Journal of Lipid Research

ObjectiveGlucokinase Regulatory Protein (GKRP) controls the activity of Glucokinase (GCK) to regulate liver glucose uptake and storage. Coding variants in GCKR, the gene encoding GKRP, strongly associate with fatty liver disease, hypertriglyceridemia, and hypercholesterolemia. Here, we sought to investigate the mechanisms by which a common GKRP variant affects hepatic lipid and cholesterol metabolism. MethodsWe developed mouse models to examine how the human GKRP P446L variant influences liver and systemic metabolism. Endogenous Gckr expression was ablated in adult mouse hepatocytes, together with re-expression of either human GKRP P446L or the reference GKRP protein. We assessed body weight, adiposity, systemic glucose homeostasis, and hepatic metabolites in mice expressing reference GKRP or GKRP P446L under multiple metabolic conditions. To determine whether the effects of GKRP P446L may result from reduced GCK activity, we analyzed mice with liver-specific deletion of Gck. ResultsHepatic expression of GKRP P446L resulted in reduced GKRP and GCK protein levels and elevated serum cholesterol. Hepatic deletion of Gck in mice recapitulated several effects of GKRP P446L, including increased hepatic cholesterol and triglyceride content. The elevated cholesterol was associated with increased cholesterogenic gene expression and cholesterol synthesis. Hepatic expression of an alternative hexokinase (HKII) normalized the effects of GCK-deficiency, suggesting that impaired glucose phosphorylation underlies the phenotype. ConclusionsThe GKRP P446L variant reduced GKRP protein abundance, and diminished GCK activity while increasing cholesterol levels. Loss of GCK elevated cholesterol and hepatic triglyceride levels. Collectively, these findings demonstrate that GCK suppresses hepatic cholesterol synthesis and lipid accumulation, suggesting that reduced GCK activity underlies the metabolic abnormalities associated with the GKRP P446L variant. HighlightsO_LIThe GKRP P446L variant reduces GKRP protein abundance and diminishes GCK activity. C_LIO_LIExpression of GKRP P446L in mouse hepatocytes increases serum cholesterol levels. C_LIO_LIHepatic GCK activity suppresses cholesterogenic gene expression and cholesterol synthesis. C_LI

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is challenging to study in vivo in humans and in vitro models are limited. Although primary human hepatocytes (PHHs) are considered the gold-standard, immortalized hepatic cell lines are utilised due to scalability. This study compared the metabolic responses of PHHs with our Huh7-based model cultured in physiologically-relevant fatty acid (FA) mixtures. MethodsPHH and Huh7 cells were treated with 2% human serum, sugars and FAs enriched in either unsaturated (OPLA) or saturated (POLA) FAs for 4 or 7 days, respectively. Stable isotope tracers investigated basal metabolic changes in response to treatment. Cell viability, media biochemistry, intracellular metabolism, lipid droplet morphology and gene expression were quantified. ResultsHuh7 cells had greater viability than PHHs, while NEFA uptake and triglyceride secretion were similar. OPLA and POLA increased large lipid droplets in Huh7 cells, whereas only OPLA produced comparable effects in PHHs. Despite higher baseline TG in PHHs, both models showed similar lipid composition, de novo lipogenic responses, and glycogen levels. Compared to Huh7 cells, PHHs exhibited higher 3-hydroxybutyrate, lower lactate, reduced glucose uptake, and donor-dependent transcriptomic variability. ConclusionsHuh7 cells are metabolically adaptable and when cultured in physiologically-relevant media, produce metabolic readouts similar PHH cells.

Age-related macular degeneration is a common ocular disease that causes vision loss in the elderly, with a complex set of risk factors and proposed mechanisms of pathogenesis. A powerful method for investigating changes in disease is metabolomics, by which small molecules can be identified and quantified simultaneously. We report here the metabolic analysis of human RPE-choroid tissue in aging and macular degeneration (AMD), as well as comparisons of human macular and extramacular RPE-choroid and neural retina. Levels of 215 metabolites were determined in young donors, AMD donors (early/intermediate, geographic atrophy, and neovascularization) and age-matched controls. The largest number of metabolite differences were observed between young and healthy aged controls, as opposed to between aged controls and any stage of AMD. Two notable metabolites found to be increased in aging choroids are trimethylamine N-oxide and uric acid, both of which were significant after Bonferroni correction. A mouse endothelial cell line treated with a high concentration of uric acid exhibited reduced migration in a wound closure assay. This study provides initial insights into the metabolome of human choroids in varying states of age and macular degeneration, as well as functional implications of these changes in the aging choroid.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central regulator of low-density lipoprotein (LDL) cholesterol metabolism, yet the functional consequences of many clinically observed PCSK9 variants remain unknown. To establish a rigorous system for quantitative variant assessment, we generated a PCSK9 knockout (KO) HepG2 cell line through CRISPR/Cas9-mediated deletion of exons 2-8, effectively removing both the prodomain and catalytic regions required for PCSK9 function. This null background enabled systematic functional mapping of wild-type (WT) PCSK9 and multiple clinically relevant variants representing well-characterized, recurrent, and previously understudied alleles. Functional assays revealed pronounced heterogeneity among variant activities. The classical gain-of-function (GOF) variants D374Y and R496W exhibited robust suppression of LDL uptake, whereas A443T--an infrequently reported and previously uncharacterized variant--demonstrated a loss-of-function (LOF)-like phenotype with significantly enhanced LDL uptake. Additional poorly characterized variants, including V4I, R104C/V114A, and R496W/N425S, displayed minimal functional profiles, providing novel mechanistic insights. Surface LDL receptor (LDLR) levels generally correlated with LDL uptake but revealed unique patterns for specific variants. This KO-based rescue system provides a high-resolution framework for mechanistic classification of both established and poorly characterized PCSK9 variants, bridging the gap between genetic discovery and functional interpretation while supporting precision lipid-lowering strategies.

Cholesterol is a key component of cellular membranes, regulating membrane organization, fluidity, and signaling. However, cholesterol analysis remains technically challenging, as no single method currently allows both accurate quantification and spatially resolved visualization. Biochemical assays provide accurate quantification but lack spatial resolution, whereas imaging strategies can perturb membrane organization or cholesterol accessibility. Here, we describe optimized protocols using fluorescent D4 probes derived from the cholesterol-binding domain of perfringolysin O (D4-mCherry and D4-GFP) to detect, visualize, and quantify cholesterol in biological samples. We detail procedures for probe production, purification, and application, and establish conditions that ensure robust and reproducible labeling of membrane-accessible cholesterol. By combining fluorescence-based imaging with quantitative analysis, this approach enables the assessment of cholesterol distribution while preserving its native membrane environment. The proposed methodology provides a versatile and reliable framework for studying cholesterol in a wide range of experimental systems.

Cannabidiol (CBD) and cannabigerol (CBG) are non-psychoactive cannabinoids that exert diverse biological activities in both normal and cancerous epithelial cells. Although autophagy plays a pivotal role in maintaining cellular homeostasis, the effects of combined CBD-CBG treatment on autophagic regulation across epithelial cell types remain largely unexplored. In this study, GFP-LC3-RFP reporter assays and ATG9-deficient cell models were employed to examine the influence of CBD and CBG on autophagy in Ca9-22 and HaCaT cells. Certain concentrations of either compound alone failed to induce autophagy and, in some cases, appeared to suppress autophagic activity. In contrast, their combined administration markedly enhanced autophagic flux in both cell lines. Low-dose CBG or high-dose CBD promoted differential greater cell survival in HaCaT-WT cells compared to their ATG9-KO counterparts. Collectively, these findings provide novel insights into the cooperative regulation of autophagy by CBD and CBG, underscoring their combined effects on cellular autophagic responses in cancer or normal epithelial cells. HighlightsO_LIIn both Ca9-22 and HaCaT cells, certain doses of CBD alone failed to induce autophagy, whereas CBG at some concentrations showed a trend toward autophagy suppression. C_LIO_LISub-effective doses of CBD and CBG in combination enhance autophagic flux in Ca9-22 and HaCaT cells, with some combinations exceeding the flux induced by higher doses of either compound alone. C_LIO_LICBD and CBG exhibit distinct dose-dependent effects on the survival of HaCaT ATG9-deficient cells compared with HaCaT-WT cells, indicating differential ATG9-dependence. C_LI

SignificanceQuantifying lipid droplet (LD) remodeling in 3D hepatic organoids is often limited to endpoint staining or phototoxic live fluorescence imaging, thereby obscuring droplet-level kinetics. AimWe aimed to develop a label-free method to track LD dynamics in living hepatic organoids under different fatty-acid loads. ApproachTime-lapse 3D refractive-index tomograms were acquired using holotomography and analyzed with a depth-adaptive, multi-threshold segmentation pipeline to quantify LD number, volume, sphericity, and refractive-index-derived concentration and dry mass at single-droplet resolution. ResultsOleic acid and linoleic acid induced LD accumulation while preserving organoid integrity, whereas palmitic acid triggered rapid structural collapse. Despite increases in total LD burden under both oleic acid and linoleic acid, droplet-level dynamics diverged: oleic acid produced volume-dominated accumulation via enlargement of fewer LDs and increased size heterogeneity, whereas linoleic acid produced number-dominated accumulation via sustained increases in LD number, yielding a more uniform population of small droplets. ConclusionsLabel-free holotomography with depth-adaptive analysis enables non-invasive, longitudinal, and multi-scale quantification of LD dynamics in intact organoids and reveals fatty-acid- dependent temporal modes of lipid storage. Statement of DiscoveryWe developed a label-free, longitudinal 3D holotomography framework with depth-adaptive lipid droplet segmentation that quantifies single-droplet dynamics in living mouse hepatic organoids. Using this platform, we found that oleic acid and linoleic acid induce LD accumulation via distinct strategies--oleic acid via droplet enlargement and linoleic acid via sustained increases in droplet number--while palmitic acid rapidly compromises organoid integrity.

The brain, though less than 10% of body mass, contains about 25% of total cholesterol (CHL), emphasizing CHLs key role in neuronal function. Many CHL actions are stereospecific, as shown by differences from its 3-hydroxy epimer, epicholesterol (epiCHL). How this minor structural change alters membrane properties and sterol transport remains unclear. Here, we compare fluorescent analogs of CHL (cholestatrienol, CTL) and epiCHL (epicholestatrienol, epiCTL), which closely mimic their natural counterparts. Biophysical membrane properties, such as flip-flop, acyl-chain ordering, and interbilayer transfer, depend on the orientation of the 3-hydroxy group. Similarly, transport by sterol transport proteins (STPs) and intracellular trafficking of the sterols in human astrocytes are stereospecific. Treatment with 25-hydroxycholesterol increases uptake of both epimers, but only CTL shows enhanced esterification and lipid droplet storage. These findings demonstrate that subtle cholesterol structural changes affect cellular homeostasis and establish epiCTL as a useful probe of sterol stereospecificity and trafficking.

Pharmacological approaches to inhibit extracellular vesicle (EV) release in vivo are increasingly used, although the effects of such compounds on local cellular environments are not fully understood. In this study, we examined the impact of intraperitoneal (i.p.) administration of the neutral sphingomyelinase inhibitor GW4869 on EV levels in the peritoneal cavity. Repeated GW4869 i.p. injections elicited a marked local inflammatory response, reduced peritoneal macrophage numbers, and paradoxically increased EV concentrations 24 hours after treatment. Independent macrophage depletion reproduced this rise in EV levels, indicating that macrophage loss and associated cellular remodeling contribute substantially to EV accumulation. These observations indicate that GW4869 can perturb local immune homeostasis in vivo, a confound that must be considered when using this compound as a putative selective inhibitor of EV release.

SNARE-mediated membrane fusion is regulated by the lipid composition of the engaged bilayers. Lipids impact fusion through direct protein-lipid interactions or through modulating the physical properties of membranes to affect protein function. Lysophospholipids (LPLs) can affect membrane curvature, fluidity and energy of deformation. Their effects are due to their head group, and the length and saturation of their single acyl chains. Here we examined how the properties of LPLs affect yeast vacuole fusion and ion transport. We found that lysophosphatidylcholine (LPC) with acyl chains containing 14-18 carbons inhibited fusion with IC50 values of {cong} 40-120 {micro}M. While acyl chain length moderately affected fusion, the head group played a major role. Unlike LPCs, Lysophosphatidic acid (LPA 18:1) failed to fully inhibit fusion, while lysophosphatidylethanolamine (LPE 18:1) had no effect. Separately we found that changes in acyl chain length and saturation differentially affected Ca2+ transport and vacuole acidification. Together these data show that the effects of LPLs on membrane fusion and ion transport were due to a combination of head group type and acyl chain length.

Rab32 and Rab38 are paralogous small GTPases involved in the biogenesis of lysosome-related organelles (LROs), yet their roles in hepatic lipid metabolism remain poorly defined. Here, Rab32 and Rab38 double-knockout (DKO) male mice exhibited an age-dependent increase in body weight accompanied by hepatic lipid accumulation, suggesting impaired hepatic lipid processing. In AML12 hepatocytes, Rab32 and Rab38 localized to ring-like, LAMP1-positive structures characteristic of LROs, whose size increased with cell confluence. Pharmacological inhibition of lysosomal acid lipase with orlistat led to the accumulation of lipid droplets (LDs) within Rab32/38-positive LROs, indicating that LD degradation occurs in these compartments. Additional treatment with bafilomycin A1 revealed invagination-like internal membrane structures within enlarged LROs. These processes were not affected by artificial inhibition of macroautophagy, highlighting the involvement of microautophagy. Ring-like signals positive for phosphatidylinositol 3-phosphate (PI3P) or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) were detected within or adjacent to LRO membranes, and LDs were frequently associated with these structures, suggesting a role for PI3P and PI(3,5)P2 in internal membrane formation. Vps4B was also required for efficient LD incorporation. Consistently, Rab32/38 double-knockdown (DKD) AML12 cells exhibited increased lipid accumulation, indicating impaired LD engulfment. Together, these findings identify Rab32/38-positive LROs as a structural platform for microautophagy-mediated lipid droplet degradation in hepatocytes.

The composition of culture medium is a major, yet frequently undercontrolled, determinant of hepatic cell state in vitro. For decades, fetal bovine serum (FBS) has been routinely incorporated into liver cell culture. Its undefined and lot-to-lot variable composition can, however, confound cell identity and experimental reproducibility. Serum-free, chemically defined media (CDM) represent an alternative approach that can improve standardization, but the consequences of transitioning from FBS-supplemented media (FBS-SM) to CDM remain insufficiently characterized in hepatic models, particularly with respect to metabolic and detoxification programs that govern xenobiotic handling and hepatotoxicity readouts. Here, we systematically assessed how replacing FBS-SM with CDM remodels transcriptomic profiles in two widely used human hepatic cell lines (HepaRG and HuH7 cells) and compared the results to that obtained from primary human hepatocytes (PHH). Global transcriptomic analyses indicated that cell type was the primary driver of variance, whereas medium induced a model-dependent secondary effect. Functional interpretation showed preferential enhancement of xenobiotic metabolism and transport-associated programs in HepaRG cells, while HuH7 cells response was dominated by lipid/sterol homeostasis and stress-linked processes. Benchmarking against PHH based on hepatic identity and detoxification gene panels further supported improved PHH alignment for HepaRG cells under CDM compared to cultures with FBS-SM, with limited improvement for HuH7 cells. Collectively, these findings address a key knowledge gap by defining how FBS-SM and CDM impact the transcriptomic profiles of HepaRG and HuH7 cells.

BackgroundElevated postprandial hypertriglyceridemia (PP-HTG) is a significant risk factor for development of cardiovascular diseases, however, the mechanisms underlying its exaggerated rise remains poorly understood. MicroRNAs (miRs) are known to be implicated in the regulation of lipid metabolism, thus identifying them as potential key players. We presently investigated whether miRs may control postprandial triglyceride (PP-TG) response. MethodsPostprandial changes in circulating miR expression as a function of the degree of postprandial TG response were evaluated in non-dyslipidemic healthy subjects (n=32). The impact of miR-100-5p on hepatic gene expression was evaluated in differentiated Caco2 and HepG2 cells by analysis of hepatic transcriptome (RNAseq), western blot and ELISA. In vivo studies were conducted in C57BL/6J mice overexpressing mimic miR-100-5p. ResultsPostprandial variation in circ-miR-100-5p levels inversely correlate with PP-TG response. Cir-miR-100-5p was preferentially associated with TGRL particles of intestinal origin in subjects exhibited a low PP TG response. Differential analysis of transcriptome from HepG2 cells transfected by either mimic miR-100-5p or scrambled mimic miR as control allowed us to identify PCSK9 as a down-regulated gene. Overexpression of miR-100-5p in HepG2 cells significantly decreased PCSK9 mRNA levels by 52% (p<0.0001), cellular protein content by 28 % (p<0.0001) as well as PCSK9 secretion by 39% (p<0.0001). In vivo systemic delivery of mimic miR-100-5p induced a two-fold reduction (p<0.0001) on PP-TG in mice, such effect being abolished by blocking the circulating form of PCSK9 with alirocumab. Finally, we revealed a significant inverse relationship between circulating miR-100-5p expression levels and both PCSK9 levels and the magnitude of postprandial hypertriglyceridemia. ConclusionTaken together, our observations reveal that miR-100-5p regulates postprandial hypertriglyceridemia by targeting PCSK9, thus enhancing hepatic triglyceride-rich lipoproteins (TGRL) uptake. Our findings allow us to propose circ-miR-100-5p as a potential biomarker for early identification of subjects at high cardiovascular risk, prior to appearance of classical clinical features of metabolic disorders. Postprandial clinical study, HDL-PP (NCT03109067) Lay summaryThis study examined whether miRs may control postprandial triglyceride response Key findingsOur data reveal that miR-100-5p regulates postprandial hypertriglyceridemia by targeting PCSK9 Our observations allow us to propose miR-100-5p as a potential biomarker for early identification of subjects at high cardiovascular risk

A recent genome mining study identified class II lanthipeptides encoded in Nostoc punctiforme PCC73102 that contain acyl groups conjugated to Lys side chains. The structure and bioactivity of these peptides, named nostolysamides, were not determined. In this study, we heterologously produced the nostolysamides by co- expression of the NpuA precursor peptide with an N-terminal SUMO tag with the class II lanthipeptide synthetase NpuM in Escherichia coli. We structurally characterized the NpuA-derived product and established the position of the thioether crosslinks. All four lanthionine and methyllanthionine residues were shown to have the DL configuration by Marfeys analysis. Tandem mass spectrometry as well as mutagenesis studies indicate an N-terminal non-overlapping methyllanthionine ring and three overlapping rings at the C-terminus for which the most likely ring pattern is proposed. After removal of the leader peptide, the resulting lanthipeptide exhibits antifungal activity against Candida species as well as antimicrobial activity against gram positive bacteria by disrupting cell membranes. The antibacterial activity is shown not to involve lipid II, consistent with the observed antifungal activity because fungi do not contain this bacterial cell wall precursor. The biosynthetic gene cluster also encodes an acetyltransferase NpuN that transfers long chain acyl groups to the side chain of a Lys residue in position 1 of the precursor peptide. In vitro studies of NpuN shows relatively broad substrate specificity with NpuN conjugating various acyl groups from acyl-CoA substrates to Lys1 in the nostolysamides. The acylation did not appreciably change the antifungal and antimicrobial activity of nostolysamide showing that it is not required for these activities.

Bifunctional diazirine lipids are valuable tools for mapping protein-lipid interactions and cellular localization by photocrosslinking. Yet, the crosslinking efficiency of these probes has not been systematically evaluated. Here, we use the lipid transfer protein STARD10, which binds phospholipids in a 1:1 stoichiometry within a hydrophobic pocket, to measure the upper limit of the photo-crosslinking efficiency of bifunctional lipid probes. We characterize reaction products using native and denaturing mass spectrometry. Our results show that approximately 5% of photoactivated lipids form covalent protein-lipid crosslinks, while the majority follow intramolecular reaction trajectories, resulting in the formation of products featuring alkene, ketone and hydroxyl moieties. These findings provide essential context for the use of bifunctional probes to uncover the cell biology of lipids and highlight the need for continuous improvement to experimental workflows. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/700185v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@15d1641org.highwire.dtl.DTLVardef@6024e0org.highwire.dtl.DTLVardef@1503dcorg.highwire.dtl.DTLVardef@1b067bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Extracellular secretion of the oncogenic sonic hedgehog signaling ligand is contingent on its release from a precursor protein through peptide bond cholesterolysis, mediated by the hedgehog C-terminal domain, SHhC. In this work, we describe the in vitro reconstitution of cholesterolysis activity for SHhC domains from vertebrate model organisms, Xenopus laevis (Xla) and Danio rerio (Dre). Cholesterolysis is assayed continuously in multi-well plates by monitoring changes in fluorescence resonance energy transfer (FRET) from an engineered precursor construct, expressed in E. coli and purified in soluble form. Using this FRET assay, we found that Xla and Dre SHhC exhibit high substrate stereospecificity, accepting cholesterol, (KM, 1-2 {micro}M, cholesterolysis t1/2 of [~]11 min) while rejecting the 3-alpha epimer, epi-cholesterol (KM > 100 {micro}M, t1/2 > 10 hr). By screening a 96-member detergent/surfactant library for compatibility with SHhC activity, we identify cationic detergents that inhibit cholesterolysis and find a shared preference for the zwitterionic n-dodecyl-phosphocholine (DPC, Fos-choline-12), which supported the fastest reaction kinetics. Lastly, we report that alanine point mutation at a conserved aspartate residue (D46A) in Xla SHhC and Dre SHhC blocks cholesterolysis; however, activity could be chemically rescued with rationally designed hyper-nucleophilic sterols. Of those sterols, 2-beta carboxy cholestanol was active as a substrate with D46A variants only; the remaining sterols were accepted by both D46A and wild-type SHhC. In summary, we have established the first in vitro kinetic assay to continuously monitor enzymatic activity of wild-type and mutant vertebrate SHhC domains in multi-well plates, a key step toward pharmacological manipulation of Sonic hedgehog protein biosynthesis in vivo.

The transbilayer distribution of plasma membrane cholesterol remains uncertain despite repeated analysis. We propose a new mechanism driving cholesterol sidedness: sterols form simple stoichiometric associations with phospholipids. Our model postulates that the phospholipids in the plasma membrane bilayer are fully complexed with cholesterol. The cholesterol in each leaflet is then the product of the abundance of its phospholipid and its sterol stoichiometry. Notably, lipid affinities are not relevant. Applying literature values for the composition, abundance and sterol stoichiometry of the phospholipid in each leaflet, the model predicts that two-thirds of the cholesterol in the human erythrocyte membrane bilayer is located in its outer leaflet, an exofacial to endofacial ratio of 2:1. The model also predicts that the overall cholesterol content of the bilayer is [~]0.75 mole/mole phospholipid, in agreement with literature values. Furthermore, our analysis suggests that the areas of the two membrane leaflets are about the same. The concordance of prediction with observation validates the model and the values used for the parameters. The sterol in the exofacial leaflet of the plasma membrane of any cell is predicted to exceed that on its contralateral side when its phospholipids have a higher sterol stoichiometry and are fully complexed. SynopsisWe propose that the transbilayer distribution of cholesterol in the plasma membrane bilayer is determined by its complexation with the phospholipids in the two leaflets. Because the complexes are homeostatically filled to stoichiometric equivalence, leaflet cholesterol is given by the abundance of its phospholipids multiplied by its sterol stoichiometry. The model predicts that two-thirds of the cholesterol in the human erythrocyte membrane bilayer resides in the outer leaflet. It also predicts the cholesterol content of the bilayer as a whole.

Metabolic dysfunction-associated steatohepatitis (MASH) is associated with increased expression of peroxisome proliferator-activated receptor gamma (PPAR{gamma}, Pparg) and reduced expression of genes involved in methionine metabolism in the liver. The nuclear receptor PPAR{gamma} is activated by fatty acids, and the knockout of Pparg in hepatocytes (Pparg{Delta}Hep) reduced the negative effects of MASH on methionine metabolism. Here, we sought to determine whether hepatocyte Pparg is required for the transcriptional regulation of genes involved in hepatic methionine metabolism in conditions with altered fatty acid flux to the liver: fasting, refeeding, and high-fat diet (HFD)-induced obesity/steatosis. Fasting induced liver steatosis and increased the expression of key genes involved in the methionine metabolism in the liver, while 6h-refeeding reversed these effects and reduced the expression of phosphatidylethanolamine N-methyltransferase (Pemt) and cystathionine beta synthase (Cbs). Overall, fasting and refeeding did not alter hepatocyte Pparg expression nor Pparg{Delta}Hep affected fasting and refeeding-mediated regulation of methionine metabolism gene expression. Diet-induced steatosis reduced hepatic Pemt expression in control (Pparg-intact) mice, and the thiazolidinedione (TZD)-mediated activation of PPAR{gamma} in diet-induced obese control (Pparg-intact) mice reduced the expression of betaine homocysteine S-methyltransferase (Bhmt) and Cbs. However, diet-induced steatosis increased hepatocyte Pparg expression, and Pparg{Delta}Hep blocked the negative effects of HFD and TZD on hepatic methionine metabolism. The PPAR{gamma}-dependent reduction of hepatic Bhmt and Cbs expression was confirmed in mouse primary hepatocytes. Taken together, hepatocyte Pparg may serve as a negative regulator of hepatic methionine metabolism in diet-induced obese mice and these actions could contribute to promoting the onset of MASH.

The liver is widely considered to be one of the most conserved organs amongst vertebrates, with it being involved in blood detoxification, bile production and the metabolism of xenobiotic compounds. Liver organoids have previously been derived from several species and used as models of drug metabolism, toxicity, and fundamental tissue biology. To date, however, these models have not been developed from ruminant species, specifically cattle and sheep. Here we present the first report of the development and comprehensive characterisation of bovine and ovine liver organoids derived from primary liver tissue. When initially established, organoids from both species were comprised of KRT19- and KRT18-positive cholangiocytes. The capacity for organoids to differentiate into hepatocyte-enriched cultures was evaluated and it was noted that there was an increase in hepatocyte markers in bovine cultures. A comparative analysis of the liver tissue and organoids of both species revealed species-specific differences in gene expression, which were conserved within organoid cultures. Most notably, bovine liver tissue and organoids had enriched expression of genes associated with fatty acid uptake and storage whereas ovine samples had higher expression of genes associated with fatty acid conversion, highlighting fundamental differences between these two ruminant species. Differences in expression of cytochrome P450 family genes were identified alongside those associated with an inflammatory response specifically in bovine samples, whereas ovine samples had higher expression of genes associated with a protective immune response. Despite this, transcriptomic analysis of organoids from both species, cultured in both growth and differentiation media, revealed preserved expression of genes associated with key liver functions, including gluconeogenesis and xenobiotic metabolism. Transcripts associated with the flavin-containing monooxygenases (FMO) family were expressed in both organoid growth media and organoid development media (OGM and ODM respectively), and both species could metabolise triclabendazole into its primary metabolite triclabendazole sulfoxide, therefore validating the potential of the organoids to be applied as in vitro models of metabolism and/or toxicity. Overall, this study provides novel insights into differences in liver composition and function between ruminant species, as well as providing novel experimental models of the liver for both cattle and sheep.

Peroxisomes are ubiquitous organelles that compartmentalize metabolic reactions including lipid catabolism and cellular detoxification. Pathogenic variants in PEX1 and PEX6 disrupt essential peroxisome functions and cause profound neurodegenerative diseases called peroxisome biogenesis disorders (PBDs). Despite retinal degeneration and blindness occurring frequently in PBDs, precisely how impaired peroxisome activity disrupts retinal function remains to be fully explored. To address this, we differentiated PEX1-/-, PEX6-/-, and wildtype human induced pluripotent stem cells into retinal pigment epithelium (iRPE) to study the consequences of peroxisome dysfunction in this disease-relevant cell type. Despite exhibiting impaired peroxisome matrix protein import, PEX1-/- and PEX6-/-iRPE had comparable morphology, tight junctions, and expression of proteins characteristic of RPE compared to wildtype iRPE. Targeted lipid profiling revealed reduced docosahexaenoic acid, a polyunsaturated fatty acid (PUFA) essential for retinal function, and elevated lipid species exclusively metabolized by peroxisomes in PEX1-/- and PEX6-/- iRPE. Following a photoreceptor outer segment (POS) challenge, PEX1-/- and PEX6-/- iRPE demonstrated disrupted PUFA retroconversion and lipid droplet accumulation. Additionally, PEX1-/- and PEX6-/-iRPE had impaired rhodopsin degradation, lysosomal dysfunction, and reduced transepithelial electrical resistance. These findings suggest that dysregulated POS metabolism in the RPE is a potential mechanism driving retinal degeneration in patients with PBDs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/701576v2_ufig1.gif" ALT="Figure 1"> View larger version (99K): org.highwire.dtl.DTLVardef@bf2389org.highwire.dtl.DTLVardef@b62e1forg.highwire.dtl.DTLVardef@8e0b21org.highwire.dtl.DTLVardef@17cb332_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic summarizing the consequences of PEX1 and PEX6 knockout on iRPE biology, including the presence of import-incompetent peroxisomes, impaired {omega}3 and {omega}6 fatty acid retroconversion, lipid droplet accumulation, and defective photoreceptor outer segment phagocytosis.