Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids

Macrophages accumulate triglycerides under certain pathological conditions such as atherosclerosis. Triglycerides are carried in the bloodstream as part of very low-density lipoproteins (VLDL) and chylomicrons. How macrophages take up and process VLDL-lipids is not very well known. Here, using VLDL-sized triglyceride-rich emulsion particles, we aimed to study the mechanism by which VLDL-triglycerides are taken up, processed, and stored in macrophages. Our results show that macrophage uptake of emulsion particles mimicking VLDL (VLDLm) is dependent on lipoproteins lipase (LPL) and requires the lipoprotein-binding C-terminal domain of LPL but not the catalytic N-terminal domain. Subsequent internalization of VLDLm-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by triglyceride hydrolysis catalyzed by lysosomal acid lipase. Transfer of lysosomal fatty acids to the ER for subsequent storage as triglycerides is mediated by Stard3, whereas NPC1 was found to promote the extracellular efflux of fatty acids from lysosomes. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete part of the internalized triglycerides as fatty acids. SummaryTriglyceride-rich lipoproteins and their remnants contribute to atherosclerosis, possibly by carrying remnant cholesterol and/or by exerting a pro-inflammatory effect on macrophage. Nevertheless, little is known about how macrophages process triglyceride-rich lipoproteins. We show that uptake by macrophages of VLDL-like particles is dependent on the enzyme lipoproteins lipase via its C-terminal domain. Subsequent internalization of VLDL-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by hydrolysis by lysosomal acid lipase. Transfer of lysosomal fatty acids to the ER for lipid storage is mediated by Stard3, while NPC1 promotes the extracellular efflux of fatty acids. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete internalized triglycerides as fatty acids. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

The two major products of intestinal triacylglycerol digestion and lipoprotein lipolysis are monoacylglycerols (MAG) and fatty acids. In the gut, these products are taken up by enterocytes and packaged into perilipin-coated cytosolic lipid droplets and then secreted as chylomicrons. We observed that fat feeding or intragastric administration of triacylglycerol oil caused the enterocyte Golgi to fragment into submicron puncta dispersed throughout the cytosol. Further, this apparent Golgi dispersion was also observed in cultured fibroblasts after treatment with fat (cream) and pancreatic lipase, but not when treated with deactivated lipase. We therefore hypothesized that a hydrolytic fat product, specifically monoacylglycerols, fatty acids or a combination of these molecules can trigger Golgi fragmentation. Disruption of coatomer function is known to cause Golgi to fuse with the ER, and blocks perilipin 2 delivery to lipid droplets. Thus, we assessed the effects of MAG on coatomer distribution, Golgi structure and perilipin 2 localization. We found that MAG, but not fatty acids, dispersed coatomer from the Golgi, fragmented the Golgi and caused perilipin 2 to accumulate on cellular membranes. Thus, our findings suggest that monoacylglycerol production during digestion disperses the Golgi, possibly by altering coatomer function, which may regulate metabolite transport between the ER and Golgi.

Starvation causes the accumulation of lipid droplets in the liver, a somewhat counterintuitive phenomenon that is nevertheless conserved from flies to humans. Much like fatty liver resulting from overfeeding, hepatic lipid accumulation (steatosis) during undernourishment can lead to lipotoxicity and atrophy of the liver. Here, we found that while surface populations of Astyanax mexicanus undergo this evolutionarily conserved response to starvation, the starvation-resistant cavefish larvae of the same species do not display an accumulation of lipid droplets upon starvation. Moreover, cavefish are resistant to liver atrophy during starvation, providing a unique system to explore strategies for liver protection. Using comparative transcriptomics between zebrafish, surface fish, and cavefish, we identified the fatty acid transporter slc27a2a/fatp2 to be correlated with the development of fatty liver. Pharmacological inhibition of slc27a2a in zebrafish rescues steatosis and atrophy of the liver upon starvation. Further, down-regulation of FATP2 in drosophila larvae inhibits the development of starvation-induced steatosis, suggesting the evolutionary conserved importance of the gene in regulating fatty liver upon nutrition deprivation. Overall, our study identifies a conserved, druggable target to protect the liver from atrophy during starvation. One-Sentence SummaryCavefish evolved protection from starvation-induced liver damage through reduction of fatty acid uptake regulated by FATP2, a mechanism conserved through 400 million years of animal evolution.

Dictyostelium discoideum is a professional phagocyte frequently used as experimental model to study cellular processes underlying the recognition, engulfment and infection course of microbial pathogens. Sphingolipids are abundant components of the plasma membrane that bind cholesterol, control vital membrane properties, participate in signal transmission and serve as adhesion molecules in recognition processes relevant to immunity and infection. While the pathway of sphingolipid biosynthesis has been well characterized in plants, animals and fungi, the identity of sphingolipids produced in D. discoideum, an organism at the crossroads between uni- and multicellular life, is not known. Combining lipidomics with a bioinformatics-based cloning strategy for key sphingolipid biosynthetic enzymes, we show here that D. discoideum produces phosphoinositol-containing sphingolipids with predominantly phytoceramide backbones. Cell-free expression of candidate inositol-phosphorylceramide (IPC) synthases from D. discoideum in defined lipid environments enabled identification of an enzyme that selectively catalyses the transfer of phosphoinositol from phosphatidylinositol onto ceramide. The corresponding IPC synthase, DdIPCS1, is non-homologous to but shares multiple sequence motifs with yeast IPC and human sphingomyelin synthases and localizes to the Golgi apparatus as well as the contractile vacuole of D. discoideum. Collectively, these findings open up important opportunities for exploring a role of sphingolipids in phagocytosis and infection across major evolutionary boundaries.

Sphingomyelin (SM) is a major component of mammalian cell membranes and particularly abundant in the myelin sheath that surrounds nerve fibers. Its production is catalyzed by SM synthases SMS1 and SMS2, which interconvert phosphatidylcholine and ceramide to diacylglycerol and SM in the Golgi and at the plasma membrane, respectively. As the lipids participating in this reaction fulfill both structural and signaling functions, SMS enzymes have considerable potential to influence diverse important cellular processes. The nematode Caenorhabditis elegans is an attractive model for studying both animal development and human disease. The organism contains five SMS homologues but none of these have been characterized in any detail. Here, we carried out the first systematic analysis of SMS family members in C. elegans. Using heterologous expression systems, genetic ablation, metabolic labeling and lipidome analyses, we show that C. elegans harbors at least three distinct SM synthases and one ceramide phosphoethanolamine (CPE) synthase. Moreover, C. elegans SMS family members have partially overlapping but also unique subcellular distributions and together occupy all principal compartments of the secretory pathway. Our findings shed light on crucial aspects of sphingolipid metabolism in a valuable animal model and opens avenues for exploring the role of SM and its metabolic intermediates in organismal development.

Structured AbstractO_ST_ABSObjectivesC_ST_ABSTriglyceride (TG) association with apolipoprotein B100 (apoB100) serves to form very low density lipoproteins (VLDL) in the liver. The repertoire of factors that facilitate this association is incompletely defined. FITM2, an integral endoplasmic reticulum (ER) protein, was originally discovered as a factor participating in cytoplasmic lipid droplets (LDs) in tissues that do not form VLDL. We hypothesized that in the liver, in addition to promoting cytosolic LD formation, FITM2 would also transfer TG from its site of synthesis in the ER membrane to nascent VLDL particles within the ER lumen. MethodsExperiments were conducted using a rat hepatic cell line (McArdle-RH7777, or McA cells), an established model of mammalian lipoprotein metabolism, and mice. FITM2 expression was reduced using siRNA in cells and by liver specific cre-recombinase mediated deletion of the Fitm2 gene in mice. Effects of FITM2 deficiency on VLDL assembly and secretion in vitro and in vivo were measured by multiple methods, including density gradient ultracentrifugation, chromatography, mass spectrometry, simulated Raman spectroscopy (SRS) microscopy, sub-cellular fractionation, immunoprecipitation, immunofluorescence, and electron microscopy. Main findings1) FITM2-deficient hepatic cells in vitro and in vivo secrete TG-depleted VLDL particles, but the number of particles is unchanged compared to controls; 2) FITM2 deficiency in mice on a high fat diet (HFD) results in decreased plasma TG levels. The number of apoB100-containing lipoproteins remains similar, but shift from VLDL to LDL density; 3) Both in vitro and in vivo, when TG synthesis is stimulated and FITM2 is deficient, TG accumulates in the ER, and despite its availability this pool is unable to fully lipidate apoB100 particles; 4) FITM2 deficiency disrupts ER morphology and results in ER stress. Principal conclusionsThe results suggest that FITM2 contributes to VLDL lipidation, especially when newly synthesized hepatic TG is in abundance. In addition to its fundamental importance in VLDL assembly, the results also suggest that under dysmetabolic conditions, FITM2 may be a limiting factor that ultimately contributes to non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH).

Loss of the prolyl endopeptidase-like (PREPL) protein causes congenital myasthenic syndrome-22 (CMS22), a rare neuromuscular and metabolic disorder. PREPL belongs to the serine hydrolase superfamily, but its physiological substrates remain unknown. Based on the predicted lipid binding pocket in its crystal structure and its in vitro esterase activity, we hypothesized that PREPL might act as a lipase in vivo and directly regulate lipid metabolism. To test this, we performed unbiased lipidomics in Prepl knockout (KO) mouse brains and CRISPR-Cas9-generated KO cell lines. Across tissue and cell types, global phospholipid composition was largely unchanged, with only modest, non-significant increases in lysophospholipids, arguing against a direct role of PREPL in (lyso)phospholipid turnover. In contrast, PREPL KO HEK293T cells exhibited a significant accumulation of triacylglycerols (TAGs) and an increased number of lipid droplets, indicating a selective shift toward lipid storage. Given the central role of peroxisomes in lipid metabolism, we assessed PREPL localization and examined peroxisome number, morphology, and levels of key peroxisomal proteins. PREPL did not localize to peroxisomes, and peroxisome number and proteins levels were largely unchanged. However, KO cells displayed elongated peroxisomes, a phenotype possibly linked to mitochondrial dysfunction. Indeed, previous studies have shown that PREPL localizes to mitochondria and is required for respiratory chain activity and oxidative phosphorylation. These mitochondrial defects are predicted to impair fatty acid {beta}-oxidation and disrupt redox balance, thereby promoting TAG synthesis and lipid droplet biogenesis as adaptive responses. Overall, our findings indicate that PREPL does not act as a canonical lipase but indirectly alters lipid homeostasis through its critical role in mitochondrial function. Elevated TAG levels and altered peroxisome morphology likely represent secondary consequences of impaired mitochondrial fatty acid metabolism in PREPL-deficient cells. These results establish a mechanistic link between mitochondrial dysfunction and lipid remodeling in PREPL deficiency, providing novel insights into the metabolic pathology of CMS22.

The liver x receptors (LXRs) are key regulators of systemic lipid metabolism. We determined whether transmembrane protein 135 (TMEM135) is an LXR target gene and its physiologic function. An LXR agonist increased TMEM135 mRNA and protein in human hepatocyte and macrophage cell lines, which was prevented by LXR knockdown. The human TMEM135 promoter contains an LXR response element that bound the LXRs via EMSA and ChIP, and mediated LXR-induced transcription in reporter assays. Knockdown of TMEM135 in HepG2 cells caused triglyceride accumulation despite reduced lipogenic gene expression, indicating a potential role in {beta}-oxidation. To determine physiologic importance, TMEM135 was knocked-down via siRNA in livers of fed and fasted C57BL/6 mice. Fasting increased hepatic fatty acid and NADH concentrations in control mice, consistent with increased fatty acid uptake and {beta}-oxidation. However, in fasted TMEM135 knockdown mice, there was a further significant increase in hepatic fatty acid concentrations and a significant decrease in NADH, indicating an impairment in {beta}-oxidation by peroxisomes and/or mitochondria. Conversely, hepatic ketones tended to increase in fasted TMEM135 knockdown compared to control mice, and because ketogenesis is exclusively dependent on mitochondrial {beta}-oxidation, this indicates peroxisomal {beta}-oxidation was impaired in knockdown mice. Localization studies demonstrated that TMEM135 co-localized with peroxisomes but not mitochondria. Mechanistically, proteomic and Western blot analyses indicated that TMEM135 regulates concentrations of matrix enzymes within peroxisomes. In conclusion, TMEM135 is a novel LXR target gene in humans that mediates peroxisomal metabolism, and thus TMEM135 may be a therapeutic target for metabolic disorders associated with peroxisome dysfunction.

miR-122 is a liver specific miRNA that plays an important role in controlling metabolic homeostasis in mammalian liver cells. Interestingly, miR-122 on exposure to lipotoxic stress is reduced in liver cells. To fight stress, miRNA processor Dicer1 is depleted to cause reduced miR-122 production and the lowering of miRNA level ensures a better stress response in hepatocytes under lipotoxic stress. Interestingly, lipid droplets, formed in the liver cells on exposure to high fat, ensure cytoplasmic phase separation of Ago2 and prevent interaction of Ago2 with Dicer1. Lipid droplets bind miRNA and enhance miRNA-Ago2 uncoupling and Ago2 phase separation. Loss of interaction between Ago2 and Dicer1 eventually facilitates export and lowering of cellular Dicer1, a process also dependent on the endosomal maturation controller protein Alix, thereby ceasing pre-miRNA processing by Dicer1 in lipid exposed cells. Depletion of lipid droplets by downregulation of Perilipins with siRNAs resulted in a rescue of cellular Dicer1 level and Ago2-Dicer1 interaction. This is a novel mechanism that liver cells adopt to restrict cellular miRNA levels under stress condition. Thus, lipid droplets prevent cell death upon exposure to high fat by reducing intra and extracellular pool of miR-122 in hepatic tissue.

Choline is an essential nutrient that is critical component of the membrane phospholipid phosphatidylcholine (PC), the neurotransmitter acetylcholine and the methylation pathway. In the liver specifically, PC is the major membrane constituent and can be synthesized by the CDP-choline or the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. With the continuing global rise in the rates of obesity and non-alcoholic fatty liver disease, we sought to explore how excess fatty acids (FA), typical of an obesity and hepatic steatosis, affect choline uptake and metabolism in primary hepatocytes. Our results demonstrate that hepatocytes chronically treated with palmitate, but not oleate or a mixture, had decreased choline uptake, which was associated with lower choline incorporation into PC and lower expression of choline transport proteins. Interestingly, a reduction in the rate of degradation spared PC levels in response to palmitate when compared to control. PE synthesis was slightly diminished; however, no compensatory changes in the PEMT pathway were observed. We next hypothesized that ER stress may be a potential mechanism by which palmitate treatment diminished choline. However, when we exposed primary hepatocytes to the common ER stress inducing compound tunicamycin, choline uptake, contrary to our expectation was augmented, concomitant with the transcript expression of choline transporters. Moreover, tunicamycin-induced ER stress divorced the observed increase in choline uptake from CDP-choline pathway flux since ER stress significantly diminished the incorporation and total PC content, similar to PE. Conclusion: Therefore, our results suggest that the altered FA milieu seen in obesity and fatty liver disease progression may adversely affect choline metabolism, but that compensatory mechanisms work to maintain phospholipid homeostasis.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC=\"FIGDIR/small/746750v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (42K):\norg.highwire.dtl.DTLVardef@1090f2aorg.highwire.dtl.DTLVardef@1c28eedorg.highwire.dtl.DTLVardef@35a80eorg.highwire.dtl.DTLVardef@ac5a53_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fatty liver is characterised by the expansion of lipid droplets and is associated with the development of many metabolic diseases, including insulin resistance, dyslipidaemia and cardiovascular disease. We assessed the morphology of hepatic lipid droplets and performed quantitative proteomics in lean, glucose-tolerant mice compared to high-fat diet (HFD) fed mice that displayed hepatic steatosis and glucose intolerance as well as high-starch diet (HStD) fed mice who exhibited similar levels of hepatic steatosis but remained glucose tolerant. Both HFD and HStD-fed mice had more and larger lipid droplets than Chow-fed animals. We observed striking differences in liver lipid droplet proteomes of HFD and HStD-fed mice compared to Chow-fed mice, with fewer differences between HFD and HStD. Taking advantage of our diet strategy, we identified a fatty liver lipid droplet proteome consisting of proteins common in HFD- and HStD-fed mice. Likewise, a proteome associated with glucose tolerance that included proteins common in Chow and HStD but not HFD-fed mice was identified. Notably, glucose intolerance was associated with changes in the ratio of adipose triglyceride lipase (ATGL) to perilipin 5 (PLIN5) in the lipid droplet proteome, suggesting dysregulation of neutral lipid homeostasis in glucose-intolerant fatty liver, which supports bioactive lipid synthesis and impairs hepatic insulin action. We conclude that our novel dietary approach uncouples ectopic lipid burden from insulin resistance-associated changes in the hepatic lipid droplet proteome.

Omega-3 polyunsaturated fatty acids (N-3 PUFA), specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are well recognized for their triacylglycerol (TAG)-lowering properties. These effects are generally attributed to reduced hepatic lipogenesis and increased {beta}-oxidation; however, the contribution of white adipose tissue (WAT) towards the hypotriglyceridemic properties of N-3 PUFA is less defined. Lipoprotein lipase (LPL) regulates TAG hydrolysis to influence fatty acid uptake into WAT, a process that can be inhibited by angiopoietin-like 4 (ANGPTL4). When re-examining a previous mouse study, we found that mice consuming a diet rich in EPA/DHA had increased WAT Angptl4 expression in the fasted state compared to a control diet. Therefore, the goal of this study was to explore the role of N-3 PUFA on the regulation of Angptl4 expression and LPL activity in adipocytes. 3T3-L1 adipocytes treated with DHA (100M), but not ALA or EPA, increased Angptl4 expression and reduced LPL activity similar to that observed with a PPAR{gamma} agonist (pioglitazone). When Ppar{gamma} expression was knocked down with siRNA, the ability of DHA and pioglitazone to induce Angptl4 expression was ablated. Further, DHA- and pioglitazone-induced reductions in LPL activity were mitigated when Angptl4 expression was silenced. Taken together, these results suggest that DHA regulates LPL activity by increasing Angptl4 expression in a PPAR{gamma}-dependent manner. Our results have uncovered a novel mechanism by which DHA regulates ANGPTL4 to influence LPL-mediated hydrolysis of circulating TAG in WAT. Future in-vivo studies are necessary to determine the relevance of DHA regulation of ANGPTL4 towards whole-body lipid homeostasis and cardiometabolic health. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/685896v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@ef2894org.highwire.dtl.DTLVardef@1513f4aorg.highwire.dtl.DTLVardef@11235cdorg.highwire.dtl.DTLVardef@8a2b68_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDHA increases Angptl4 gene expression and reduces LPL activity in 3T3-L1 adipocytes C_LIO_LIChanges in FOXO1 do not explain DHA-induced changes in Angptl4 gene expression C_LIO_LISilencing Ppar{gamma} ablates DHA-induced increases in Angptl4 gene expression C_LIO_LISilencing Angptl4 partially attenuates DHA-induced reductions in LPL activity C_LI

The liver controls plasma lipids by secreting lipid-rich very low density lipoproteins (VLDL) into blood. Inside hepatocytes in the liver, Lipid Droplets (LDs) are transported to the smooth Endoplasmic Reticulum (sER) by kinesin-1 motors, and then catabolized in the sER to supply lipids for VLDL assembly. LDs are the only cellular organelle bounded by a phospholipid monolayer, and are thus distinct from all other (bilayer-bounded) organelles. It is therefore plausible that a given protein can bind to the LD membrane using mechanisms that are completely different from all other organelles. Indeed, here we find that kinesin-1 uses its tail domain to bind LDs, but alternative mechanisms to bind other organelles. A peptide corresponding to kinesins tail domain therefore competes with, and removes kinesin-1 selectively from LDs with minimal effect on other organelles. Delivery of lipids for VLDL assembly is consequently reduced, causing a remarkable reduction of [~]50% of secreted lipids (triglycerides and cholesterol) in cell culture. We further develop Orally fed Egg-liposomes as a method to deliver kinesin tail domain peptide to the liver of Zebrafish. The peptide reverses diet-induced hyperlipidaemia in Zebrafish larvae and brings the larvae back to a normolipidaemic state, thus confirming the effectiveness of our method in a physiologically relevant in-vivo situation. Strikingly, the peptide causes no unwanted accumulation of lipids in the liver, no toxicity and no developmental or behavioural defects in Zebrafish. Using a peptide to displace proteins (e.g. kinesin) selectively from LDs provides a conceptually novel and radically different approach against hyperlipidaemia. This monolayer-versus-bilayer strategy can be potentially extended to target other LD-bound proteins that function as key regulators of Lipid metabolism.

Pyrimidine catabolism is implicated in hepatic steatosis. Dihydropyrimidine Dehydrogenase (DPYD) is an enzyme responsible for uracil and thymine catabolism, and DPYD human genetic variability affects clinically observed toxicity following 5-Fluorouracil (5-FU) administration. In an in vitro model of diet-induced steatosis, the pharmacologic inhibition of DPYD resulted in protection from lipid accumulation. Additionally, a gain-of-function mutation of DPYD, created through clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) engineering, led to an increased lipid burden, which was associated with altered mitochondrial functionality in a hepatocarcionma cell line. The studies presented herein describe a novel role for DPYD in hepatocyte metabolic regulation as a modulator of hepatic steatosis.

AbstractSpondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal recessive disorder of the skeleton and the retina caused by biallelic variants in PCYT1A, encoding the nuclear enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes the rate-limiting step in phosphatidylcholine (PC) biosynthesis by the Kennedy pathway. As a first step in understanding the consequences of PCYT1A variants on SMD-CRD pathophysiology, we generated and characterized a series of cellular models for SMD-CRD, including CRISPR-edited PCYT1A-null HEK293 and ATDC5 cell lines. Immunoblot and PC synthesis assays of cultured skin fibroblasts from SMD-CRD patient cell lines revealed patient genotype-specific reductions in CCT steady state levels (10-75% of wild-type) and choline incorporation into PC (22-54% of wild-type). While PCYT1A-null HEK293 cells exhibited fewer and larger lipid droplets in response to oleate loading than their wild-type counterparts, SMD-CRD patient fibroblasts (p.Ser323Argfs*38 homozygotes) failed to show significant differences in lipid droplet numbers or sizes as compared to controls. Lipid droplet phenotypes in PCYT1A-null HEK293 cells were rescued by transfection with wild-type, p.Ala99Val, and p.Tyr240His human PCYT1A cDNAs. While both edited cellular models had normal morphology and proliferation rates compared to unedited controls, Pcyt1a-null ATDC5 cells demonstrated accelerated rates of chondrocyte differentiation as compared to their wild-type counterparts. Lipidomics revealed changes in 75-200 lipid levels in PCYT1A-null HEK293 and ATDC5 cells or in SMD-CRD patient fibroblasts as compared to wild-type controls. The specific lipids altered and extent of change varied by cell type. Importantly, both PCYT1A-null HEK293 cells and SMD-CRD patient fibroblast cell lines had decreased phosphatidylcholine:phosphatidylethanolamine (PC:PE) ratios and decreased levels of several lysophosphatidylcholine (LPC) species as compared to wild-type controls, suggesting compensatory PC production through increased LPC remodeling by LPCAT or decreased conversion of PC to LPC by phospholipase A2. Our results show that all tested PCYT1A alleles associated with SMD-CRD are hypomorphic and suggest involvement of PCYT1A in chondrocyte differentiation, PC:PE ratio maintenance and LPC metabolism, and lipid droplet formation. Author SummaryRare genetic disorders can reveal the function of genes on an organismal scale. When normal gene activity is lost, patients can experience a range of symptoms, often dependent on the residual activity of the encoded protein. Rare variants in the gene PCYT1A can cause multiple inherited disorders, including a disorder of the skeleton and the retina characterized by short stature, bone abnormalities, and blindness. PCYT1A is required for normal cellular function, particularly lipid metabolism, but the role of this gene in human disease is still poorly understood. To determine consequences of genetic variants in patients with this disorder, we made and studied a series of cellular models, including cells cultured from patients and CRISPR-edited cell lines lacking normal copies of PCYT1A. Here we show that patient variants lead to reduced PCYT1A expression and/or function and have adverse consequences on cell biology and lipid metabolism that are often cell-type specific. This work advances understanding of the role of lipid metabolism in skeletal and eye development.

Acute liver failure is a life-threatening condition during infancy. Biallelic pathogenic variants in LARS1 cause infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute hepatic failure in infants. LARS functions as a protein associated with mTORC1 and plays a crucial role in amino acid-triggered mTORC1 activation and autophagy regulation. A previous study demonstrated that larsb-knockout zebrafish show a condition resembling ILFS. However, a comprehensive analysis of larsb-knockout zebrafish has not yet been performed because of early mortality. We herein generated a long-term viable zebrafish model carrying a LARS1 variant identified in an ILFS1 patient (larsb-I451F zebrafish) and analyzed the pathogenesis of the affected liver of ILFS1. Hepatic dysfunction is most prominent in ILFS1 patients during infancy; correspondingly, the larsb-I451F zebrafish manifested hepatic anomalies during the developmental stages. The larsb-I451F zebrafish demonstrates augmented lipid accumulation within the liver under autophagy activation. Inhibition of DGAT1, which converts fatty acids to triacylglycerols, improved lipid droplets in the liver of larsb-I451F zebrafish. Notably, treatment with an autophagy inhibitor ameliorated hepatic lipid accumulation in this model. Our findings suggested that enhanced autophagy caused by biallelic LARS1 variants contributes to ILFS1-associated hepatic dysfunction. Furthermore, the larsb-I451F zebrafish model, which has a prolonged survival rate compared to the larsb-knockout model, highlights its potential utility as a tool for investigating the pathophysiology of ILFS1-associated liver dysfunction. Author SummaryInfantile liver failure (ALF) is a rare but life-threatening condition primarily caused by various genetic and infectious factors during infancy. Comprehensive research into its causes is crucial for treatment decisions, including liver transplantation and supportive interventions. While specific therapies exist for some conditions, a significant proportion of infant ALF cases remains unresolved. Recent advances in genetic sequencing have identified congenital disorders, particularly involving the LARS1 gene, as contributors to ALF. LARS1 is essential for regulating processes related to amino acids and autophagy. To better understand this condition, we created a zebrafish model carrying specific LARS1 gene variants seen in ALF patients. These zebrafish displayed liver abnormalities similar to those observed in infants with ALF. Our study revealed that enhanced autophagy, triggered by biallelic LARS1 variants, plays a significant role in liver dysfunction associated with ALF. Notably, inhibiting specific enzymes involved in fat metabolism and autophagy showed promising results in reducing hepatic lipid accumulation in our zebrafish model. This research provides insights that may lead to improved understanding and potential treatments for this devastating condition.

Yeast ceramide synthesis is regulated by two homologous genes, LAC1 and LAG1, each with distinct physiological roles, especially under ER stress conditions. This study examines their specific functions in lipid homeostasis and the ER stress response. Deleting LAG1 enhances cell growth and survival under ER stress, whereas LAC1 deletion does not provide similar resistance. In contrast, LAG1 deletion significantly impacts phospholipid and neutral lipid metabolism without altering the expression of key ER stress response genes. In comparison, LAG1 overexpression, unlike LAC1 overexpression, severely impairs cell growth and viability, induces ER stress responses, disrupts phospholipid biosynthesis, alters membrane morphology, modifies neutral lipid synthesis, and reduces lipid droplet numbers. Overall, LAG1 uniquely regulates ER stress and lipid homeostasis, independent of its function in ceramide synthesis. Understanding the specific contributions of LAG1 to lipid homeostasis and ER stress provides valuable insight into cellular stress mechanisms.

Obesity caused by overnutrition is a major risk factor for non-alcoholic fatty liver disease (NAFLD). Several lipid intermediates such as fatty acids, glycerophospholipids and sphingolipids are implicated in NAFLD, but detailed characterization of lipids and their functional links to proteome and phosphoproteome remain to be elucidated. To characterize this complex molecular relationship, we used multi-omics approach by conducting comparative proteomic, phoshoproteomic and lipidomic analyses of high fat (HFD) and low fat (LFD) diet fed mice livers. We quantified 2447 proteins and 1339 phosphoproteins containing 1650 class I phosphosites (with localization probability > 0.75), of which 669 phosphosites were significantly different between HFD and LFD mice livers. We detected alterations of proteins associated with cellular metabolic processes such as small molecule catabolic process, monocarboxylic acid, long- and medium-chain fatty acid, and ketone body metabolic processes, and peroxisome organization. We observed significant downregulation of protein phosphorylation in HFD fed mice liver in general. Untargeted lipidomics identified upregulation of triacylglycerols, glycerolipids and ether glycerophosphocholines and downregulation of glycerophospholipids such as lysoglycerophospholipids, as well as ceramides and acylcarnitines. Analysis of differentially regulated phosphosites revealed phosphorylation dependent deregulation of insulin signaling as well as lipogenic and lipolytic pathways during HFD induced obesity. Thus, this study reveals a molecular connection between decreased protein phosphorylation and lipolysis, as well as lipid-mediated signaling in diet-induced obesity.

Lipids have been implicated as mediators of insulitis and {beta}-cell death in type 1 diabetes development, but the mechanisms underlying this association are poorly understood. Here, we investigated the changes in islet/{beta}-cell lipid composition using three models of insulitis: human islets and EndoC-{beta}H1 {beta}-cells treated with the cytokines IL-1{beta} and IFN-{gamma}, and islets from non-obese diabetic mice. Across all three models, lipidomic analyses showed a consistent change in abundance of the lysophosphatidylcholine, phosphatidylcholine and triacylglycerol species. We also showed that lysophosphatidylcholine and its biosynthetic enzyme PLA2G6 are enriched in murine islets. We determined that the ADP-ribosyl-acceptor glycohydrolase ARH3 is regulated by cytokines downstream of PLA2G6, which in turn regulates proteins involved in apoptosis, lipid metabolism, antigen processing and presentation and chemokines. ARH3 reduced cytokine-induced apoptosis, which may represent a negative feedback mechanism. Overall, these data show the importance of lipid metabolism in regulating {beta}-cell death in type 1 diabetes. HighlightsO_LILipidomics of 3 insulitis models revealed commonly regulated lipid classes. C_LIO_LIIdentification of 35 proteins regulated by cytokines via PLA2G6 signaling. C_LIO_LIARH3 reduces cytokine-induced apoptosis via PLA2G6 regulation. C_LIO_LIARH3 regulates the levels of proteins related to insulitis and type 1 diabetes. C_LI

ObjectiveAdrenocortical hormone levels increase in obesity, potentially contributing to development of obesity-associated pathologies. Here we explored whether lipidomic remodeling of the adrenal gland could mediate altered adrenocortical steroidogenesis during obesity. MethodsLipidomic analysis was performed in adrenal glands using shotgun mass spectrometry (MS), and steroid profiling of sera by liquid chromatography tandem mass spectrometry (LC-MS/MS) from lean and obese mice. Gene expression analysis was performed in adrenal glands and adrenocortical cell populations. The role of Fatty Acid Desaturase 2 (FADS2) and arachidonic acid on steroid hormone production was studied in primary adrenal gland cell cultures. ResultsAdrenal glands of obese mice displayed a distinct lipidomic profile, encompassing longer and more unsaturated storage lipids and phospholipids compared to adrenal glands of lean mice. Arachidonoyl acyl chains were abundant in the adrenal gland phospholipidome and increased upon obesity. This was accompanied by increased Fads2 expression, the rate-limiting enzyme of arachidonic acid synthesis, and enhanced plasma adrenocortical hormone levels. Inhibition of FADS2 in primary adrenal gland cell cultures abolished steroidogenesis, which was restored by arachidonic acid supplementation. ConclusionsOur data suggest that the FADS2 - arachidonic acid axis regulates adrenocortical hormone synthesis, while alterations in the content of arachidonoyl chains in the adrenal gland phopsholipidome could account for disturbed adrenocortical hormone production. HighlightsO_LIThe adrenal gland lipidome is remodeled in obesity. C_LIO_LIArachidonoyl groups are abundant in the adrenal gland phospholipidome and increase in obesity. C_LIO_LIFADS2 is highly expressed in the adrenal gland and its expression is further increased in obesity. C_LIO_LIFADS2 inhibition blunts adrenocortical steroidogenesis in primary adrenal gland cell cultures, while arachidonic acid supplementation restores it. C_LI