirrhosis. Patients with NASH are more susceptible to more rapid progression to cirrhosis and hepatocellular carcinoma. There is no single factor that drives proceeding from simple steatosis to NASH. However, a combination of multi parameters such as genetic background, gut microflora, intake of high fat/ fructose dietary contents or methionine/choline-deficient diet, and consequently accumulated hepatocellular lipids mainly including triglycerides and also other bio-analytes, such as free fatty acids, cholesterol, and phospholipids display a crucial role in disease promotion. NAFLD is related to overweight and insulin resistance (IR) and is regarded as the hepatic presentation of the metabolic syndrome, an amalgamation of medical statuses such as hyperlipidemia, hypertension, type 2 diabetes, and visceral obesity. Despite the increasing prevalence of this disease, which imposes a remarkable clinical burden, most affected patients remain undiagnosed in a timely manner, largely related to the asymptomatic entity of NAFLD patients and the unavailability of accurate and efficient noninvasive diagnostic tests. However, liver biopsy is considered a gold standard for NAFLD diagnosis, but due to being expensive and invasiveness is inappropriate for periodic disease screening. Some noninvasive monitoring approaches have been established recently for NAFLD assessment. In addition to the problem of correct disease course prediction, no effective therapeutic modalities are approved for disease treatment. Imaging techniques can commonly validate the screening and discrimination of NAFLD; nevertheless, staging the disease needs a liver biopsy. The present therapeutic approaches depend on weight loss, sports activities, and dietary modifications, although different insulin-sensitizing drugs, antioxidants, and therapeutic agents seem hopeful. This review aims to focus on the current knowledge concerning epidemiology, pathogenesis, and different biochemical experiments and imaging modalities applied to diagnose the different grades of NAFLD and its management, as well as new data about pharmacological therapies for this disorder.

Effects of Heat Stress on Breast Muscle Metabolomics and Lipid Metabolism Related Genes in Growing Broilers

With global warming and worsening climatic conditions, heat stress (HS) has become a significant challenge affecting the development of poultry production. In this study, we aimed to determine the effects of HS on breast muscle metabolomics and lipid metabolism-related genes in growing broilers. One hundred twenty 29-day-old Arbor Acres broilers were randomly divided into normal temperature (NT; 21 ± 1 °C) and heat stress (HS; 31 ± 1 °C) groups, with six replicates (ten birds in each replicate) in each group, raised for 14 days in two environment chambers at 60 ± 7% relative humidity. Compared with the broilers in the NT group, the average daily food intake, average daily gain and breast muscle yield in the HS group were significantly lower (p &lt; 0.05). The feed conversion ratio was significantly higher in the HS group (p &lt; 0.05). The concentrations of serum corticosterone, free fatty acids and cholesterol and the percentage of abdominal fat of broilers in the HS group were significantly higher (p &lt; 0.05) than the values of the broilers in the NT group. Untargeted breast muscle metabolome analysis revealed 14 upregulated differential metabolites, including glycerophosphocholine, and 27 downregulated differential metabolites, including taurine, in the HS group compared to the NT group; the HS group also displayed significant effects on six metabolic pathways compared to the NT group (p &lt; 0.05). The mRNA expression levels of peroxisome proliferator-activated receptor gamma coactivator-1-alpha, peroxisome proliferator-activated receptor alpha (PPARα) and ATP-binding cassette transporter A1 in the liver and breast muscles were significantly decreased in the HS group compared with the NT group (p &lt; 0.05). The collective findings reveal that HS can cause disorders in breast muscle lipid metabolism in broilers. The PPARα gene might be the key gene in the mechanism of the lipid metabolism that is induced by HS in breast muscle of broilers. These findings provide novel insights into the effects of HS on chicken growth.

Shotgun lipidomics of human subretinal fluids under rod-dominant retina reveals cone-dominated lipids

Subretinal fluid (SRF) accumulates between photoreceptor outer segments and retinal pigment epithelium during rhegmatogenous retinal detachment. Biomolecular components such as lipids originate from cells surrounding the SRF. Knowledge of the composition of these molecules in SRF potentially provides mechanistic insight into the physiologic transfer of lipids between retinal tissue compartments. Using mass spectrometry and tandem mass spectrometry analysis on an electrospray ionization quadrupole-time-of-flight mass spectrometer, we identified a total of 115 lipid molecular species of 11 subclasses and 9 classes in two samples from two patients with rhegmatogenous retinal detachment. These included 47 glycerophosphocholines, 6 glycerophosphoethanolamines, 1 glycerophosphoinositol, 18 sphingomyelins, 9 cholesteryl esters, free cholesterol, 3 ceramides, 22 triacylglycerols and 8 free fatty acids. Glycerophosphocholines were of the highest intensity. By minimizing the formation of different adduct forms or clustering ions of different adducts, we determined the relative intensity of lipid molecular species within the same subclasses. The profiles were compared with those of retinal cells available in the published literature. The glycerophosphocholine profile of SRF was similar to that of cone outer segments, suggesting that outer segment degradation products are constitutively released into the interphotoreceptor matrix, appearing in SRF during detachment. This hypothesis was supported by the retinal distributions of corresponding lipid synthases’ mRNA expression obtained from an online resource based on publicly available single-cell sequencing data. In contrast, based on lipid profiles and relevant gene expression in this study, the sources of free cholesterol and cholesteryl esters in SRF appeared more ambiguous, possibly reflecting that outer retina takes up plasma lipoproteins. Further studies to identify and quantify lipids in SRF will help better understand etiology of diseases relevant to outer retina.

Discrimination of lipid composition and cellular localization in human liver tissues by stimulated Raman scattering microscopy.

The molecular mechanisms driving the progression from nonalcoholic fatty liver (NAFL) to fibrosing steatohepatitis (NASH) are insufficiently understood. Techniques enabling the characterization of different lipid species with both chemical and spatial information can provide valuable insights into their contributions to the disease progression. We extend the utility of stimulated Raman scattering (SRS) microscopy to characterize and quantify lipid species in liver tissue sections from patients with NAFL and NASH. We applied a dual-band hyperspectral SRS microscopy system for imaging tissue sections in both the C-H stretching and fingerprint regions. The same sections were imaged with polarization microscopy for detecting birefringent liquid crystals in the tissues. Our imaging and analysis pipeline provides accurate classification and quantification of free cholesterol, saturated cholesteryl esters (CEs), unsaturated CE, and triglycerides in liver tissue sections. The subcellular resolution enables investigations of the heterogeneous distribution of saturated CE, which has been under-examined in previous studies. We also discovered that the birefringent crystals, previously found to be associated with NASH development, are predominantly composed of saturated CE. Our method allows for a detailed characterization of lipid composition in human liver tissues and enables further investigation into the potential mechanism of NASH progression.

Mobilization of cholesterol induces the transition from quiescence to growth in Caenorhabditis elegans through steroid hormone and mTOR signaling

Recovery from the quiescent developmental stage called dauer is an essential process in C. elegans and provides an excellent model to understand how metabolic transitions contribute to developmental plasticity. Here we show that cholesterol bound to the small secreted proteins SCL-12 or SCL-13 is sequestered in the gut lumen during the dauer state. Upon recovery from dauer, bound cholesterol undergoes endocytosis into lysosomes of intestinal cells, where SCL-12 and SCL-13 are degraded and cholesterol is released. Free cholesterol activates mTORC1 and is used for the production of dafachronic acids. This leads to promotion of protein synthesis and growth, and a metabolic switch at the transcriptional level. Thus, mobilization of sequestered cholesterol stores is the key event for transition from quiescence to growth, and cholesterol is the major signaling molecule in this process.

Filipin complex-reactive brain lesions: a cautionary tale.

Filipin complex is an autooxidation-prone fluorescent histochemical stain used in the diagnosis of Niemann-Pick Disease Type C (NP-C), a neurodegenerative lysosomal storage disorder. It is also widely used by researchers examining the distribution and accumulation of unesterified cholesterol in cell and animal models of neurodegenerative diseases including NP-C and Sanfilippo syndrome (mucopolysaccharidosis IIIA; MPS IIIA). Recently, it has been suggested to be useful in studying Alzheimer's and Huntington's disease. Given filipin's susceptibility to photobleaching, we sought to establish a quantitative biochemical method for free cholesterol measurement. Brain tissue from mice with MPS IIIA was stained with filipin. Total and free cholesterol in brain homogenates was measured using a commercially available kit and a quantitative LC-MS/MS assay was developed. Gangliosides GM1, GM2 and GM3 were also quantified using LC-MS/MS. As anticipated, the MPS IIIA mouse brain displayed large numbers of filipin-positive intra-cytoplasmic inclusions, presumptively endo-lysosomes. Challenging the prevailing dogma, however, we found no difference in the amount of free cholesterol in MPS IIIA mouse brain homogenates cf. control tissue, using either the fluorometric kit or LC-MS/MS assay. Filipin has previously been reported to bind to GM1 ganglioside, however, this lipid does not accumulate in MPS IIIA cells/tissues. Using a fluorometric assay, we demonstrate for the first time that filipin cross-reacts with both GM2 and GM3 gangliosides, explaining the filipin-reactive inclusions observed in MPS IIIA brain cells. Filipin is not specific for free cholesterol, and positive staining in any setting should be interpreted with caution.

Abstract B081: SOAT1 as a targetable KRAS dependency in PDAC

Abstract Metabolic rewiring is an emerging hallmark of neoplasia. In pancreatic ductal adenocarcinoma (PDAC), KRAS alters myriad metabolic programs to promote cellular transformation. Among them, the cholesterol synthesis pathway has emerged as a vulnerability of cancer cells due to the importance of cholesterol in membrane fluidity and the production of isoprenoid intermediates that enable KRAS membrane localization and activation in pancreatic cancer. We recently reported that the non-essential gene SOAT1 (Sterol-O-Acyl transferase 1), encoding an enzyme that esterifies free cholesterol into an insoluble form for storage and transport, is markedly upregulated in metastatic PDAC cells. SOAT1 promotes the hyperactivation of the mevalonate synthesis pathway due to loss of negative feedback by free cholesterol, thereby leading to increased production of isoprenoids such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are required for RAS and RHO membrane localization and activity, respectively. Genetic ablation of Soat1 reduced RAS and RHO membrane localization and inhibited cellular proliferation and mitochondrial function in vitro, and these effects were largely rescued by exogenous FPP and GGPP. Therefore, our study nominated SOAT1 as a new candidate gene for therapeutic targeting. Based on SuFEx Click Chemistry (ASCC) technology, we have designed and synthesized a panel of drug-like candidates. The first series of Soat1 probes selectively impair the proliferation of PDAC cells that express SOAT1, reduce SOAT1 protein content in cells, and covalently bind to SOAT1. In mice, the most active SOAT1 probes were tolerated in a 28-day daily dosing study up to 120 mg/kg. Marked tumor growth inhibition was noted in mice bearing PDAC xenografts and GEMM mice treated as monotherapy with 15 mg/kg/d of the most active SOAT1 probe, and tissue analysis demonstrated tumor necrosis, decreased proliferation, and increased apoptosis. Mechanistically, SOAT1 loss or inhibition targets multiple important components of cellular transformation in PDAC including KRAS, RHO, and mitochondrial biology, and our work suggests that it is an attractive target to pursue for further drug development in this cancer. Citation Format: Wenjun Lan, Shoujun Sun, Jeremy Nigri, Josh Homer, Young Park, Astrid Deschenes, Paolo Cifani, Justin Cross, Victoria Gaeth, Sunny Kim, Shawn Ting, Rachel Rubino, John Moses, David Tuveson. SOAT1 as a targetable KRAS dependency in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B081.

Gut microbiota in combination with blood metabolites reveals characteristics of the disease cluster of coronary artery disease and cognitive impairment: a Mendelian randomization study.

The coexistence of coronary artery disease (CAD) and cognitive impairment has become a common clinical phenomenon. However, there is currently limited research on the etiology of this disease cluster, discovery of biomarkers, and identification of precise intervention targets. We explored the causal connections between gut microbiota, blood metabolites, and the disease cluster of CAD combined with cognitive impairment through two-sample Mendelian randomization (TSMR). Additionally, we determine the gut microbiota and blood metabolites with the strongest causal associations using Bayesian model averaging multivariate Mendelian randomization (MR-BMA) analysis. Furthermore, we will investigate the mediating role of blood metabolites through a two-step Mendelian randomization design. We identified gut microbiota that had significant causal associations with cognitive impairment. Additionally, we also discovered blood metabolites that exhibited significant causal associations with both CAD and cognitive impairment. According to the MR-BMA results, the free cholesterol to total lipids ratio in large very low density lipoprotein (VLDL) was identified as the key blood metabolite significantly associated with CAD. Similarly, the cholesteryl esters to total lipids ratio in small VLDL emerged as the primary blood metabolite with a significant causal association with dementia with lewy bodies (DLB). For the two-step Mendelian randomization analysis, we identified blood metabolites that could potentially mediate the association between genus Butyricicoccus and CAD in the potential causal links. Our study utilized Mendelian randomization (MR) to identify the gut microbiota features and blood metabolites characteristics associated with the disease cluster of CAD combined with cognitive impairment. These findings will provide a meaningful reference for the identification of biomarkers for the disease cluster of CAD combined with cognitive impairment as well as the discovery of targets for intervention to address the problems in the clinic.