Intracellular Lipid Research Articles

Three positive charge nonapoptotic-induced photosensitizer with excellent water solubility for tumor therapy

Photodynamic therapy (PDT) has emerged as a significant cancer therapy option. Currently, cation-based organic small molecule aggregation-induced emission (AIE) photosensitizers (PSs) attract the wide attention of many scientists, due to improved reactive oxygen species (ROS) production after cationization. However, such PSs tend to localize only the mitochondria, limiting the death way of tumor cells (usually apoptosis) during PDT process, which may affect the therapeutic effect under some circumstances. Herein, we designed a novel water-soluble three positive charge PS, TPAN-18F, which could be distributed uniformly in cell cytoplasm and had distribution in different sub-organelles (mitochondria, endoplasmic reticulum, lysosome). The experimental results showed that TPAN-18F-based PDT process can not only disrupt mitochondrial functions (reducing ATP production and destroying mitochondrial membrane potential), but also elevate the intracellular lipid peroxides (LPOs) level, which evoke the non-apoptotic death manner of tumor cells. Further, in vivo studies showed that TPAN-18F-based PDT could effectively inhibit tumor growth. Accordingly, we believe that the construction of TPAN-18F is suggestive for tumor non-apoptotic therapy.

Localization and phenotyping of tuberculosis bacteria using a combination of deep learning and SVMs

Successful treatment of pulmonary tuberculosis (TB) depends on early diagnosis and careful monitoring of treatment response. Identification of acid-fast bacilli by fluorescence microscopy of sputum smears is a common tool for both tasks. Microscopy-based analysis of the intracellular lipid content and dimensions of individual Mycobacterium tuberculosis (Mtb) cells also describe phenotypic changes which may improve our biological understanding of antibiotic therapy for TB. However, fluorescence microscopy is a challenging, time-consuming and subjective procedure. In this work, we automate examination of fields of view (FOVs) from microscopy images to determine the lipid content and dimensions (length and width) of Mtb cells. We introduce an adapted variation of the UNet model to efficiently localising bacteria within FOVs stained by two fluorescence dyes; auramine O to identify Mtb and LipidTox Red to identify intracellular lipids. Thereafter, we propose a feature extractor in conjunction with feature descriptors to extract a representation into a support vector multi-regressor and estimate the length and width of each bacterium. Using a real-world data corpus from Tanzania, the proposed method i) outperformed previous methods for bacterial detection with a 8% improvement (Dice coefficient) and ii) estimated the cell length and width with a root mean square error of less than 0.01%. Our network can be used to examine phenotypic characteristics of Mtb cells visualised by fluorescence microscopy, improving consistency and time efficiency of this procedure compared to manual methods.

Iron, Ferroptosis, and Head and Neck Cancer.

Ferroptosis is an iron-dependent regulatory form of cell death characterized by the accumulation of intracellular reactive oxygen species and lipid peroxidation. It plays a critical role not only in promoting drug resistance in tumors, but also in shaping therapeutic approaches for various malignancies. This review aims to elucidate the relationship between ferroptosis and head and neck cancer treatment by discussing its conceptual framework, mechanism of action, functional aspects, and implications for tumor therapy. In addition, this review consolidates strategies aimed at improving the efficacy of head and neck cancer treatment through modulation of ferroptosis, herein serving as a valuable reference for advancing the treatment landscape for this patient population.

Macrophages originated IL-33/ST2 inhibits ferroptosis in endometriosis via the ATF3/SLC7A11 axis

Endometriosis is a gynecological inflammatory disease that is linked with immune cells, specifically macrophages. IL-33 secreted from macrophages is known to accelerate the progression of endometriosis. The periodic and repeated bleeding that occurs in women with endometriosis leads to excess iron in the microenvironment that is conducive to ferroptosis, a process related to intracellular ROS production, lipid peroxidation and mitochondrial damage. It is suggested that eESCs may specifically be able to inhibit ferroptosis. However, it is currently unclear whether IL-33 directly regulates ferroptosis to influence the disease course in endometriosis. In this study, eESCs co-cultured with macrophages or stimulated with IL-33/ST2 were observed to have increased cell viability and migration. Additionally, IL-33/ST2 decreased intracellular iron levels and lipid peroxidation in eESCs exposed to erastin treatment. Furthermore, IL-33/ST2 treatment resulted in a notable upregulation in SLC7A11 expression in eESCs due to the downregulation of negative transcription factor ATF3, thereby suppressing ferroptosis. The P38/JNK pathway activated by IL-33/ST2 was also found to inhibit the transcription factor ATF3. Therefore, we concluded that IL-33/ST2 inhibits the ATF3-mediated reduction in SLC7A11 transcript levels via the P38/JNK pathway. The findings reveal that macrophage-derived IL-33 upregulates SLC7A11 in eESCs through the p38/JNK/ATF3 pathway, ultimately resulting in protection against ferroptosis in eESCs. Moreover, we conducted an experiment using endometriosis model mice that showed that a combination of IL-33-Ab and erastin treatment alleviated the disease, showing the promise of combining immunotherapy and ferroptosis therapy.

Transcriptional landscape of human trophoblast cells treated with calcitriol and TGF-β1

Calcitriol and transforming growth factor beta 1 (TGF-β1) are unrelated molecules that regulate biological processes according to the genetic target, cell type, and context. Several studies have shown independent effects of calcitriol and TGF-βs on the placenta, but there is no information regarding the impact of their combination on these cells. Therefore, this study analyzed the effects of calcitriol, TGF-β1, and their combination in primary cultures of human trophoblast cells using a whole genome expression microarray. Data analysis revealed a set of differentially expressed genes induced by each treatment. Enrichment pathway analysis identified modulatory effects of calcitriol on genes related to metabolic processes such as vitamin D, steroid, and fat-soluble vitamins as well as antimicrobial and immune responses. In relation to TGF-β1, the analysis showed a few differentially expressed genes that were mainly associated with the neutrophil immune response. Lastly, the analysis revealed that the combination of calcitriol and TGF-β1 up-regulated genes involving both immunologic processes and the biosynthesis of unsaturated fatty acids, eicosanoids, and lipoxins, among others. In contrast, pathways down-regulated by the combination were mostly associated with the catabolic process of acylglycerols and peptides, PPAR signaling pathway, cellular response to low-density lipoprotein stimulus, renin angiotensin system and digestion, mobilization and transport of lipids. Consistent with these results, the combined treatment on human trophoblast cells induced the accumulation of intracellular neutral lipid droplets and stimulated both gene and protein expression of 15-hydroxyprostaglandin dehydrogenase. In conclusion, the results revealed that differentially expressed genes induced by the combination modified the transcriptional landscape compared to each treatment alone, mainly altering the storage, activity and metabolism of lipids, which might have an impact on placental development.

MRP1-Dependent Extracellular Release of Glutathione Induces Cardiomyocyte Ferroptosis After Ischemia-Reperfusion.

The membrane components of cardiomyocytes are rich in polyunsaturated fatty acids, which are easily oxidized. Thus, an efficient glutathione-based lipid redox system is essential for maintaining cellular functions. However, the relationship between disruption of the redox system during ischemia-reperfusion (IR), oxidized lipid production, and consequent cell death (ferroptosis) remains unclear. We investigated the mechanisms underlying the disruption of the glutathione-mediated reduction system related to ferroptosis during IR and developed intervention strategies to suppress ferroptosis. In vivo fluctuations of both intra- and extracellular metabolite levels during IR were explored via microdialysis and tissue metabolome analysis. Oxidized phosphatidylcholines were assessed using liquid chromatography high-resolution mass spectrometry. The areas at risk following IR were assessed using triphenyl-tetrazolium chloride/Evans blue stain. Metabolomic analysis combined with microdialysis revealed a significant release of glutathione from the ischemic region into extracellular spaces during ischemia and after reperfusion. The release of glutathione into extracellular spaces and a concomitant decrease in intracellular glutathione concentrations were also observed during anoxia-reperfusion in an in vitro cardiomyocyte model. This extracellular glutathione release was prevented by chemical inhibition or genetic suppression of glutathione transporters, mainly MRP1 (multidrug resistance protein 1). Treatment with MRP1 inhibitor reduced the intracellular reactive oxygen species levels and lipid peroxidation, thereby inhibiting cell death. Subsequent in vivo evaluation of endogenously oxidized phospholipids following IR demonstrated the involvement of ferroptosis, as levels of multiple oxidized phosphatidylcholines were significantly elevated in the ischemic region 12 hours after reperfusion. Inhibition of the MRP1 transporter also alleviated intracellular glutathione depletion in vivo and significantly reduced the generation of oxidized phosphatidylcholines. Administration of MRP1 inhibitors significantly attenuated infarct size after IR injury. Glutathione was released continuously during IR, primarily in an MRP1-dependent manner, and induced ferroptosis. Suppression of glutathione release attenuated ferroptosis and reduced myocardial infarct size following IR.

Short Communication: The oleaginous Candida tropicalis isolated from mangrove soil in eastern Thailand and the fatty acid composition profile of its intracellular lipids

Abstract. Wongchamrearn S, Boontanom P, Ungwiwatkul S, Emnin N, Chantarasiri A. 2023. Short Communication: The oleaginous Candida tropicalis isolated from mangrove soil in eastern Thailand and the fatty acid composition profile of its intracellular lipids. Biodiversitas 24: 5088-5095. Oleaginous yeasts are microorganisms that can accumulate intracellular lipids at a rate of more than 20% of their cell dry weight. They are considered ideal microbes for a sustainable bioeconomy and a promising source for biotechnological applications. Thus, the exploration of efficient oleaginous yeasts from various environments is necessitated. At present, the number of oleaginous yeasts that have been discovered from mangrove environments remains scarce. Therefore, this study isolated and screened for lipid-accumulating yeast strains in mangrove soil samples collected from Rayong Province and Chonburi Province, Thailand. The intracellular lipids of isolated yeasts were extracted and quantitatively analyzed to evaluate the efficient oleaginous strains. Fifteen yeast strains were considered lipid-accumulating yeasts based on the Sudan IV staining method and lipid content determination, with intracellular lipid accumulation ranging from 5.71±1.89% (w/w) to 33.83±1.41% (w/w). The isolated yeast strain MJ13 was designated the most efficient oleaginous strain and subsequently genetically identified as Candida tropicalis. The fatty acid composition of its intracellular lipids was analyzed by gas chromatography, which consisted of five saturated fatty acids and five unsaturated fatty acids with 10-22 carbon atoms. The predominant fatty acids were palmitic (C16:0) and stearic (C18:0) acids. Some fatty acids essential to the human body and pharmacological sciences were detected, involving a-linolenic (C18:3n3) and cis-13,16-docosadienoic (C22:2) acids. This oleaginous yeast strain could be applied in lipid-related biofuel and high-value bioeconomy applications.

The Thermal Properties and Nutritional Value of Biomass of Oleaginous Yeast Rhodotorula sp. during Glucose Fed-Batch Cultivation in Medium with Waste Nitrogen

The biotechnological processing of oleaginous yeast biomass should be comprehensively managed using the zero-waste policy. This study focused on the biomass of the red yeast Rhodotorula obtained from a medium containing waste nitrogen. The cells accumulate lipids in intracellular lipid droplets; however, they are also rich in protein. Therefore, the nutritional value of lipid and protein, according to their fatty acid and amino acid composition, is a necessary step for practical application. For the very first time, this study focused on understanding the influence of temperature on powdered red yeast biomass to study components phase transition or chemical reactions by using DSC. Rhodotorula glutinis var. rubescens was cultivated in a glucose fed-batch in a potato wastewater medium, where the biomass yield was powdered, and lipid and fatty acid, protein, and amino acid compositions were determined. The DSC diagrams of red yeast biomass were characterized by two small and mild endothermic peaks, indicating the presence of fat and the presence of low molecular weight carbohydrates and a distinct peak associated with the presence of crystalline sugars. The nutritional quality of the lipid fraction as atherogenicity (0.223), thrombogenicity index (0.438), PUFA/SFA (0.24), and the n-6/n-3 ratio (3.275) was adequate for the recommendation and resulted from the fatty acid composition. The yeast protein was characterized by a high content of glutamic acid (99 mg/1 g of protein), and a value of essential amino acid index of protein suggested a superior amino acid composition compared to the FAO/WHO standard. Despite a high essential amino acid index (>120), yeast protein was characterized by a low content of Lys or Met.

Melatonin targets ferroptosis through bimodal alteration of redox environment and cellular pathways in NAFLD model.

Ferroptosis is a non-conventional cellular death caused by lipid peroxide induced iron deposition. Intracellular lipid accumulation followed by generation of lipid peroxides is an Hallmark of Non Alcoholic Fatty Liver Disease (NAFLD). Melatonin (MLT) is an important pineal hormone with tremendous antioxidant and anti-inflammatory properties. Various studies targeted ferroptosis in different diseases using melatonin. However, none of them focused the intrinsic mechanism of MLT's action to counteract ferroptosis in NAFLD. Hence, the present study investigated the role of MLT in improvement of NAFLD induced ferroptosis. &#160;HepG2 cells were treated with Free Fatty Acids (FFAs) to induce in vitro NAFLD state and C57BL/6 mice were fed with HFD followed by MLT administration 1.</p> &#160;The results indicated that MLT administration caused the recovery from both FFA and HFD induced ferroptotic state via increasing GSH and SOD level, decreasing lipid ROS and MDA (Malondialdehyde) level, increasing Nrf2 and HO-1 level to defend cells against an oxidative environment. MLT also altered the expression of two key proteins GPX4 and SLC7A11 back to their normal levels, which would otherwise cause ferroptosis. MLT also protected against histopathological damage of both liver tissue and HepG2 cells as depicted by Oil Red O, HE staining and immunofluorescence microscopy. MLT also had control over pAMPKα as well as PPARγ and PPARα responsible for lipid homeostasis and lipogenesis.</p> &#160;In brief, MLT exerted its multifaceted effect in FFA and HFD induced NAFLD by retrieving cellular oxidative environment, reducing lipogenesis and lipid peroxidation and modulating Nrf2/HO-1 and GPX4/SLC7A11 axis to combat ferroptosis.</p>.

FRI018 A Novel Truncating Variant Of EBF2 Disrupts Human Adipocyte Differentiation In Lipodystrophy Syndromes: An Example Of A Discovery From A Clinical Translational Pipeline

Abstract Disclosure: M.C. Foss de Freitas: Advisory Board Member; Self; PTC Therapeutics. N. Wys: None. M. Udler: None. L. Pais: None. A. Monteiro da Rocha: None. O.A. MacDougald: None. E.A. Oral: Consulting Fee; Self; Regeneron Pharmaceuticals, Aegerion Pharmaceuticals, Ionis Pharmaceuticals Inc., Third Rock Ventures, Rejuvenate Inc. Grant Recipient; Self; Regeneron Pharmaceuticals, Aegerion Pharmaceuticals, Ionis Pharmaceuticals Inc., Novo Nordisk, Fractyl Laboratories, Rhythm pharmaceuticals, GID Dynamics. Other; Self; Aegerion Pharmaceuticals. T. Chun: None. Objective/Goals: Partial Lipodystrophy is characterized by the gradual loss of adipose tissues, which progresses after birth. The genetic and molecular underpinning of many cases of partial lipodystrophy have not been fully understood. Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy, we initiated deep sequencing for our patients with partial lipodystrophy syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel truncating EBF2 variant. Methods: We performed loss-of-function and gene rescue experiments using 3T3-L1 and human primary subcutaneous preadipocytes. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen). After lentivirus transfection (Full-length EBF2 or truncated variant EBF2), cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone, insulin and isobutyl methylxanthine. Total RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was synthesized using IScript (Bio-Rad). Real-time qPCR was performed using TaqMan probes for Pparg and Fabp4, two key adipogenesis markers. Results: The patient was found to carry a heterozygous nonsense mutation in exon 6 of 16 (NM_022659.4) of EBF2, causing the premature termination of the protein at amino acid position 165 (EBF2 8:26033143 C&amp;gt;A (hg38), c.493G&amp;gt;T, p.Glu165Ter). Adipogenesis was significantly suppressed in 3T3-L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed a marked reduction of intracellular lipid content and Pparg and Fabp4 expression (&amp;gt;80% reduction). With lentiviral gene transfer, EBF2 fully rescued adipogenic potential, whereas the truncated variant EBF2 p.Glu165Ter did not. Of note, 3T3-L1 cells transfected with the EBF2 variant impaired adipogenesis, suggesting an inhibitory effect of the EBF2 variant on adipogenesis. We next confirmed the inhibition in adipogenesis with the EBF2 variant in primary human preadipocytes from subcutaneous adipose tissues. Using lentiviral gene transfer in human preadipocytes, we observed that the full-length EBF2 increased the gene expression of PPARg and FABP4 while the truncated EBF2 variant did not. Conclusion: Our data suggest that EBF2 is indispensable for adipogenesis of mouse and human preadipocytes. The truncating variant of EBF2 p.Glu165Ter likely plays a pathogenic role in the partial lipodystrophy presentation of this patient. Whole genome (or exome) sequencing undertaken in patients with partial lipodystrophy and subsequent biological functional analysis may lead to identification of novel mechanisms driving novel discoveries. Presentation: Friday, June 16, 2023

Fatty acid binding protein 5 regulates docetaxel sensitivity in taxane-resistant prostate cancer cells.

Prostate cancer is a leading cause of cancer-related deaths in men in the United States. Although treatable when detected early, prostate cancer commonly transitions to an aggressive castration-resistant metastatic state. While taxane chemotherapeutics such as docetaxel are mainstay treatment options for prostate cancer, taxane resistance often develops. Fatty acid binding protein 5 (FABP5) is an intracellular lipid chaperone that is upregulated in advanced prostate cancer and is implicated as a key driver of its progression. The recent demonstration that FABP5 inhibitors produce synergistic inhibition of tumor growth when combined with taxane chemotherapeutics highlights the possibility that FABP5 may regulate other features of taxane function, including resistance. Employing taxane-resistant DU145-TXR cells and a combination of cytotoxicity, apoptosis, and cell cycle assays, our findings demonstrate that FABP5 knockdown sensitizes the cells to docetaxel. In contrast, docetaxel potency was unaffected by FABP5 knockdown in taxane-sensitive DU145 cells. Taxane-resistance in DU145-TXR cells stems from upregulation of the P-glycoprotein ATP binding cassette subfamily B member 1 (ABCB1). Expression analyses and functional assays confirmed that FABP5 knockdown in DU145-TXR cells markedly reduced ABCB1 expression and activity, respectively. Our study demonstrates a potential new function for FABP5 in regulating taxane sensitivity and the expression of a major P-glycoprotein efflux pump in prostate cancer cells.

Perilipin 2-positive mononuclear phagocytes accumulate in the diabetic retina and promote PPARγ-dependent vasodegeneration.

Type 2 diabetes mellitus (T2DM), characterized by hyperglycemia and dyslipidemia, leads to nonproliferative diabetic retinopathy (NPDR). NPDR is associated with blood-retina barrier disruption, plasma exudates, microvascular degeneration, elevated inflammatory cytokine levels, and monocyte (Mo) infiltration. Whether and how the diabetes-associated changes in plasma lipid and carbohydrate levels modify Mo differentiation remains unknown. Here, we show that mononuclear phagocytes (MPs) in areas of vascular leakage in DR donor retinas expressed perilipin 2 (PLIN2), a marker of intracellular lipid load. Strong upregulation of PLIN2 was also observed when healthy donor Mos were treated with plasma from patients with T2DM or with palmitate concentrations typical of those found in T2DM plasma, but not under high-glucose conditions. PLIN2 expression correlated with the expression of other key genes involved in lipid metabolism (ACADVL, PDK4) and the DR biomarkers ANGPTL4 and CXCL8. Mechanistically, we show that lipid-exposed MPs induced capillary degeneration in ex vivo explants that was inhibited by pharmaceutical inhibition of PPARγ signaling. Our study reveals a mechanism linking dyslipidemia-induced MP polarization to the increased inflammatory cytokine levels and microvascular degeneration that characterize NPDR. This study provides comprehensive insights into the glycemia-independent activation of Mos in T2DM and identifies MP PPARγ as a target for inhibition of lipid-activated MPs in DR.

Apolipoprotein A1 Inhibits Adipogenesis Progression of Human Adipose-Derived Mesenchymal Stem Cells.

According to the reports, the most vital characteristic of obesity is an aberrant accumulation of triglycerides (TG) in the adipocyte. On the other hand, circulating concentrations of apolipoprotein A1 (apoA1) have been demonstrated to be strongly correlated with the prevalence and the pathological development of obesity. Nevertheless, the underlying mechanisms whereby apoA1 modulates the pathogenesis of obesity is still not fully elucidated. Adipose-derived mesenchymal stem cells (AMSCs, isolated from the hospitalized patients were combined with 15 μg/ml recombined human apoA1 protein. The effects of apoA1 on modulating the intracellular levels of TG and the expression contents of adipogenic related cytokines were also analyzed. Furthermore, whether apoA1 modulated the adipogenesis progression via sortilin was also explored in the current research. During the adipogenesis progression, apoA1 could significantly lower the quantity of intracellular lipid droplets (LDs). Meanwhile, apoA1 could decrease the intracellular levels of TG and down-regulate the expression contents of several vital adipogenic related cytokines, such as CCAAT enhancer-binding proteins α/β (C/EBPα/β), fatty acid synthetase (FAS), and fatty acid-binding protein 4 (FABP4). Moreover, the inhibitory effect of apoA1 was further verified to be induced through upregulating the SORT1 gene expression which subsequently increased sortilin protein. Consistent with these findings, silencing the SORT1 gene expression could induce the loss-of-function (LOF) of apoA1 in modulating the adipogenesis progression of AMSCs. In conclusion, apoA1 could suppress the adipogenesis progression of human AMSCs through, at least partly, up-regulating the SORT1 gene expression which subsequently increases the sortilin protein content. Thereby, the present research sheds light on a novel pathogenic mechanism by which apoA1 regulates adipogenesis progression and proposes that apoA1 embraces the function to treat obesity in clinical practice.

Oxidized Low-Density Lipoprotein Accumulation Suppresses Glycolysis and Attenuates the Macrophage Inflammatory Response by Diverting Transcription from the HIF-1α to the Nrf2 Pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our invitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

Risk factors for intracellular fatty accumulation in rotator cuff muscle: a histologic analysis

Fatty accumulation in rotator cuff muscles has been associated with shoulder dysfunction, risk of repair failure, and poor postoperative outcomes. This study sought to assess risk factors associated with true fatty accumulation based on histologic analysis and determine whether preoperative function directly correlated with this fatty rotator cuff accumulation. Supraspinatus muscle biopsy specimens obtained prospectively from patients undergoing arthroscopic rotator cuff repair were stained with LipidTOX to quantify lipid accumulation. Two-step cluster analysis with Goutallier classification was used to define the fatty and non-fatty rotator cuff groups. We further performed a receiver operating characteristic curve analysis to confirm the group cutoff values. In total, 51 patients (aged 60.1±10.5 years) were included. There were 19 high-grade partial tears, 10 small tears, 7 medium tears, 10 large tears, and 5 massive tears. Both cluster and receiver operating characteristic curve analyses yielded a cutoff value of 30% LipidTOX/4',6-diamidino-2-phenylindole (DAPI) separating the fatty vs. non-fatty groups. In the univariate analysis, patients with fatty rotator cuffs were aged 63.2 years on average compared with 59.7 years in the non-fatty group (P=.038). Female patients made up 57.1% of the fatty cohort, which was statistically higher than the non-fatty group (P=.042). Massive and large tears were more likely to occur in the fatty group (P=.005). In the multivariate analysis, full tendon tears had the largest predictive status of falling into the fatty group (odds ratio, 15.4; P=.008), followed by female sex (odds ratio, 4.9; P=.036). Patients in the fatty group had significantly higher American Shoulder and Elbow Surgeons scores (P=.048) and lower visual analog scale scores (P=.002). This prospective histologic assessment revealed that full-thickness rotator cuff tears and female sex were the largest risk factors for intracellular lipid accumulation. Although tear size correlated with fatty accumulation, the sex disparity is a noteworthy finding that warrants further research.

Extracellular Vimentin Alters Energy Metabolism And Induces Adipocyte Hypertrophy.

Previous studies have reported that oxidative stress contributes to obesity characterized by adipocyte hypertrophy. However, mechanism has not been studied extensively. In the current study, we evaluated role of extracellular vimentin secreted by oxidized low-density lipoprotein (oxLDL) in energy metabolism in adipocytes. We treated 3T3-L1-derived adipocytes with oxLDL and measured vimentin which was secreted in the media. We evaluated changes in uptake of glucose and free fatty acid, expression of molecules functioning in energy metabolism, synthesis of adenosine triphosphate (ATP) and lactate, markers for endoplasmic reticulum (ER) stress and autophagy in adipocytes treated with recombinant vimentin. Adipocytes secreted vimentin in response to oxLDL. Microscopic evaluation revealed that vimentin treatment induced increase in adipocyte size and increase in sizes of intracellular lipid droplets with increased intracellular triglyceride. Adipocytes treated with vimentin showed increased uptake of glucose and free fatty acid with increased expression of plasma membrane glucose transporter type 1 (GLUT1), GLUT4, and CD36. Vimentin treatment increased transcription of GLUT1 and hypoxia-inducible factor 1α (Hif-1α) but decreased GLUT4 transcription. Adipose triglyceride lipase (ATGL), peroxisome proliferator-activated receptor γ (PPARγ), sterol regulatory element-binding protein 1 (SREBP1), diacylglycerol O-acyltransferase 1 (DGAT1) and 2 were decreased by vimentin treatment. Markers for ER stress were increased and autophagy was impaired in vimentin-treated adipocytes. No change was observed in synthesis of ATP and lactate in the adipocytes treated with vimentin. We concluded that extracellular vimentin regulates expression of molecules in energy metabolism and promotes adipocyte hypertrophy. Our results show that vimentin functions in the interplay between oxidative stress and metabolism, suggesting a mechanism by which adipocyte hypertrophy is induced in oxidative stress.

Comparison of different lipid staining methods in human meibomian gland epithelial cells

This study aimed to compare the utility of four different dyes for intracellular lipid detection in immortalized human meibomian gland epithelial cells (IHMGECs). IHMGECs were cultured in a serum-containing medium for 10 days in the presence or absence of Roxadustat (Roxa), a known inducer of IHMGEC differentiation. Cells were then fixed and stained with Oil Red O (ORO), Sudan III (SIII), LipidTOX green (LT), or Nile Red (NR). IHMGECs were evaluated for the number, size, and area of stained intracellular lipid vesicles or the intensity of staining using bright field (ORO, SIII) or fluorescence (LT, NR) microscopy. Data were captured with ImageJ and analyzed with Student's two-tailed t-test. Our findings demonstrate that different staining methods can yield significantly different patterns of intracellular lipid quantity and/or distribution in IHMGECs. ORO and SIII significantly increased the size and area of lipid-containing vesicles in Roxa-treated cells. Neither stain showed a change in the number of vesicles during IHMGEC differentiation. Vesicle size was significantly greater in cells stained with ORO, as compared to SIII. In addtion, LT, but not NR, showed a significant increase in intracellular lipid intensity in IHMGECs following Roxa -induced differentiation. Our results demonstrate significant differences in the distribution patterns and intensities of lipid-containing vesicles in IHMGECs after staining with ORO, SIII, LT, and NR. ORO, SIII, and LT, but not NR staining, are helpful methods to help identify and quantitate the extent of intracellular lipid accumulation during IHMGEC differentiation.

Ablation of Dual-Specificity Phosphatase 6 Protects against Nonalcoholic Fatty Liver Disease via Cytochrome P450 4A and Mitogen-Activated Protein Kinase

Dual-specificity phosphatase 6 (DUSP6) is a specific phosphatase for mitogen-activated protein kinase (MAPK). This study used a high-fat diet (HFD)-induced murine nonalcoholic fatty liver disease model to investigate the role of DUSP6 in this disease. Wild-type (WT) and Dusp6-haploinsufficiency mice developed severe obesity and liver pathology consistent with nonalcoholic fatty liver disease when exposed to HFD. In contrast, Dusp6-knockout (KO) mice completely eliminated these phenotypes. Furthermore, primary hepatocytes isolated from WT mice exposed to palmitic and oleic acids exhibited abundant intracellular lipid accumulation, whereas hepatocytes from Dusp6-KO mice showed minimal lipid accumulation. Transcriptome analysis revealed significant down-regulation of genes encoding cytochrome P450 4A (CYP4A), known to promote ω-hydroxylation of fatty acids and hepatic steatosis, in Dusp6-KO hepatocytes compared with that in WT hepatocytes. Diminished CYP4A expression was observed in the liver of Dusp6-KO mice compared with WT and Dusp6-haploinsufficiency mice. Knockdown of DUSP6 in HepG2, a human liver-lineage cell line, also promoted a reduction of lipid accumulation, down-regulation of CYP4A, and up-regulation of phosphorylated/activated MAPK. Furthermore, inhibition of MAPK activity promoted lipid accumulation in DUSP6-knockdown HepG2 cells without affecting CYP4A expression, indicating that CYP4A expression is independent of MAPK activation. These findings highlight the significant role of DUSP6 in HFD-induced steatohepatitis through two distinct pathways involving CYP4A and MAPK.

Effect of fatty acids on intracellular pneumocandin B0 storage in the fermentation of Glarea lozoyensis

The natural product pneumocandin B0 is the precursor of the antifungal drug caspofungin. To explore the relationship between pneumocandin B0 and oil. We found that the addition of 1 g/L of oil to the fermentation medium is more conducive to the production of pneumocandin B0. The metabolic reaction mechanism was explored using different fatty acids and the results showed that stearic acid and acetic acid increased the total production of pneumocandin B0 by 22.98% and 9.08%, respectively, as well as increasing the content of intracellular lipid droplets. We also analyzed gene expression and pathway differences between the two different fatty acids using transcriptome analyses. The addition of both acetic acid and stearic acid promoted an active pentose phosphate pathway, providing cells with higher intracellular reducing power. We found that the addition of fatty acids can lead to lipid accumulation, and lipid droplets can sequester lipophilic secondary metabolites such as pneumocandin B0 to reduce cell damage. These results provide novel insights into the relationship between pneumocandin B0 biosynthesis and fatty acids in G. lozoyensis. In addition, this study provides important genetic information for improving the yield of pneumocandin B0 through a strategy of metabolic engineering in the future.Graphical

Chlorogenic acid enhances PPARγ-mediated lipogenesis through preventing Lipin 1 nuclear translocation in Staphylococcus aureus-exposed bovine mammary epithelial cells

Chlorogenic acid (CGA) as one of the most ubiquitously dietary polyphenolic compounds, has been reported to have various antimicrobial effects and exhibit strong anti-inflammatory ability. Staphylococcus aureus is a gram-positive bacterium that can induce mastitis. However, the mechanism through which S. aureus infection affects lipid synthesis and whether CGA have protective effect on S. aureus reduced lipid synthesis is not fully understood. In this study, the internalization of S. aureus reduced intracellular lipid droplet formation, decreased the levels of intracellular triacylglycerol, total cholesterol and 7 types of fatty acid and downregulated the expression of lipogenic genes FAS, ACC, and DGAT1 in bovine mammary epithelial cells (BMECs). In addition, we found that S. aureus intracellular infection attenuated mTORC1 activation resulting in Lipin 1 nuclear localization. Remarkablely, S. aureus infection-mediated repression of lipid synthesis related to the mTORC1 signaling and Lipin 1 nuclear localization can be alleviated by CGA. Thus, our findings provide a novel mechanism by which lipid synthesis is regulated under S. aureus infection and the protective effects of CGA on lipid synthesis in BMECs.