Cytoplasmic Lipid Droplets Research Articles

Zebrafish are resilient to the loss of major diacylglycerol acyltransferase enzymes

In zebrafish, maternally deposited yolk is the source of nutrients for embryogenesis prior to digestive system maturation. Yolk nutrients are processed and secreted to the growing organism by an extra-embryonic tissue, the yolk syncytial layer (YSL). Export of lipid from the YSL occurs through the production of triacylglycerol-rich lipoproteins. Here we report that mutations in the triacylglycerol synthesis enzyme, diacylglycerol acyltransferase-2 (Dgat2), cause yolk sac opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. Though triacylglycerol synthesis continues, it is not properly coupled to lipoprotein production as dgat2 mutants produce fewer, smaller, ApoB-containing lipoproteins. Unlike DGAT2-null mice, which are lipopenic and die soon after birth, zebrafish dgat2 mutants are viable, fertile and exhibit normal mass and adiposity. Residual Dgat activity cannot be explained by the activity of other known Dgat isoenzymes, as dgat1a;dgat1b;dgat2 triple mutants continue to produce YSL lipid droplets and remain viable as adults. Further, the newly identified diacylglycerol acyltransferase, Tmem68, is also not responsible for the residual triacylglycerol synthesis activity. Unlike overexpression of Dgat1a and Dgat1b, monoacylglycerol acyltransferase-3 (Mogat3b) overexpression does not rescue yolk opacity, suggesting it does not possess Dgat activity in the YSL. However, mogat3b;dgat2 double mutants exhibit increased yolk opacity and often have structural alterations of the yolk extension. Quadruple mogat3b;dgat1a;dgat1b;dgat2 mutants either have severely reduced viability and stunted growth, or do not survive past 3 days post fertilization, depending on the dgat2 mutant allele present. Our study highlights the remarkable ability of vertebrates to synthesize triacylglycerol through multiple biosynthetic pathways.

Unlocking the Secrets of Adipose Tissue: How an Obesity-Associated Secretome Promotes Osteoblast Dedifferentiation via TGF-β1 Signaling, Paving the Path to an Adipogenic Phenotype.

Obesity poses a significant global health challenge, given its association with the excessive accumulation of adipose tissue (AT) and various systemic disruptions. Within the adipose microenvironment, expansion and enrichment with immune cells trigger the release of inflammatory mediators and growth factors, which can disrupt tissues, including bones. While obesity's contribution to bone loss is well established, the direct impact of obese AT on osteoblast maturation remains uncertain. This study aimed to explore the influence of the secretomes from obese and lean AT on osteoblast differentiation and activity. SAOS-2 cells were exposed to the secretomes obtained by culturing human subcutaneous AT from individuals with obesity (OATS) or lean patients, and their effects on osteoblasts were evaluated. In the presence of the OATS, mature osteoblasts underwent dedifferentiation, showing an increased proliferation accompanied by a morphological shift towards a mesenchymal phenotype, with detrimental effects on osteogenic markers and the calcification capacity. Concurrently, the OATS promoted the expression of mesenchymal and adipogenic markers, inducing the formation of cytoplasmic lipid droplets in SAOS-2 cells exposed to an adipogenic differentiation medium. Additionally, TGF-β1 emerged as a key mediator of these effects, as the OATS was enriched with this growth factor. Our findings demonstrate that obese subcutaneous AT promotes the dedifferentiation of osteoblasts and increases the adipogenic profile in these cells.

Species-specific histological characterizations of renal tubules and collecting ducts in the kidneys of cats and dogs.

The histomorphological features of normal kidneys in cats and dogs have been revealed despite the high susceptibility of cats to tubulointerstitial damage. Herein, the histological characteristics of the two species were compared. Cytoplasmic lipid droplets (LDs) were abundant in the proximal convoluted tubules (PCTs) of cats aged 23-27 months but scarce in dogs aged 24-27 months. LDs were rarely observed in the distal tubules (DTs) and collecting ducts (CDs) of either species, as visualized by the expression of Tamm-Horsfall protein 1, calbindin-D28K, and aquaporin 2. The occupational area ratio of proximal tubules (PTs) in the renal cortex was higher, but that of DTs or CDs was significantly lower in adult cats than in dogs. Single PT epithelial cells were larger, but PCT, DT, and CD lumens were significantly narrower in adult cats than in dogs. Unlike adults, young cats at 6 months exhibited significantly abundant cytoplasmic LDs in proximal straight tubules, indicating lipid metabolism-related development. Histochemistry of the 21 lectins also revealed variations in glycosylation across different renal tubules and CDs in both species. Sodium-glucose cotransporter 2 was expressed only in PTs, excluding the proximal straight tubules with few LDs in adult cats or the PCTs of young cats and adult dogs. These findings are crucial for understanding species-specific characteristics of renal histomorphology and pathogenesis.

O-102 From oocytes to embryos

Abstract Title: Oxidative stress and DNA damage in oocytes: clinical relevance and diagnosis Study question: Is it possible to detect signs of oxidative stress (OS) in a living oocyte? Summary answer: OS-driven changes in lipid profile can be detected in human oocytes. What is known already: Oxidative stress and DNA damage are associated with increasing age of women. From all cellular molecules, lipids are most prone to OS-driven damage. DNA is more stable, so its damage may occur only later, probably as a result of cytoplasmic changes caused by damage to lipids and proteins. Initially, peroxidation of cellular lipids causes damage to lipid bilayers i.e. biological membranes and membranous organelles. Accumulation of lipid peroxidation products - toxic aldehydes - causes DNA damage and mutations. Screening of DNA damage in the oocyte is not feasible in human ART. However, oocyte lipid damage can be detected using live-cell label-free spectroscopies. So far, changes in the lipid profile of human oocytes have not been investigated. Here we propose the screening of lipid profile as a reliable method of evaluation of OS-driven damage of the oocyte. Study design, size, duration: This study aims to thoroughly discuss effects of OS in female gamete. Special consideration is given to current limits in DNA damage detection and oocyte quality screening. Then, strategies to evaluate OS-driven damage of cellular components are proposed. Finally, obstacles and future perspectives in clinical diagnosis and treatment of OS-damaged oocytes are discussed. Participants/materials, setting, methods: To investigate if it is possible to detect differences in oocyte LF, we examined a total of 48 GV oocytes derived from 26 women under 33 years of age and with no history of infertility, enrolled in an oocyte donation program. Furthermore, to evaluate the effect of lipid profile changes in oocytes on embryo development, 167 C57BL/6 mice were used. Modifications of cytoplasmic lipid droplets (LDs) profile in oocytes were evaluated by Coherent anti-stokes Raman Spectroscopy (CARS) and further validated by Fourier Transform Infrared Spectroscopy (FTIR) and Transmission Electron Microscopy (TEM). The influence of oocyte lipid changes on embryo development was verified using mouse model. Main results and the role of chance: Here we show woman-age-dependent progressive changes in LF of oocytes consisting of increased LDs size, area and number, assessed by label-free imaging (CARS). LF variations in oocytes were detectable also within individual donors. We next confirmed woman-age-dependent changes in oocytes by demonstration of lipid peroxidation and composition changes (FTIR) and alterations of structural properties of lipid bilayers (TEM). Finally, using a mouse model we showed that LF changes in oocytes negatively affect preimplantation embryo development. Limitations, reasons for caution: The idea of this study was to propose an original approach to assess the quality of oocytes based on changes in the lipid profile. The information presented should not be viewed as directly applicable, because human oocytes at the GV stage were assessed, and not at the MII stage. Wider implications of the findings: Our findings highlight functional connections between lipids and other molecules building the oocyte, including DNA. Furthermore, this original study open the possibility to develop an innovative tool for oocyte assessment. Study funding/competing interest(s): This research was funded by National Science Centre of Poland Grants 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547.

Targeting aberrant fatty acid synthesis and storage in endocrine resistant breast cancer cells.

Lipid metabolic reprogramming is an emerging characteristic of endocrine therapy (ET) resistance in estrogen receptor-positive (ER+) breast cancer. We explored changes in lipid metabolism in ER+ breast cancer cell lines following acquired resistance to common endocrine treatments and tested efficacy of an inhibitor in current clinical trials. We derived ER+ breast cancer cell lines resistant to Tamoxifen (TamR), Fulvestrant (FulvR), and long-term estrogen withdrawal (EWD). Parental and ET resistant cells were subjected to global gene expression and unbiased lipidomic profiling. Lipid storage changes were assessed via neutral lipid staining with Oil Red O (ORO). The impact of the fatty acid synthase (FASN) inhibitor TVB-2640 on the growth and lipid storage of these cell lines was evaluated. Additionally, 13 C 2 -acetate tracing was used to examine FASN activity in parental and ET resistant cells in the absence or presence of TVB-2640. Compared to parental cells, lipid metabolism and processing pathways were notably enriched in ET resistant cells, which exhibited distinct lipidomes characterized by increased triglyceride and polyunsaturated FA (PUFA) species. ET-resistant cells displayed enhanced cytoplasmic lipid droplets. Increased FASN protein levels were observed in ET-resistant cells, and TVB-2640 effectively inhibited FASN activity. FASN inhibition reduced cell growth in some but not all cell lines and ET resistance types and did not correlate to lipid storage reduction. 13 C 2 -acetate tracing confirmed reduced palmitate synthesis and enhanced PUFA synthesis in ET-resistant cells, especially when combined with FulvR. ET resistant breast cancer cells exhibit a shift towards enhanced triglyceride storage and complex lipids enriched with PUFA acyl chains. While targeting FASN alongside ET may not fully overcome ET resistance in our models, focusing on the unique lipid metabolic dependencies, such as PUFA pathways, may present a promising alternative strategy for treating ET resistant breast cancer.

Could Cytoplasmic Lipid Droplets be Linked to Inefficient Oxidative Phosphorylation in Cancer?

Could Cytoplasmic Lipid Droplets be Linked to Inefficient Oxidative Phosphorylation in Cancer?

Cancer radioresistance is characterized by a differential lipid droplet content along the cell cycle

BackgroundCancer radiation treatments have seen substantial advancements, yet the biomolecular mechanisms underlying cancer cell radioresistance continue to elude full understanding. The effectiveness of radiation on cancer is hindered by various factors, such as oxygen concentrations within tumors, cells’ ability to repair DNA damage and metabolic changes. Moreover, the initial and radiation-induced cell cycle profiles can significantly influence radiotherapy responses as radiation sensitivity fluctuates across different cell cycle stages. Given this evidence and our prior studies establishing a correlation between cancer radiation resistance and an increased number of cytoplasmic Lipid Droplets (LDs), we investigated if LD accumulation was modulated along the cell cycle and if this correlated with differential radioresistance in lung and bladder cell lines.ResultsOur findings identified the S phase as the most radioresistant cell cycle phase being characterized by an increase in LDs. Analysis of the expression of perilipin genes (a family of proteins involved in the LD structure and functions) throughout the cell cycle also uncovered a unique gene cell cycle pattern.ConclusionsIn summary, although these results require further molecular studies about the mechanisms of radioresistance, the findings presented here are the first evidence that LD accumulation could participate in cancer cells’ ability to better survive X-Ray radiation when cells are in the S phase. LDs can represent new players in the radioresistance processes associated with cancer metabolism. This could open new therapeutic avenues in which the use of LD-interfering drugs might enhance cancer sensitivity to radiation.

Cytoskeletal rearrangement precedes nucleolar remodeling during adipogenesis

Differentiation of adipose progenitor cells into mature adipocytes entails a dramatic reorganization of the cellular architecture to accommodate lipid storage into cytoplasmic lipid droplets. Lipid droplets occupy most of the adipocyte volume, compressing the nucleus beneath the plasma membrane. How this cellular remodeling affects sub-nuclear structure, including size and number of nucleoli, remains unclear. We describe the morphological remodeling of the nucleus and the nucleolus during in vitro adipogenic differentiation of primary human adipose stem cells. We find that cell cycle arrest elicits a remodeling of nucleolar structure which correlates with a decrease in protein synthesis. Strikingly, triggering cytoskeletal rearrangements mimics the nucleolar remodeling observed during adipogenesis. Our results point to nucleolar remodeling as an active, mechano-regulated mechanism during adipogenic differentiation and demonstrate a key role of the actin cytoskeleton in defining nuclear and nucleolar architecture in differentiating human adipose stem cells.

Nuclear lipid droplets in Caco2 cells originate from nascent precursors and in situ at the nuclear envelope

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.

Abstract 365: Proteomic analysis of cytoplasmic lipid droplets and whole cell lysates reveal ferroptosis regulation by fatty acid synthase-derived lipid droplets in metastatic breast cancer

Abstract Breast cancer remains a serious public health concern, where metastasis accounts for the majority of patient mortality. Lipid accumulation in metastases is associated with reduced treatment response and poorer patient outcomes, although the mechanisms linking lipid accumulation and metastasis are not clear. We previously demonstrated that metastatic MCF10CA1a breast cancer cells have higher de novo lipogenesis and cytoplasmic lipid droplets (CLDs) compared to non-metastatic MCF10A-ras cells. Additionally, fatty acid synthase (FASN)-derived TAG stores sustained key metastatic processes in the MCF10CA1a cells. The proteome of CLDs is diverse and plastic, reflecting differing CLD functions; therefore, we sought to identify proteins differentially abundant on CLDs from vehicle-treated and FASN-inhibited MCF10CA1a breast cancer cells. We performed untargeted, global proteomics analysis on whole cell lysates (WCLs) and CLDs from vehicle-treated and FASN-inhibited (TVB-3166) MCF10CA1a cells to determine protein pathways differentially enriched on CLDs. Together, 2,416 and 3,539 proteins were identified within CLD fractions and WCLs, respectively. Of the proteins identified in CLD fractions, ~30% (717) were commonly identified within both conditions. Interestingly, 1,571 proteins were unique to the CLD fraction from vehicle-treated cells, whereas only 19 proteins were unique to the CLD fraction from FASN-inhibited cells. Proteins unique to CLDs from vehicle-treated cells include those known to reduce cancer cell survival and progression, such as proteins involved in cadherin-binding and cell cycle regulation. Proteins known to reduce cancer progression that associate with CLDs may prevent them from functioning at their normal site of action. Interestingly, an inhibitor of ferroptosis, glutathione peroxidase 4 (GPX4), was only detected in the vehicle-treated CLD and WCL samples, whereas proteins that promote ferroptosis were more abundant on CLDs of the more migratory cells, although their levels between WCLs were similar. Taken together, these data suggested that the machinery for ferroptosis may be mislocalized in TAG-rich cells, thereby protecting them from ferroptosis. Indeed, FASN-inhibited cells had nearly 30% more intracellular iron compared to the vehicle-treated cells, an important prerequisite for ferroptosis, thus indicating higher levels of ferroptotic stress. Functionally, the vehicle-treated cells were 25% more readily rescued by N-acetylcysteine from ferroptosis induced by erastin than were FASN-inhibited cells, despite comparable sensitivity to erastin. These preliminary findings suggest that FASN-derived CLDs in metastatic cells may aid in protecting against ferroptosis to promote breast cancer progression. Citation Format: Chaylen Andolino, Kimberly K. Buhman, Michael F. Coleman, Stephen D. Hursting, Marjorie Layosa, Michael K. Wendt, Dorothy Teegarden. Proteomic analysis of cytoplasmic lipid droplets and whole cell lysates reveal ferroptosis regulation by fatty acid synthase-derived lipid droplets in metastatic breast cancer [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 365.

Abstract 781: Impact of vitamin D and pyruvate carboxylase on hypoxia inducible lipid droplet associated (HILPDA) protein in metastatic breast cancer cells

Abstract Metastatic breast cancer, a critical health issue for women globally, is closely linked to aberrant lipid metabolism. One feature of this dysregulated metabolism is the increase in lipid storage in the form of cytoplasmic lipid droplets (CLDs), although what drives their accumulation or their role in metastasis is not clear. In prior studies, we demonstrated that the active form of vitamin D (1,25-dihydroxyvitamin D, 1,25(OH)2D) downregulates pyruvate carboxylase (PC), a key metabolic enzyme, thereby reducing lipid accumulation in metastatic breast cancer cells (MCF10CA1a). Additionally, we have demonstrated that both 1,25(OH)2D treatment and PC-inhibition reduce breast cancer cell migration. Therefore, the goal of the current research is to identify the proteins associated with CLDs in metastatic MCF10CA1a breast cancer cells following 1,25(OH)2D treatment or reduction in PC expression (shPC). Proteins from CLDs and whole cell lysates (WCL) were processed for untargeted proteomic analysis. In the CLD fractions, 1562 identified enriched proteins were common to both MCF10CA1a and 1,25(OH)2D treated cells, while 160 proteins were higher in the MCF10CA1a cells, and 550 proteins were higher in the 1,25(OH)2D-treated cells compared to MCF10CA1a CLD fraction. In the CLD fractions, MCF10CA1a and shPC cells had a similar level of 380 enriched proteins, although 65 were higher in the shPC compared with MCF10CA1a, whereas 1268 were more enriched in the MCF10CA1a CLD fraction. Hypoxia Inducible Lipid Droplet Associated (HILPDA, known to inhibit triacylglycerol lipolysis) was only enriched in the CLD fraction of the metastatic MCF10CA1a cells. HILPDA was not identified in the CLDs of the 1,25(OH)2D treated or the shPC cells, nor in any WCL fraction. Further investigation of HILPDA is important to determine if HILPDA plays a role in dysregulated cellular metabolism, including CLD accumulation, or migration, and may potentially be a target for future therapies to prevent breast cancer progression. Citation Format: Yazhen Song, Chaylen Andolino, Dorothy Teegarden. Impact of vitamin D and pyruvate carboxylase on hypoxia inducible lipid droplet associated (HILPDA) protein in metastatic breast cancer cells [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 781.

Zebrafish ApoB-Containing Lipoprotein Metabolism: A Closer Look.

Zebrafish have become a powerful model of mammalian lipoprotein metabolism and lipid cell biology. Most key proteins involved in lipid metabolism, including cholesteryl ester transfer protein, are conserved in zebrafish. Consequently, zebrafish exhibit a human-like lipoprotein profile. Zebrafish with mutations in genes linked to human metabolic diseases often mimic the human phenotype. Zebrafish larvae develop rapidly and externally around the maternally deposited yolk. Recent work revealed that any disturbance of lipoprotein formation leads to the accumulation of cytoplasmic lipid droplets and an opaque yolk, providing a visible phenotype to investigate disturbances of the lipoprotein pathway, already leading to discoveries in MTTP (microsomal triglyceride transfer protein) and ApoB (apolipoprotein B). By 5 days of development, the digestive system is functional, making it possible to study fluorescently labeled lipid uptake in the transparent larvae. These and other approaches enabled the first in vivo description of the STAB (stabilin) receptors, showing lipoprotein uptake in endothelial cells. Various zebrafish models have been developed to mimic human diseases by mutating genes known to influence lipoproteins (eg, ldlra, apoC2). This review aims to discuss the most recent research in the zebrafish ApoB-containing lipoprotein and lipid metabolism field. We also summarize new insights into lipid processing within the yolk cell and how changes in lipid flux alter yolk opacity. This curious new finding, coupled with the development of several techniques, can be deployed to identify new players in lipoprotein research directly relevant to human disease.

Evaluating the hypolipidemic effect of garlic essential oil encapsulated in a novel double-layer delivery system

The limited application of garlic essential oil (GEO) is attributed to its pungent taste, poor water solubility and low bioavailability. Liposomes are nontoxic, biodegradable and biocompatible, and β-cyclodextrin can inhibit undesirable odors and improve the stability and bioavailability. Thus a promising dual-layer GEO β-cyclodextrin inclusion compound liposome (GEO-DCL) delivery system with both advantages was designed and prepared in this study. Experimental results indicated that the encapsulation efficiency of GEO-DCLs was 5% higher than that of GEO liposomes (GEO-CLs), reaching more than 88%. In vitro release experiment showed that the release rate of GEO in GEO-DCLs was 40% lower than that of GEO-CLs after incubation in gastric juice for 6-h, indicating that the stability of GEO-DCLs was better than GEO-CLs. Evaluation of the effects of GEO-DCLs on lowering blood lipid levels in hypercholesterolemia mice. GEO-DCLs could reduce the weight and fat deposition in hypercholesterolemia mice. Inhibiting the increase of TC, LDL-C, and decrease of HDL-C in mice. The degree of liver injury was decreased, the number of round lipid droplets in liver cytoplasm was reduced, and the growth of fat cells was inhibited. The lipid-lowering effects of GEO-DCLs were dose-dependent. GEO-DCL can improve the bioavailability of GEO and improve dyslipidemia. Based on GEO's efficacy in lowering blood lipids, this study developed a kind of GEO-DCL compound pomegranate juice beverage with good taste, miscibility and double effect of reducing blood lipids. This study lays a foundation for the application of GEO in the field of functional food.

Abstract 17218: CES1 Deficiency is Associated With Metabolic Reprograming and Endothelial Dysfunction in Pulmonary Arterial Hypertension

Introduction: PAH is associated with loss of pulmonary microvessels & vascular remodeling. Metabolic reprogramming(MR) characterized by high glycolysis & reduced FAO is hallmark feature of PAH linked to oxidative stress, endothelial cell(EC) dysfunction & reduced angiogenesis. BMPR2 gene mutations promote MR; however, due to low penetrance, alterations in other genes are likely necessary for PAH development. We found Carboxylesterase 1 (CES1) is an ER enzyme involved in lipid metabolism & cellular detoxification with a potentially important role in MR & EC dysfunction in PAH. Hypothesis: Loss of CES1 leads to EC dysfunction through MR, lipotoxicity & oxidative stress & serves as a genetic modifier of BMPR2 in ECs Methods: Healthy/PAH ECs & lung tissues were obtained from transplants and commercial sources. Healthy ECs were transfected with siRNA/plasmid to knockdown/overexpress CES1. Mitochondrial & ER structure & function was assessed by confocal microscopy & proteomic assays. Metabolic flux in ECs was analyzed using the SeahorseXF. CES1 HET KO mice exposed to normoxia and hypoxia were studied & lungs were processed for scRNA-seq. Results: CES1 mRNA/protein expression was reduced in PAH ECs & lung vascular lesions. Compared to controls, siCES1 ECs have high apoptosis,reduced angiogenesis,increased ROS,mitochondrial fragmentation & ER stress. Lipid staining of siCES1 PMVECs demonstrates cytoplasmic lipid droplet accumulation correlating with reduced FAO and switch to glycolysis. Restoring CES1 expression reverses MR, reduces ROS & improves functional status of siCES1/PAH ECs. CES1 is a downstream target of BMPR2 under NRF2 transcriptional regulation & its loss exacerbates MR & dysfunction associated with BMPR2 loss that improves via CES1 restoration. Hemodynamic & scRNA-seq studies show that hypoxic CES1 HETKO mice develop more severe PH & demonstrate dysregulation of genes in key metabolic & angiogenic pathways. Conclusions: CES1 is essential for maintenance & repair of the pulmonary endothelium & acts as a key modifier of BMPR2 signaling. Limitations of current therapies to reverse EC dysfunction and prevent small vessel loss warrant therapeutic interventions that can restore CES1 expression and serve as a novel treatment approach for PAH.

Comparison of two Phaeodactylum tricornutum ecotypes under nitrogen starvation and resupply reveals distinct lipid accumulation strategies but a common degradation process.

Phaeodactylum tricornutum is a model species frequently used to study lipid metabolism in diatoms. When exposed to a nutrient limitation or starvation, diatoms are known to accumulate neutral lipids in cytoplasmic lipid droplets (LDs). Those lipids are produced partly de novo and partly from the recycle of plastid membrane lipids. Under a nitrogen resupply, the accumulated lipids are catabolized, a phenomenon about which only a few data are available. Various strains of P. tricornutum have been isolated around the world that may differ in lipid accumulation patterns. To get further information on this topic, two genetically distant ecotypes of P. tricornutum (Pt1 and Pt4) have been cultivated under nitrogen deprivation during 11 days followed by a resupply period of 3 days. The importance of cytoplasmic LDs relative to the plastid was assessed by a combination of confocal laser scanning microscopy and cell volume estimation using bright field microscopy pictures. We observed that in addition to a basal population of small LDs (0.005 μm3 to 0.7 μm3) present in both strains all along the experiment, Pt4 cells immediately produced two large LDs (up to 12 μm3 after 11 days) while Pt1 cells progressively produced a higher number of smaller LDs (up to 7 μm3 after 11 days). In this work we showed that, in addition to intracellular available space, lipid accumulation may be limited by the pre-starvation size of the plastid as a source of membrane lipids to be recycled. After resupplying nitrogen and for both ecotypes, a fragmentation of the largest LDs was observed as well as a possible migration of LDs to the vacuoles that would suggest an autophagic degradation. Altogether, our results deepen the understanding of LDs dynamics and open research avenues for a better knowledge of lipid degradation indiatoms.

Visualization of invisible cell-death sign by electric-double-layer modulation

Leakage and self-spreading of a cytoplasmic lipid droplet in association with cell death, which was totally invisible in a conventional optical microscope, was discovered by using the recently developed electric-double-layer modulation imaging (EDLMI). The method allowed real-time imaging of the leaked lipid droplet with the thickness of a few nanometers. We demonstrate that the discovered phenomenon is useful as a new cellular sign for diagnosis of cell death.

HyperCKemia: An early sign of childhood-onset neutral lipid storage disease with myopathy

Neutral lipid-storage disease with myopathy (NLSDM) is an autosomal recessive neuromuscular disorder caused by mutations in PNPLA2, and the average age at onset is 30 years. To date, only eight patients with childhood-onset NLSDM have been reported in detail. We investigated 3 unreported patients with NLSDM detected in childhood and reviewed 8 childhood-onset and 82 adult-onset patients with NLSDM documented in the literature. In the childhood-onset cohort, NLSDM presented initially as asymptomatic or paucisymptomatic hyperCKemia in 6/11 patients, and follow-up data showed onset of muscle weakness in 6/11 childhood-onset patients. In the adult-onset cohort, 95.1% (78/82) of patients showed muscle weakness. Cardiac involvement developed in 6/11 childhood-onset patients. Hepatomegaly was observed in 3/11 childhood-onset patients. Serum creatine kinase levels were elevated greater than five-fold of the upper limit of normal (ULN) in most childhood-onset patients and were elevated to less than ten-fold of the ULN in most adult-onset patients. Peripheral blood smears and muscle biopsies showed cytoplasmic lipid droplets in leukocytes and myocytes. NLSDM can present in children with asymptomatic or paucisymptomatic hyperCKemia before the onset of muscle weakness. The presence of lipid droplets in leucocytes (Jordans' anomaly) aids in diagnosing and confirming the pathogenicity of PNPLA2 variants of uncertain significance. There were no clear genotype-phenotype correlations in patients with NLSDM.

Cytoplasmic lipid droplets (LDs) occurrence in leaf tissues of different Coffea species: A new physiological trait in coffee

The present investigation aims to ascertain the presence of cytoplasmic lipid droplets (LDs) in different Coffea sp. leaf tissues. A detailed morpho-anatomical characterization, in addition to highlighting that the overall internal leaf tissue organization is independent of the coffee botanical species, put in evidence for the first time the presence of LDs in Coffea sp. leaves. Leaves from plant accessions of several coffee species, kept under greenhouse conditions, have been sampled in two different periods (January and June). Cytoplasmic LDs have been observed in all samples and they are mainly present as a single body, ranging from a minimum diameter of 1 µm to a maximum of 10 µm, detected in both palisade and spongy parenchyma cells of mesophyll tissue. In some case LDs are also sporadically observed in some epidermal cells in both abaxial or adaxial surface or in the subsidiary cells of stomata. Depending on season as well as coffee species, LDs show variability in terms of quantity, size, clustering and tissue distribution (presence in the mesophyll and/or epidermal tissue). The occurrence of LDs in a particular coffee leaf tissue is the prerequisite to further explore the physiological role of these organelles and reveal their possible functional benefits.

CES1 Releases Oxylipins from Oxidized Triacylglycerol (oxTAG) and Regulates Macrophage oxTAG/TAG Accumulation and PGE2/IL-1β Production.

Triacylglycerols (TAGs) are storage forms of fat, primarily found in cytoplasmic lipid droplets in cells. TAGs are broken down to their component free fatty acids by lipolytic enzymes when fuel reserves are required. However, polyunsaturated fatty acid (PUFA)-containing TAGs are susceptible to nonenzymatic oxidation reactions, leading to the formation of oxylipins that are esterified to the glycerol backbone (termed oxTAGs). Human carboxylesterase 1 (CES1) is a member of the serine hydrolase superfamily and defined by its ability to catalyze the hydrolysis of carboxyl ester bonds in both toxicants and lipids. CES1 is a bona fide TAG hydrolase, but it is unclear which specific fatty acids are preferentially released during lipolysis. To better understand the biochemical function of CES1 in immune cells, such as macrophages, its substrate selectivity when it encounters oxidized PUFAs in TAG lipid droplets requires study. We sought to identify those esterified oxidized fatty acids liberated from oxTAGs by CES1 because their release can activate signaling pathways that enforce the development of lipid-driven inflammation. Gaining this knowledge will help fill data gaps that exist between CES1 and the lipid-sensing nuclear receptors, PPARγ and LXRα, which are important drivers of lipid metabolism and inflammation in macrophages. Oxidized forms of triarachidonoylglycerol (oxTAG20:4) or trilinoleoylglycerol (oxTAG18:2), which contain physiologically relevant levels of oxidized PUFAs (<5 mol %), were incubated with recombinant CES1 to release oxylipins and nonoxidized arachidonic acid (AA) or linoleic acid (LA). CES1 hydrolyzed each oxTAG, yielding regioisomers of hydroxyeicosatetraenoic acids (5-, 11-, 12-, and 15-HETE) and hydroxyoctadecadienoic acids (9- and 13-HODE). Furthermore, human THP-1 macrophages with deficient CES1 levels exhibited a differential response to extracellular stimuli (oxTAGs, lipopolysaccharide, and 15-HETE) as compared to those with normal CES1 levels, including enhanced oxTAG/TAG lipid accumulation and altered cytokine and prostaglandin E2 profiles. This study suggests that CES1 can metabolize oxTAG lipids to release oxylipins and PUFAs, and it further specifies the substrate selectivity of CES1 in the metabolism of bioactive lipid mediators. We suggest that the accumulation of oxTAGs/TAGs within lipid droplets that arise due to CES1 deficiency enforces an inflammatory phenotype in macrophages.

#6086 NEUTRAL LIPID STORAGE DISEASE WITH RENAL INVOLVEMENT: A CASE REPORT

Abstract Background and Aims Neutral lipid storage diseases (NLSD) are rare conditions caused by an inborn error of neutral lipid metabolism that results in a deficit in the degradation of cytoplasmic triglycerides and a consequent abnormal storage of the neutral lipids. Their accumulation in cytoplasmic lipid droplets in most tissues of the body results in a very heterogeneous phenotype: myopathic syndrome, intellectual deficit, cataract, neuropathy, cardiomyopathy, hepatomegaly are the main clinical manifestations. Less frequent conditions are diabetes, chronic pancreatitis and renal involvement. Method A 27-year-old woman visited our hospital on November 2022 because of progressive onset of edema, dyspnea, nausea and loss of appetite. The medical history showed that 4 years before she was diagnosed with diabetes mellitus and she has been suffering from severe muscle weakness (she was helping herself with the wheelchair) for two years. In 2017 a proctological evaluation showed an anal hypotonia from likely neuropathy. Blood chemistry tests revealed a metabolic acidosis, a decreased renal function, anaemia and nephrotic range proteinuria at the urinalysis. A bilateral pleural effusion was described at the chest radiograph and a circumferential pericardial effusion at a first echocardiogram. The ultrasound of the abdomen described a small size of the right kidney (90 mm) and an angiomyolipoma on the left kidney. Neurological examination showed a mild intellectual disability, facies with dysmorphic appearance and hypotonia of proximal and distal muscular masses of the upper limbs. Lower limbs showed a more pronounced strength deficit on the left and a hypotonia to proximal muscle masses. Electromyography showed a condition of severe sensitivity polyneuropathy: signs of chronic denervation and morphological alterations of the motor unit especially in the proximal districts of the lower limbs. Ophthalmological examination showed an advanced cataract. Cardiological evaluation through heart echo scanshowed a dilated heart disease with ventricular function and dilation of the pulmonary artery. A quadriceps muscle biopsy finally revealed myogenic features consistent with neutral lipid storage disease. Fabry test came back negative. Despite several drug treatments, the patient continued to be asthenic, inappetent and the oxygen therapy could not be removed because frequent episodes of desaturation. The deterioration of the patient's clinical conditions required the onset of dialysis immediately. Unfortunately, despite adequate information on the risks of possible refusal, the patient denied consent for central venous catheter placement and hence the beginning of renal replacement therapy. Sadly, the patient was lost to follow up and it was not possible to carry out other diagnostic investigations. Results Our patient developed an end-stage renal disease in the framework of a neutral lipid storage disease. Since cytoplasmic lipid droplets are ubiquitous organelles, we can assume that in this case, kidney involvement is due to a pathogenetic mechanism similar to that of other storage diseases such as Fabry disease (Table 1). Conclusion Here we describe a 27-year-old NLSD female patient showing late onset myopathy and difficulties in mobilization in association with severe cardiac and ocular involvement, mild intellectual disability, diabetes mellitus type 2 and end stage renal disease. Our data expand the clinical manifestations of NLSD, providing further evidence for clinical NLSD heterogeneity and mainly, further evidence for storage diseases with renal involvement.