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Nutrients Mar 2024The liver plays a crucial role in glucose metabolism. Obesity and a diet rich in fats (HFD) contribute to the accumulation of intracellular lipids. The aim of the study...
The liver plays a crucial role in glucose metabolism. Obesity and a diet rich in fats (HFD) contribute to the accumulation of intracellular lipids. The aim of the study was to explore the involvement of acyl-CoA synthetase 1 (ACSL1) in bioactive lipid accumulation and the induction of liver insulin resistance (InsR) in animals fed an HFD. The experiments were performed on male C57BL/6 mice divided into the following experimental groups: 1. Animals fed a control diet; 2. animals fed HFD; and 3. HFD-fed animals with the hepatic ACSL1 gene silenced through a hydrodynamic gene delivery technique. Long-chain acyl-CoAs, sphingolipids, and diacylglycerols were measured by LC/MS/MS. Glycogen was measured by means of a commercially available kit. The protein expression and phosphorylation state of the insulin pathway was estimated by Western blot. HFD-fed mice developed InsR, manifested as an increase in fasting blood glucose levels (202.5 mg/dL vs. 130.5 mg/dL in the control group) and inhibition of the insulin pathway, which resulted in an increase in the rate of gluconeogenesis (0.420 vs. 0.208 in the control group) and a decrease in the hepatic glycogen content (1.17 μg/mg vs. 2.32 μg/mg in the control group). Hepatic ACSL1 silencing resulted in decreased lipid content and improved insulin sensitivity, accounting for the decreased rate of gluconeogenesis (0.348 vs. 0.420 in HFD) and the increased glycogen content (4.3 μg/mg vs. 1.17 μg/mg in HFD). The elevation of gluconeogenesis and the decrease in glycogenesis in the hepatic tissue of HFD-fed mice resulted from cellular lipid accumulation. Inhibition of lipid synthesis through silencing ACSL1 alleviated HFD-induced hepatic InsR.
Topics: Male; Animals; Mice; Mice, Inbred C57BL; Insulin Resistance; Tandem Mass Spectrometry; Liver; Diglycerides; Glycogen; Insulins
PubMed: 38613036
DOI: 10.3390/nu16071003 -
Frontiers in Veterinary Science 2024Pulmonary hypertension (PH) is a common complication in dogs with myxomatous mitral valve disease (MMVD), characterized by elevated blood pressure in pulmonary artery....
BACKGROUND
Pulmonary hypertension (PH) is a common complication in dogs with myxomatous mitral valve disease (MMVD), characterized by elevated blood pressure in pulmonary artery. Echocardiography is a reliable technique for PH diagnosis in veterinary medicine. However, it is limited to use as an early detection method. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has found extensive application in the discovery of serum protein biomarkers for various diseases. The objective of this study was to identify serum proteins in healthy control dogs and MMVD dogs both with and without PH using LC-MS/MS.
MATERIALS AND METHODS
In this research, a total of 81 small-breed dogs participated, and they were categorized into three groups: the control ( = 28), MMVD ( = 24) and MMVD+PH ( = 29) groups. Serum samples were collected and analyzed by LC-MS/MS.
RESULTS
Differentially expressed proteins were identified, and the upregulated and downregulated proteins in MMVD+PH group including Myomesin 1 (MYOM1) and Histone deacetylase 7 (HDAC7), Pleckstrin homology domain containing M3 (PLEKHM3), Diacylglycerol lipase alpha (DAGLA) and Tubulin tyrosine ligase like 6 (TTLL6) were selected as proteins of interest in MMVD dogs with PH.
CONCLUSION
Different types of proteins have been identified in healthy dogs and MMVD dogs with and without PH. Additional studies are needed to investigate the potential of these proteins as biomarkers for PH in dogs with MMVD.
PubMed: 38596466
DOI: 10.3389/fvets.2024.1327453 -
Journal of Ethnopharmacology Jul 2024Hepatic steatosis, a hallmark of non-alcoholic fatty liver disease (NAFLD), represents a significant global health issue. Liver lipidomics has garnered increased focus...
ETHNOPHARMACOLOGICAL RELEVANCE
Hepatic steatosis, a hallmark of non-alcoholic fatty liver disease (NAFLD), represents a significant global health issue. Liver lipidomics has garnered increased focus recently, highlighting Traditional Chinese Medicine's (TCM) role in mitigating such conditions through lipid metabolism regulation. The Zuogui Jiangtang Qinggan Formula (ZGJTQGF), a longstanding TCM regimen for treating Type 2 Diabetes Mellitus (T2DM) with NAFLD, lacks a definitive mechanism for its lipid metabolism regulatory effects.
AIM OF THE STUDY
This research aims to elucidate ZGJTQGF's mechanism on lipid metabolism in T2DM with NAFLD.
MATERIALS AND METHODS
The study, utilized db/db mice to establish T2DM with NAFLD models. Evaluations included Hematoxylin-Eosin (HE) and Oil Red O stainedstaining of liver tissues, alongside biochemical lipid parameter analysis. Liver lipidomics and Western blotting further substantiated the findings, systematically uncovering the mechanism of action mechanism.
RESULTS
ZGJTQGF notably reduced body weight, and Fasting Blood Glucose (FBG), enhancing glucose tolerance in db/db mice. HE, and Oil Red O staining, complemented by biochemical and liver lipidomics analyses, confirmed ZGJTQGF's efficacy in ameliorating liver steatosis and lipid metabolism anomalies. Lipidomics identified 1571 significantly altered lipid species in the model group, primarily through the upregulation of triglycerides (TG) and diglycerides (DG), and the downregulation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Post-ZGJTQGF treatment, 496 lipid species were modulated, with increased PC and PE levels and decreased TG and DG, showcasing significant lipid metabolism improvement in T2DM with NAFLD. Moreover, ZGJTQGF's influence on lipid synthesis-related proteins was observed, underscoring its anti-steatotic impact through liver lipidomic alterations and offering novel insights into hepatic steatosis pathogenesis.
CONCLUSIONS
Liver lipidomics analysis combined with protein verification further demonstrated that ZGJTQGF could ameliorate the lipid disturbance of TG, DG, PC, PE in T2DM with NAFLD, as well as improve fatty acid and cholesterol synthesis and metabolism through De novo lipogenesis pathway.
Topics: Animals; Non-alcoholic Fatty Liver Disease; Lipidomics; Diabetes Mellitus, Type 2; Drugs, Chinese Herbal; Liver; Male; Lipid Metabolism; Mice; Mice, Inbred C57BL; Blood Glucose
PubMed: 38588985
DOI: 10.1016/j.jep.2024.118160 -
MBio May 2024Systemic infections by spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug...
UNLABELLED
Systemic infections by spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from is a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an nucleotidase-coupled malachite-green-based high throughput assay for purified Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average ' score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects against cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting Cho1 function by inducing phenotypes consistent with the mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound.
IMPORTANCE
Fungal phosphatidylserine synthase (Cho1) is a logical antifungal target due to its crucial role in the virulence and viability of various fungal pathogens, and since it is absent in humans, drugs targeted at Cho1 are less likely to cause toxicity in patients. Using fungal Cho1 as a model, there have been two unsuccessful attempts to discover inhibitors for Cho1 homologs in whole-cell screens prior to this study. The compounds identified in these attempts do not act directly on the protein, resulting in the absence of known Cho1 inhibitors. The significance of our research is that we developed a high-throughput target-based assay and identified the first Cho1 inhibitor, CBR-5884, which acts both on the purified protein and its function in the cell. This molecule acts as a competitive inhibitor with a value of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase.
Topics: Candida albicans; Antifungal Agents; CDPdiacylglycerol-Serine O-Phosphatidyltransferase; Enzyme Inhibitors; High-Throughput Screening Assays; Small Molecule Libraries; Microbial Sensitivity Tests; Fungal Proteins; Phosphatidylserines; Furans; Thiophenes
PubMed: 38587428
DOI: 10.1128/mbio.00633-24 -
BioRxiv : the Preprint Server For... Mar 2024The protein kinase C (PKC) family of serine/threonine kinases, which consist of three distinctly regulated subfamilies, have long been established as critical for a...
The protein kinase C (PKC) family of serine/threonine kinases, which consist of three distinctly regulated subfamilies, have long been established as critical for a variety of cellular functions. However, how PKC enzymes are regulated at different subcellular locations, particularly at emerging signaling hubs such as the ER, lysosome, and Par signaling complexes, is unclear. Here, we present a sensitive Excitation Ratiometric (ExRai) C Kinase Activity Reporter (ExRai-CKAR2) that enables the detection of minute changes in subcellular PKC activity. Using ExRai-CKAR2 in conjunction with an enhanced diacylglycerol (DAG) biosensor capable of detecting intracellular DAG dynamics, we uncover the differential regulation of PKC isoforms at distinct subcellular locations. We find that G-protein coupled receptor (GPCR) stimulation triggers sustained PKC activity at the ER and lysosomes, primarily mediated by Ca sensitive conventional PKC (cPKC) and novel PKC (nPKC), respectively, with nPKC showing high basal activity due to elevated basal DAG levels on lysosome membranes. The high sensitivity of ExRai-CKAR2, targeted to either the cytosol or Par-complexes, further enabled us to detect previously inaccessible endogenous atypical PKC (aPKC) activity in 3D organoids. Taken together, ExRai-CKAR2 is a powerful tool for interrogating PKC regulation in response to physiological stimuli.
PubMed: 38586003
DOI: 10.1101/2024.03.29.587373 -
Cell Chemical Biology Mar 2024Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are...
Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCβ (opto-PLCβ). Opto-PLCβ uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCβ triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCβ can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCβ offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo.
PubMed: 38582083
DOI: 10.1016/j.chembiol.2024.03.001 -
Lipids in Health and Disease Apr 2024Pulmonary fibrosis (PF) is a severe pulmonary disease with limited available therapeutic choices. Recent evidence increasingly points to abnormal lipid metabolism as a... (Review)
Review
Pulmonary fibrosis (PF) is a severe pulmonary disease with limited available therapeutic choices. Recent evidence increasingly points to abnormal lipid metabolism as a critical factor in PF pathogenesis. Our latest research identifies the dysregulation of low-density lipoprotein (LDL) is a new risk factor for PF, contributing to alveolar epithelial and endothelial cell damage, and fibroblast activation. In this study, we first integrative summarize the published literature about lipid metabolite changes found in PF, including phospholipids, glycolipids, steroids, fatty acids, triglycerides, and lipoproteins. We then reanalyze two single-cell RNA-sequencing (scRNA-seq) datasets of PF, and the corresponding lipid metabolomic genes responsible for these lipids' biosynthesis, catabolism, transport, and modification processes are uncovered. Intriguingly, we found that macrophage is the most active cell type in lipid metabolism, with almost all lipid metabolic genes being altered in macrophages of PF. In type 2 alveolar epithelial cells, lipid metabolic differentially expressed genes (DEGs) are primarily associated with the cytidine diphosphate diacylglycerol pathway, cholesterol metabolism, and triglyceride synthesis. Endothelial cells are partly responsible for sphingomyelin, phosphatidylcholine, and phosphatidylethanolamines reprogramming as their metabolic genes are dysregulated in PF. Fibroblasts may contribute to abnormal cholesterol, phosphatidylcholine, and phosphatidylethanolamine metabolism in PF. Therefore, the reprogrammed lipid profiles in PF may be attributed to the aberrant expression of lipid metabolic genes in different cell types. Taken together, these insights underscore the potential of targeting lipid metabolism in developing innovative therapeutic strategies, potentially leading to extended overall survival in individuals affected by PF.
Topics: Humans; Pulmonary Fibrosis; Single-Cell Gene Expression Analysis; Lipid Metabolism; Endothelial Cells; Phospholipids; Cholesterol; Phosphatidylcholines
PubMed: 38570797
DOI: 10.1186/s12944-024-02062-8 -
Journal of Lipid Research May 2024Intestinal epithelial cells convert excess fatty acids into triglyceride (TAG) for storage in cytoplasmic lipid droplets and secretion in chylomicrons. Nuclear lipid...
Intestinal epithelial cells convert excess fatty acids into triglyceride (TAG) for storage in cytoplasmic lipid droplets and secretion in chylomicrons. Nuclear lipid droplets (nLDs) are present in intestinal cells but their origin and relationship to cytoplasmic TAG synthesis and secretion is unknown. nLDs and related lipid-associated promyelocytic leukemia structures (LAPS) were abundant in oleate-treated Caco2 but less frequent in other human colorectal cancer cell lines and mouse intestinal organoids. nLDs and LAPS in undifferentiated oleate-treated Caco2 cells harbored the phosphatidate phosphatase Lipin1, its product diacylglycerol, and CTP:phosphocholine cytidylyltransferase (CCT)α. CCTα knockout Caco2 cells had fewer but larger nLDs, indicating a reliance on de novo PC synthesis for assembly. Differentiation of Caco2 cells caused large nLDs and LAPS to form regardless of oleate treatment or CCTα expression. nLDs and LAPS in Caco2 cells did not associate with apoCIII and apoAI and formed dependently of microsomal triglyceride transfer protein expression and activity, indicating they are not derived from endoplasmic reticulum luminal LDs precursors. Instead, undifferentiated Caco2 cells harbored a constitutive pool of nLDs and LAPS in proximity to the nuclear envelope that expanded in size and number with oleate treatment. Inhibition of TAG synthesis did affect the number of nascent nLDs and LAPS but prevented their association with promyelocytic leukemia protein, Lipin1α, and diacylglycerol, which instead accumulated on the nuclear membranes. Thus, nLD and LAPS biogenesis in Caco2 cells is not linked to lipoprotein secretion but involves biogenesis and/or expansion of nascent nLDs by de novo lipid synthesis.
Topics: Humans; Caco-2 Cells; Nuclear Envelope; Lipid Droplets; Animals; Mice; Cell Differentiation; Choline-Phosphate Cytidylyltransferase; Oleic Acid; Triglycerides
PubMed: 38570093
DOI: 10.1016/j.jlr.2024.100540 -
Scientific Reports Apr 2024Alzheimer's disease (AD) is a neurodegenerative disease that commonly causes dementia. Identifying biomarkers for the early detection of AD is an emerging need, as brain...
Alzheimer's disease (AD) is a neurodegenerative disease that commonly causes dementia. Identifying biomarkers for the early detection of AD is an emerging need, as brain dysfunction begins two decades before the onset of clinical symptoms. To this end, we reanalyzed untargeted metabolomic mass spectrometry data from 905 patients enrolled in the AD Neuroimaging Initiative (ADNI) cohort using MS-DIAL, with 1,304,633 spectra of 39,108 unique biomolecules. Metabolic profiles of 93 hydrophilic metabolites were determined. Additionally, we integrated targeted lipidomic data (4873 samples from 1524 patients) to explore candidate biomarkers for predicting progressive mild cognitive impairment (pMCI) in patients diagnosed with AD within two years using the baseline metabolome. Patients with lower ergothioneine levels had a 12% higher rate of AD progression with the significance of P = 0.012 (Wald test). Furthermore, an increase in ganglioside (GM3) and decrease in plasmalogen lipids, many of which are associated with apolipoprotein E polymorphism, were confirmed in AD patients, and the higher levels of lysophosphatidylcholine (18:1) and GM3 d18:1/20:0 showed 19% and 17% higher rates of AD progression, respectively (Wald test: P = 3.9 × 10 and 4.3 × 10). Palmitoleamide, oleamide, diacylglycerols, and ether lipids were also identified as significantly altered metabolites at baseline in patients with pMCI. The integrated analysis of metabolites and genomics data showed that combining information on metabolites and genotypes enhances the predictive performance of AD progression, suggesting that metabolomics is essential to complement genomic data. In conclusion, the reanalysis of multiomics data provides new insights to detect early development of AD pathology and to partially understand metabolic changes in age-related onset of AD.
Topics: Humans; Alzheimer Disease; Neurodegenerative Diseases; Multiomics; Neuroimaging; Biomarkers; Lipids; Cognitive Dysfunction; Disease Progression
PubMed: 38565541
DOI: 10.1038/s41598-024-56837-1 -
Journal of Oleo Science 2024High-performance size exclusion chromatography (HPSEC) equipped with an evaporative light scattering detector (ELSD) was utilized for characterization of palm fatty acid...
High-performance size exclusion chromatography (HPSEC) equipped with an evaporative light scattering detector (ELSD) was utilized for characterization of palm fatty acid distillate (PFAD) and its esterified products, with a particular focus on lipid profiles and diacylglycerol (DAG) regioisomers. The separation of triacylglycerol (TAG), DAG, monoacylglycerol (MAG), and free fatty acid (FFA) was achieved through a single 100-Å Phenogel column, coupled with a 2-cm C18 guard, utilizing toluene/acetic acid (100:0.25, v/v) as the mobile phase. This separation was based on size sieving principles and the interactions between the hydroxyl group(s) and the Phenogel matrix. The limit of detection (LOD) and limit of quantification (LOQ) for the esterified PFAD products analyzed by this method fell within the range of 4.8-5.5 μg/mL and 14.7-16.7 μg/mL, respectively. Additionally, the same column, paired with a 2-cm silica guard and a mobile phase comprised of toluene/isooctane/acetic acid (35:65:0.15, v/v/v), was used for the characterization of DAG regioisomers within the esterified PFAD. LODs and LOQs for sn-1,3-DAG and sn- 1,2-DAG were determined to be 39.2 and 118.7 μg/mL, and 32.8 and 99.5 μg/mL, respectively. Investigation of esterified PFAD products prepared using 4% HSO at 120°C. After 2 h, the analysis revealed the highest MAG content at 31.85%, accompanied by 51.54% DAG, 2.35% TAG, and a residual 14.27% FFA. Notably, as the reaction time extended, the MAG content decreased, while both DAG and TAG levels exhibited an increasing trend. Further examination of DAG regioisomers during PFAD esterification, under varying catalyst concentrations (2-10%) and reaction temperatures (80-140°C), demonstrated a significant increase in the percentage of sn-1,3-DAG, inversely correlated with the reduction in FFA from 2% H SO and 80°C onwards. Remarkably, the percentage of sn-1,2-DAG remained relatively stable regardless of changes in catalyst concentrations or temperatures, confirming its susceptibility to isomerization into the thermodynamically more stable sn-1,3-DAG form. This study provides valuable insights into the composition and behavior of esterified PFAD products.
Topics: Diglycerides; Esterification; Triglycerides; Fatty Acids, Nonesterified; Fatty Acids; Monoglycerides; Chromatography, Gel; Acetates; Toluene
PubMed: 38556279
DOI: 10.5650/jos.ess23196