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

This report identifies a bidirectional signaling axis connecting lipid metabolism to nuclear mechanotransduction, with the potential to control fatty acid/triglyceride metabolism. The sterol regulatory element-binding (SREBP) family of transcription factors control fatty acid, triglyceride and cholesterol synthesis and metabolism. The family consists of three members: SREBP1a, SREBP1c, and SREBP2, that are regulated by intracellular cholesterol levels and insulin signaling. The SREBP2-dependent control of the LDL receptor gene is a well-established target for cholesterol-lowering therapeutics and the activity of SREBP1c is an attractive target in metabolic disease. In the current report, we identify SYNE4 (nesprin-4), a component of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, as a direct target of the SREBP family of transcription factors, and show that nesprin-4 in turn supports SREBP1c function. We identify functional SREBP binding sites in the human SYNE4 promoter and demonstrate that these are required for the sterol- and SREBP-dependent regulation of the promoter. Furthermore, we show that the endogenous SYNE4 gene is also regulated by SREBP1/2 and intracellular sterol levels. Interestingly, SREBP2 is responsible for the sterol regulation of the SYNE4 gene in HepG2 cells, while SREBP1 is the major regulator in MCF7 cells, demonstrating that diberent cell types use diberent SREBP paralogs to regulate the same promoter/gene. Importantly, we find that nesprin-4 is a positive regulator of SREBP1c expression and function in HepG2 cells and during the diberentiation of human adipose-derived stem cells. In summary, the current report identifies a novel regulatory interaction between lipid metabolism and the LINC complex. Importantly, we demonstrate that this signaling axis is bidirectional, forming a closed loop that has the potential to control SREBP1c activity and thereby fatty acid and triglyceride synthesis/metabolism. Based on our data, we propose that the nesprin-4-dependent regulation of SREBP1c could represent a novel therapeutic target in metabolic disease.

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

Phosphatidylinositol (4,5)-bisphosphate (PIP2), the most abundant cellular poly-phosphoinositide (PPI) class of phospholipid, is a central plasma membrane (PM)-associated signaling hub that controls many cellular processes. In this study, we demonstrate that either deletion of the gene encoding actin-binding protein profilin1 (Pfn1) or disruption of Pfn1-actin interaction leads to downregulation of PM PIP2 content in cells. This is also phenocopied when F-actin is depolymerized implying that Pfn1-dependent PIP2 alteration is related to its actin-regulatory function. Phospholipase C (PLC) activity is critical for Pfn1-deficient cells to exhibit the PIP2-related phenotype. These findings, taken together with biochemical signatures of elevated PIP2 hydrolysis (higher baseline PM diacylglycerol-to PIP2 ratio and protein kinase C activity) exhibited by Pfn1-deficient cells, imply that PLC-mediated PIP2 hydrolysis plays a role in Pfn1-dependent regulation of PM PIP2. Furthermore, we unexpectedly found that Pfn1 loss leads to dramatic alterations in several other important forms of lipids, revealing a previously unrecognized role of Pfn1 as a broad regulator of cellular lipid environment that extends beyond PPI control. In conclusion, our study establishes Pfn1 as an important regulator of cellular lipid homeostasis. SUMMARY STATEMENTThis study uncovers a mechanism of how functional loss of Profilin1, a key regulator of actin cytoskeleton, can trigger downregulation of plasma membrane content of PIP2, an important class of phospholipid, in cells.

IP3R-Grp75-VDAC1 protein complex at the mitochondria-ER contact sites (MERCS) is involved in response to nutrients and control of glucose and energy metabolism, however, early alterations of the complex and MERCS in response to increased fat intake remain inconclusive. We investigated early effects of high-fat diet (HFD) on IP3R-Grp75-VDAC1 protein expression in correlation with ER-mitochondrial interaction in the liver of mice. Five-week-old mice were fed an HFD or a standard diet (SD) for 2 weeks (2W) or 8 weeks (8W). MERCS fractionation by a gradient ultracentrifugation, Western blot, transmission electron microscopy (TEM), Oroboros high-resolution respirometry were used to analyse liver tissues, while real-time PCR was used to profile genes responsive to HFD. No macroscopic morphological or functional alterations were observed in mice at 2W, while, expectedly, at 8W of HFD mice gained weight and glucose intolerance. Total IP3R protein was reduced at both 2W and 8W points by a post-transcriptional mechanism, while in MERCS, IP3R, VDAC1 and Grp75 were reduced at 8W time-point. TEM analysis revealed a significant reduction of mitochondrial coverage by MERCS, mitochondrial fragmentation and shortening of ER-mitochondria distance already at 2W time-point. Mitochondrial function and metabolism were largely spared. Markers of altered protein homeostasis such as Lmp2, Mecl-1 and Lmp7 showed an early upregulation. In conclusion, HFD induces early alterations in liver MERCS that precede gain of weight and glucose intolerance, suggesting their primary role in obesity and metabolic diseases and as potential therapeutic target.

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.

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.

Lipophagy is an important microautophagic process that degrades lipid droplets (LDs) to mobilize stored lipids as an energy source during nutrient starvation. However, the molecular mechanisms regulating lipophagy in response to nutrient starvation remain poorly understood. We found that budding yeast mutants defective in glycosylphosphatidylinositol (GPI) lipid remodeling exhibited aberrant accumulation of lipid droplets (LDs) and neutral lipids under glucose starvation. Our data suggest that the accumulation results from a failure of vacuolar liquid-ordered (Lo) domain-mediated lipophagy. Furthermore, we demonstrated that glycosylphosphatidylinositol-anchored proteins (GPI-APs) localize to vacuoles in response to glucose depletion and that a mutant defective in endocytosis has defects in both vacuolar Lo domain formation and lipophagy. These results imply that GPI lipid remodeling is required for Lo domain-mediated lipophagy upon glucose starvation. We propose that endocytosis functions to supply the lipid portion of GPI-APs, remodeled to C26 diacylglycerol, to the vacuolar membrane for Lo domain formation. Summary StatementOur data suggest that the endocytic transport of GPI-APs remodeled with C26 diacylglycerol to the vacuole is required for vacuolar Lo domain formation and subsequent lipophagy in response to glucose deprivation. This reveals the essential role of GPI lipid remodeling in ensuring lipophagy to adapt to changes in nutrient availability.

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

Staphylococcus aureus (S. aureus) is an opportunistic pathogen that is a global health concern for its ability to cause a wide spectrum of clinical infections. Due to the emergence of resistance to commonly used antibiotics, there has been interest in exploring the use of antimicrobial peptides to treat S. aureus infections. However, changes in the lipid composition of the lipid bilayer membrane can alter the activity of peptides, and S. aureus is able to induce variations in lipid composition in response to environmental stress. Here, we explore how the main lipid components in S. aureus are altered when exposed to LL-37, a human cathelicidin involved in primary immune response, and ATRA-1, a short antimicrobial peptide derived from the snake Naja atra venom. A lipidomic study is conducted through HPLC-MS-MS (LC-ESI-MS/MS) to quantify phosphatidylglycerol, cardiolipin, lysyl-phosphatidylglycerol, monogalacto- and digalacto-diacylglycerol, and carotenoids. In addition, menaquinones, responsible for electron transport during oxidative phosphorylation, were also quantified. Biophysical properties such as membrane electric surface potential and lipid packing were assessed. We find that lipid adaptation is specific to the type of antimicrobial peptide, where ATRA-1 mainly induces changes in the electric surface potential through variations in Lysyl-PG, while exposure to LL-37 changes carotenoid levels, inducing an increase in membrane rigidity as measured by FTIR. In addition, both peptides induce a reduction in menaquinone and DGDG levels. These findings highlight the role of membrane lipid remodeling as a peptide-specific response mechanism in S. aureus, with implications for the development of AMP-based therapies. HighlightsO_LIStaphylococcus aureus responds through shifts in lipid composition and membrane biophysical properties to exposure to the antimicrobial peptides LL-37 and ATRA-1. C_LIO_LIBoth LL-37 and ATRA-1 lead to shifts in the glycolipids MGDG and DGDG; two lipids involved in regulating negative membrane curvature stress and responsible for shifting resistance to antimicrobial peptide activity in Staphylococcus aureus. C_LIO_LILL-37 treatment leads to an overall reduction in carotenoid content in Staphylococcus aureus, including the carotenoid end-product staphyloxanthin and the precursor 4,4-diaponeurosporenoic acid. Both lipids regulate membrane biophysical properties and protect Staphylococcus aureus from oxidative stress. C_LIO_LIBoth LL-37 and ATRA-1 lead to a reduction in menaquinone levels, which are involved in the electron transport chain during oxidative phosphorylation. Reduction in these menaquinones have been associated to the formation of small colony variants that are often observed in chronic Staphylococcus aureus infections. C_LI

Staphylococcus aureus (S. aureus) is a clinically relevant pathogen capable of adapting its membrane composition in response to environmental stress. In this adaptive process, bacterial carotenoids play a crucial role. Although staphyloxanthin (STX) is the main carotenoid produced by the bacterium, S. aureus also synthesizes other pigmented intermediates that play an unknown role in regulating membrane biophysical properties. In this study, we purified 4,4-diaponeurosporenoic acid (4,4'-DNPA) from S. aureus carotenoid extracts and evaluated its effect on the thermotropic and biophysical properties of representative membrane models. The highly rigid triterpenoid 4,4'-DNPA is one of the last precursors in the biosynthesis of STX and is found in high concentrations in the stationary phase of S. aureus. Phase transition temperatures were determined using infrared spectroscopy, while interfacial hydration and hydrophobic core dynamics were investigated using fluorescence spectroscopy through Laurdan generalized polarization and DPH anisotropy. The results show that 4,4'-DNPA increases the main phase transition temperature of lipid bilayers in a concentration-dependent manner. This is in contrast to STX that decreases the transition temperature. This difference is consistent with the additional fatty acid present in STX that changes its effect on the phase behavior. Furthermore, 4,4'-DNPA reduced the interfacial hydration levels and restricted hydrophobic-core dynamics at higher concentrations, consistent with increased molecular order and stability. 4,4'-DNPA therefore complements STX in increasing membrane order and lipid packing. These findings support the notion that the production of bacterial carotenoids functions as a biophysical regulatory mechanism of lipid packing in S. aureus membranes.

Background and AimsFenofibrate is widely prescribed for hyperlipidaemia and has been associated with rare but severe cases of drug-induced liver injury (DILI), yet its effects on liver sinusoidal endothelial cells (LSECs) remain to be investigated. LSECs maintain a highly permeable specialized sinusoidal barrier characterized by transcellular pores (fenestrations), regulating the bidirectional transfer of circulating compounds to and from the hepatocytes. As drug-induced alterations in fenestration architecture could influence xenobiotic access to hepatocytes, these changes may modulate pathways associated with DILI. Understanding the effects of fenofibrate on LSEC ultrastructure may therefore provide insights into previously underexplored endothelial contributions to hepatic drug responses. MethodsBoth fenofibrate and its active metabolite, fenofibric acid, were evaluated for their effects on LSEC ultrastructure, mechanical properties, and functional markers. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) and were used to quantify fenestration architecture. AFM was additionally used to measure cellular mechanical properties, which were interpreted in the context of fluorescence-based quantification of cytoskeletal organization. Gene expression, viability, and cytotoxicity were assessed using PCR-based and biochemical assays. ResultsFenofibrate reduced fenestration number and porosity at both tested concentration (10, and 25 {micro}M). It also decreased the apparent Youngs modulus of LSECs, accompanied by changes in tubulin and actin architecture, without detectable cytotoxicity. In contrast, treatment with fenofibric acid did not result in significant structural or mechanical effects on LSECs, even at higher concentrations. ConclusionsTogether, these data identify LSECs as a drug-responsive hepatic cell type for fenofibrate, suggesting that LSECs could represent an underrecognized contributor to the complex, multifactorial processes underlying DILI. This work provides a framework for evaluating endothelial contributions to fenofibrate-associated liver effects in more complex models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/718907v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1f9ec6forg.highwire.dtl.DTLVardef@11174a1org.highwire.dtl.DTLVardef@1000e2borg.highwire.dtl.DTLVardef@a23b00_HPS_FORMAT_FIGEXP M_FIG Fenofibrate reduces LSEC fenestrations and metabolic activity at higher concentrations, while its metabolite, fenofibric acid, does not affect LSEC, regardless of its concentration. C_FIG

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent condition that progresses from simple steatosis to advanced fibrosis, significantly affecting liver function and systemic health. Despite its widespread impact, therapeutic options are limited, highlighting the urgent need for comprehensive exploration to identify potential therapeutic targets. In this study, we created an analysis pipeline anchored on liver gene expression, integrating differential meta-analysis of transcriptomic data across three MASLD stages, transcriptome-wide Mendelian randomization (MR), and transcriptome-wide association studies (TWAS), to identify 39 candidate genes potentially involved in MASLD progression. Furthermore, we prioritized these genes using a scoring system that incorporated gene expression-clinical phenotype correlation meta-analysis, proteome-wide association studies (PWAS), and external genetic data from the GWAS Catalog and ExPheWAS. Single-nucleus RNA sequencing (snRNA-seq) analysis of liver cells from healthy to cirrhotic stages revealed stage- and cell-type-specific expression patterns of these prioritized genes. Through experimental validation in a lipid overload hepatocyte model, we confirmed the role of MLIP in lipid metabolism. These findings, available through an interactive web portal (masldportal.net), provide valuable insights into MASLD mechanisms and offer an easy-accessible resource for the research community. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/706502v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1cec9f8org.highwire.dtl.DTLVardef@12df220org.highwire.dtl.DTLVardef@1734cecorg.highwire.dtl.DTLVardef@bf5b52_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

The secretion of glucagon from the pancreatic alpha () cell within the islets of Langerhans is physiologically regulated by nutrients (glucose, amino acids, fatty acids), neurotransmitters, and paracrine hormones. Insulin and somatostatin form an intra-islet paracrine network to control glucagon secretion through direct inhibitory effects on cell secretory granule exocytosis. In a potential new cellular pathway for the regulation of glucagon secretion, we have previously identified the neuronal trafficking protein Stathmin-2 (Stmn2) as a negative regulator of glucagon trafficking and secretion by directing glucagon to degradative lysosomes. In this study, we examined if insulin and somatostatin direct glucagon to lysosomes in a Stmn2-dependent manner as part of their paracrine mechanisms. Using the TC1-6 glucagon-secreting cell line and confocal microscopy of both fixed and live cells, we show that insulin and somatostatin direct glucagon, glucagon+LAMP1+ vesicles, and LAMP1-RFP to the intracellular region, away from sites of exocytosis. As visualized in live cells, insulin treatment resulted in the rapid retrograde transport of lysosomes from the cell periphery, and this effect was lost under siRNA-mediated silencing of Stmn2. Somatostatin appeared to enhance the intracellular retention of lysosomes, also in a Stmn2-dependent manner. We determined a possible mechanism for Stmn2 in the regulation of lysosome transport in TC1-6 cells through the Arf-like small GTPase Arl8, indicating that Stmn2 may function in lysosomal positioning along microtubules. We propose that Stmn2-mediated lysosomal transport may be a potential new pathway, in addition to inhibition of secretory granule exocytosis, through which insulin and somatostatin regulate glucagon secretion.

Metabolic-dysfunction associated steatotic liver disease (MASLD) is the most common chronic liver disease. Fracture risk is increased among people with MASLD, however, the genetic contribution to risk is undetermined. PNPLA3I148M is a common SNP which accounts for most MASLD heritability and increases MASLD morbidity and mortality. However, PNPLA3I148M impact on bone is unexplored. To bridge this gap, we used a validated murine model of MASLD (DIAMOND mice) which received human PNPLA3 transgenes via adeno-associated vector serotype 8 (AAV8) and assessed bone morphology, cellularity, and transcriptomics. PNPLA3I148M was expressed in bone and associated with bone loss, decreased bone formation, increased bone resorption, and increased bone marrow adiposity. PNPLA3I148M reprogrammed the transcriptome in bone, enriching expression of pathways associated with fatty acid metabolism and hampering bone turnover. Notably, these findings occurred in the absence of MASLD. These findings suggest PNPLA3I148M possesses an intrinsic deleterious skeletal role.

Parasitic flatworms, including cestodes and trematodes, are covered by a specialized syncytial tegument that mediates nutrient uptake and host-parasite interactions. While the tegument of trematodes has been extensively characterized, its molecular composition in cestodes remains largely unknown. In this work, we performed a comparative proteomic analysis of the tegument of three cestode species, including larval and adult stages: Hymenolepis microstoma, Mesocestoides corti (syn. M. vogae) and Echinococcus multilocularis. Using stringent enrichment criteria relative to whole-worm extracts, we identified hundreds of tegument-enriched proteins in each species. Comparative analyses revealed a conserved core of tegumental proteins shared among all three species, including members of the Tegument Allergen-Like (TAL) family, vesicular trafficking components and calcium-sensing proteins, and identified candidates for nutrient uptake activities such as glucose and nucleoside transporters. Further comparative analyses revealed a set of shared tegumental proteins with the trematode Schistosoma mansoni, including conserved proteins that are specific to parasitic flatworms, supporting the existence of a conserved ancestral tegumental proteome. Finally, we confirmed tegumental expression of several candidate genes in H. microstoma and E. multilocularis, and demonstrated regionally restricted gene expression among tegumental cytons, suggesting functional specialization within the syncytial tegument. Altogether, these results reveal an evolutionarily conserved composition of the tegument of parasitic flatworms, providing a foundation for future work targeting this critical host-parasite interface.

Membrane lipid synthesis is globally coordinated by a limited set of master transcription factors that regulate broad gene networks encoding lipid-metabolic enzymes and their regulators. Here, we identify the C2H2 zinc-finger transcription factor Com2 as a regulator of sphingolipid homeostasis in Saccharomyces cerevisiae that promotes transcription of downstream targets, including the protein kinase Ypk1, a key activator of sphingolipid synthesis. Com2 protein abundance increased upon treatment with myriocin, an inhibitor of sphingolipid synthesis, but rapidly decreased after addition of phytosphingosine (PHS), a precursor of complex sphingolipids; this decrease was blocked by proteasome inhibitors. These results suggest that Com2 is regulated in a sphingolipid-dependent manner through proteasome-mediated degradation. Moreover, a Com2 mutant in which lysine residues putatively involved in ubiquitination were replaced with arginine exhibited attenuated PHS-dependent degradation and elevated phosphorylation. Likewise, a mutant in which putative phosphorylation sites were replaced with alanine showed reduced PHS-dependent degradation. Together, these findings indicate that Com2 undergoes phosphorylation-dependent degradation via the ubiquitin-proteasome system in response to sphingolipid levels.

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.

Type 1 diabetes (T1D) is a consequence of {beta}-cell death. ER stress precedes T1D onset and prolonged ER stress in {beta}-cells can lead to {beta}-cell apoptosis. We reported that lipid signaling generated by the Ca2+-independent phospholipase A2{beta} (iPLA2{beta}), encoded by Pla2g6, participates in ER stress-mediated {beta}-cell apoptosis. {beta}-Cell membranes are enriched in arachidonic acid containing glycerophospholipids and the iPLA2{beta} catalyzes the hydrolysis of arachidonic acid in ER stressed {beta}-cells. Metabolism of arachidonic acid leads to the generation of various proinflammatory lipids, raising the possibility that they contribute to ER stress and {beta}-cell death leading to T1D. However, molecular mechanisms by which such {beta}-cell-iPLA2{beta}-derived lipid (iDL) signaling contributes to {beta}-cell apoptosis are not understood. It is well known that ER stress-mediated {beta}-cell apoptosis is associated with induction of transcription factors, NF{kappa}B and STAT1. We report here that both induce Pla2g6 and, unexpectedly, we find that iPLA2{beta}, which lacks DNA-binding motifs, associates with NFkB, Stat1, and Pla2g6 promoter regions. Consistently, p65-NF{kappa}B and pSTAT1 induction is reduced with select inhibition or knockdown of iPLA2{beta}. Surprisingly, iPLA2{beta} expression is also reduced by select inhibition of iPLA2{beta}, raising the possibility of feedback regulation by iDLs. In support, we find that select iDLs, recognized to be proinflammatory, enhance association of iPLA2{beta} with Pla2g6, Nfkb, and Stat1 promoter regions leading to induction of all three gene products and {beta}-cell apoptosis. Our findings reveal previously unrecognized transcriptional regulation by iDL signaling and, iPLA2{beta} itself, that leads to gene products that promote {beta}-cell apoptosis. Analogous findings in human islets validate this mechanism raising the possibility that targeting select lipid signaling can reduce ER stress in {beta}-cells and ameliorate T1D development.

Milk microRNAs are believed to play gene regulatory functions in the consumers cells. Milk from different species is enriched in microRNAs predicted to influence immunity, metabolism, and intestinal homeostasis. For milk microRNAs to regulate gene expression in the consumer, they must survive digestion and be present at sufficient levels to influence intestinal cells and potentially beyond-intestinal cells. Milk microRNAs are proposed to be protected from degradation through their association with milk extracellular vesicles (EVs), which might also deliver them to cells. Studies on milk microRNA oral transfer and tissue bioavailability are limited by interspecies sequence homology, making it difficult to distinguish endogenous from exogenous microRNAs. Here, we used a transgenic (TG) cow model expressing four unique microRNA sequences (AmiRs) in its milk to study their association with milk EVs, their resistance to in vitro digestion, and AmiR uptake and regulatory activity in vitro. We confirmed the presence of the four milk EV populations in raw wild-type (WT) and TG cow milk, similar to those previously reported in commercial (pasteurized) cow milk, and confirmed their association with AmiRs and classical milk microRNAs. AmiRs showed differential resistance to simulated adult digestion. In vitro uptake studies showed a modest gene regulatory effect of AmiRs in Caco-2 cells incubated with TG milk EVs. The intent of using this model was to perform in vitro analysis which could lay the groundwork for later in vivo bioavailability studies, taking advantage of the uniqueness of the AmiRs sequences and bypassing the limitation of microRNA sequence homology. HighlightsO_LINew milk EV populations identified previously in commercial bovine milk were also identified in raw milk, indicating that they are not merely the result of processing C_LIO_LIThe routinely discarded EVs (12K and 35K) seem to be preferentially enriched with microRNAs in raw cow milk as was previously shown for commercial cow milk C_LIO_LIAmiRs in transgenic milk resist differentially to simulated digestion C_LI