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

Lipopolysaccharide (LPS), a ubiquitous bacterial component of food, is neutralized by a variety of mechanisms that help to establish a threshold, which when exceeded results in an inflammatory TLR4 mediated response. Notably both CEACAM1 and CD36 affect downstream signaling of TLR4 to LPS. Furthermore, CEACAM1 associates with CD36 in hepatocytes, regulating lipid storage and bile acid (BA) secretion that includes reverse transport of LPS to the intestine. Direct binding of LPS-Ra micelles to soluble CEACAM1 or soluble CD36 was analyzed by surface plasmon resonance (SPR), size exclusion chromatography (SEC) and transmission electron microscopy (TEM). Direct binding of CEACAM1 to CD36 was analyzed by SPR and proximity ligation assays. Molecular models were generated by Alpha Fold and Molecular Dynamics. LPS Binding: SPR binding constants of KD= 1.04 x 10-10 M and KD= 3.38 x 10-10 M were obtained for LPS-Ra micelle binding to sCEACAM1 and sCD36, respectively. On SEC, the molecular sizes of LPS-Ra micelles bound to sCEACAM1 and sCD36 were approximately 500 and 800 kDa, respectively. In addition, LPS binding to both was reduced by sodium cholate and sodium deoxycholate. Alpha Fold predicted a binding site of LPS-Ra to CD36, while Molecular Dynamic studies of an N-domain mutant of CEACAM1, that breaks a conserved salt bridge, revealed the presence of an open form that is predicted to bind LPS. sCEACAM1 to sCD36 Binding: A KD of 5.28 x 10-8 M was obtained for sCEACAM1 binding to immobilized sCD36 by SPR. Antibody-based-proximity ligation demonstrated the association of the ectodomains of CEACAM1 and CD36 on hepatic cells and when co-expressed in HEK cells. In addition, biotin-based proximity ligation demonstrated association of the cytoplasmic domains of CEACAM1 and a CD36-BioID2 fusion protein when co-expressed in HEK cells. Alpha Fold predicted both head-to-head (trans) and side-to-side (cis) binding of the N-domain of CEACAM1 to CD36, from which a membrane model of their cis-interaction could account for the proximity ligation results. Both CEACAM1 and CD36 share a common LPS micelle binding function, as well as binding to each other, and together, may regulate uptake and excretion of micellar LPS.

In yeast, transcriptional adaptor 2 (ADA2; SAGA complex subunit ADA2), a member of histone acetyltransferase (HAT) complex, regulates transcription through cell signalling, but its precise role in cellular metabolism remains unclear. In this study, genetic loss of ADA2 (ada2{Delta}) induces squalene (SQ) accumulation, indicating aberrant triterpene metabolism, coupled with endoplasmic reticulum (ER)/nuclear ER (nER) expansion. Lipid analyses of ada2{Delta} revealed elevated phosphatidic acid (PA) and phosphatidylcholine (PC) levels, indicating disrupted phospholipid metabolism. The expanded ER causes basal autophagy elevation, cellular recycling, and nER phagy, suggesting a regulatory role for ADA2 in autophagy. Downregulation of phosphatidate cytidylyltransferase (CDS1) and inositol-3-phosphate synthase (INO1), coupled with elevated PA and PC in ada2{Delta}, points to a significant disruption in cytidine-diphosphate-diacylglycerol and phosphatidylinositol pathway. Overexpression of CDS1 or INO1, or the inositol supplementation, in ada2{Delta} restores SQ, basal autophagy and ER phagy. The observed target of rapamycin Ser/Thr kinase complex (TORC1) activity in ada2{Delta} is due to the high PA content. Rapamycin-mediated inhibition of TORC1 reduced SQ, PA and ER expansion while increasing lipid droplets. In contrast, a rapamycin-treated ada2{Delta}pah1{Delta} strain retained high PA, SQ and ER expansion, underscoring the functional role of TORC1-nuclear envelope morphology protein 1 (Nem1)/sporulation-specific protein SPO7 (Spo7)-Pah1 axis. Notably, SQ levels remained unchanged in a rapamycin-treated ada2{Delta}atg39{Delta} strain, suggesting that loss of nER-phagy receptor, Atg39, impairs the effectiveness of TORC1 inhibition. In conclusion, our data unveiled a critical role for Ada2 in maintaining the intricate relationship between lipid and triterpene/sterol metabolism and connecting autophagy and ER homeostasis.

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

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.

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.

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.

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

The enzyme nitric oxide synthase (NOS) breaks down the semi-essential amino acid L-arginine (L-Arg) in the cell to produce citrulline and nitric oxide (NO). NO is a crucial signalling molecule in cells that controls the metabolism of fats and carbohydrates. The aim of this study was to investigate two important genes in the L-Arg-NOS-NO signalling pathway, AMPK and ACC-1, as markers of the molecular mechanisms that are triggered when liver cells sense elevated L-Arg. Mouse liver epithelial insulin-sensitive BNL CL2 cells were used as a model system and cultured with 0, 400 or 800 {micro}M L-Arg. Cell growth parameters were analysed alongside qRT-PCR based analysis of target transcripts involved in lipid and glucose metabolic pathways. In a further experiment, NOS inhibitor; L-NAME (40 mM) and external NO donor; SNAP (100 {micro}M) were added and the effect on target gene expression analysed. L-Arg addition impacted culture viability and cell growth. AMP-activated protein kinase (AMPK) was regulated in response to L-Arg addition with increasing extracellular concentrations elevating AMPK mRNA and protein expressions. L-NAME decreased target gene expression in an L-Arg addition dependent manner. SNAP (100 {micro}M) addition increased target gene expression after 6 and 24 h. NO, produced as a result of L-Arg addition and the factors L-NAME and SNAP, that regulate NO bioavailability, impacted BNL CL2 cell NO/AMPK/ACC-1 signalling pathways via regulating mRNA expression and subsequently protein expression.

The acceleration of hepatic lipid disposal during acute exercise has been proposed as a contributor to the anti-steatotic effects of exercise training. Ketogenesis, which produces acetoacetate (AcAc) and {beta}-hydroxybutyrate ({beta}OHB) from fatty acids, is among the lipid disposal pathways stimulated by exercise. This study tested the hypothesis that hepatic ketogenesis is necessary for exercise training to lower liver lipids. Liver-specific 3-hydroxymethylglutaryl-CoA synthase 2 knockout (HMGCS2 KO) mice and wild type (WT) littermates underwent sedentary, acute exercise, and exercise training protocols. Liver ketone bodies and lipids were determined via mass spectrometry platforms. Stable isotope infusions in conscious, unrestrained mice defined mitochondrial oxidative fluxes at rest and during exercise. Loss of hepatic HMGCS2 decreased liver AcAc and {beta}OHB concentrations and impaired their increase during exercise. Liver triacylglycerides (TAGs) were comparable between genotypes at rest (i.e., ad libitum fed and short fasted conditions). In contrast, liver TAGs were elevated in HMGCS2 KO mice following acute, non-exhaustive exercise. Liver TCA cycle flux was higher in KO mice at rest. During exercise, TCA cycle flux increased in both WT and KO mice but was not different between genotypes with greater exercise duration. This suggests that enhanced disposal of lipids via the TCA cycle may prevent liver lipid accumulation in HMGCS2 KO mice under sedentary conditions, but not during exercise. Unexpectedly, exercise training decreased liver TAGs similarly in both HMGCS2 KO and WT mice. In conclusion, hepatic ketogenesis supports liver lipid homeostasis during acute exercise, but is not required for exercise training to lower liver lipids. NEW & NOTEWORTHYExercise training has been proposed to mitigate liver steatosis partly through enhanced hepatic lipid disposal. During acute exercise, the disposal of fatty acids to ketone bodies is stimulated. This study tested the hypothesis that hepatic ketogenesis was required for exercise training to reduce liver fat in mice. The results show that hepatic ketogenesis is needed to prevent lipid accumulation during acute exercise, but is not necessary for exercise training to lower liver lipids.

It is well established that Staphylococcus aureus can incorporate straight-chain unsaturated fatty acids (SCUFAs) into its lipids in addition to the normally biosynthesized branched-chain and straight-chain saturated fatty acids. Incorporation of oleic acid into S. aureus lipids has recently been shown to significantly enhance S. aureus growth at low temperatures due to the greater fluidity imparted to the membrane. Here, we show that low-temperature growth of S. aureus is not limited to oleic acid but enhanced also by various antimicrobial SCUFAs when present at low concentrations. A fakA-deficient strain did not show SCUFA-induced growth stimulation, which indicates that the fatty acid kinase is necessary for SCUFA incorporation into membrane lipids to promote low-temperature growth. Determination of total lipid fatty acid composition showed that incorporated SCUFAs make up [~]12% or less of the total fatty acids. Lipidomic investigations revealed elevated synthesis of diglucosyldiglyceride in the absence or presence of SCUFAs. SCUFAs were incorporated into diglucosyldiglyceride to a greater extent than phosphatidyglycerol at both 12 {degrees}C and 37 {degrees}C. The presence of SCUFAs at low temperatures also enhanced production of the carotenoid staphyloxanthin. The results suggest that multiple strategies are at play in the membrane adaptation of S. aureus to low temperatures. Inclusion of oleic acid in media decreased the minimum growth temperature of S. aureus, suggesting that the presence of SCUFAs in food may facilitate the growth of S. aureus at low temperature. Also, incorporation of SCUFAs into lipids may promote the disruption of the membrane by SCUFAs.

Mouse models of metabolic dysfunction-associated steatotic liver disease (MASLD) are valuable tools for identifying novel molecular mechanisms that drive progression from MASLD to metabolic dysfunction-associated steatohepatitis (MASH). However, generating a clinically relevant MASLD/MASH mouse model with obesity and peripheral metabolic dysfunction remains a challenge. In this study, we fed two different MASH-inducing diets to male mice with pre-existing high-fat (HF) diet-induced obesity. While a HF diet containing 40% Kcal from fat (mostly corn-oil shortening), 2% cholesterol, and 22% fructose reduced adiposity in these mice, a high-fat diet with 60% Kcal from fat (mostly lard), containing 2% cholesterol and supplemented with 10% fructose in the drinking water (HFC+Fr diet) promoted body weight and fat mass gain. Of note, 24 weeks of the HFC+Fr diet induced obesity, metabolic dysfunction, and liver steatosis in male and female mice, and promoted MASH with fibrosis in male mice. Furthermore, the HFC+Fr diet increased the expression of hepatocyte peroxisome proliferator-activated receptor {gamma} (Pparg), but the knockout of Pparg in hepatocytes (Pparg{Delta}Hep) reduced the development of MASH and fibrosis in male mice. In addition, the expression of key hepatic genes involved in methionine metabolism was downregulated by the HFC+Fr diet and upregulated by Pparg{Delta}Hep only in male mice. Overall, the HFC+Fr diet is obesogenic and promotes MASLD in both male and female mice. However, the HFC+Fr diet promotes MASH in a sex- and hepatocyte Pparg-specific manner, which may be associated with downregulation of hepatic methionine metabolism. New & NoteworthyWe explored how a new dietary intervention with fructose in the drinking water and added cholesterol to a high-fat diet extensively used to induce obesity and insulin resistance, promotes the onset of MASLD with obesity and metabolic dysfunction in male and female mice. This clinically relevant model of MASLD shows increased expression of hepatocyte PPAR{gamma} in both male and female mice, but only male mice have PPAR{gamma}-dependent impaired methionine metabolism and develop MASH with fibrosis.

Whole-body estrogen receptor (ER) knockout mice develop hepatic steatosis; however, liver-specific ER knockout (LERKO) mice fail to recapitulate this susceptibility and maintain normal hepatic mitochondrial function. However, estrogen-mediated protection against hepatic steatosis is lost in LERKO mice following ovariectomy (OVX). Here, we tested whether loss of hepatic ER blunts estrogen modulation of hepatic mitochondrial respiratory capacity and mitochondrial proteome following ovariectomy (OVX). Sham or ovariectomy (OVX) surgery was performed in middle-aged female mice (36-40 weeks), followed by AAV injection to generate Control (Con; GFP) or LERKO mice (Cre). All mice were placed on a high-fat diet (HFD) for 10 weeks following surgery. Half of the OVX mice received 17-beta estradiol (E2) replacement (OVX+E2) for the last 4 weeks of HFD. OVX mice had greater body mass and adiposity, which was reversed by E2 replacement in both Con and LERKO mice. While E2 replacement reduced steatosis in both Con and LERKO OVX mice, the LERKO OVX mice maintained greater hepatic triglyceride content. E2 replacement promoted greater basal and ADP-stimulated (State 3) mitochondrial respiration in Con OVX but not in LERKO OVX mice under palmitate-supported conditions. Changes in mitochondrial respiration could not be attributed to altered responses to changes in energy demand (GATP) or to alterations in mitochondrial H2O2 production. Conversely, maximal coupled branched-chain amino acid-supported respiration was universally suppressed by E2 replacement. Proteomics analysis revealed E2-mediated reductions in hepatic mitochondrial energy transduction, with relatively minimal differences between Con and LERKO mice. In conclusion, post-ovariectomy estrogen treatment reduces steatosis in the absence of hepatic ER; however, triglyceride levels remain higher, and mitochondrial respiratory deficits persist despite similar proteomic signatures, suggesting that ER signaling is required for optimal estrogen hepatic responsiveness.

Sphingomyelin is a critical sphingolipid found in plasma membranes of metazoa that provides structural and communicative functions. Sphingomyelin synthases are key enzymes that generate sphingomyelin but their precise functions in animal development and function are not fully understood. The Caenorhabditis elegans model encodes five sphingomyelin synthases (sms-1-5). Previously, egg-laying and locomotion phenotypes were observed in an sms-1(ok2399) deletion mutant. In this study, we attempted to replicate these findings to enable mechanistic dissection of sphingomyelin function. We indeed found that the sms-1(ok2399) mutant exhibited egg-laying and locomotion defects, however, we were unable to rescue this phenotype. Further, we generated two additional sms-1 deletion mutants (rp398 and rp399) and found that their egg-laying and locomotion behavior is not different to wild-type animals. We suggest that the sms-1(ok2399) contains a background mutation that causes behavioral deficits, and that SMS-1 loss does not overtly affect C. elegans egg-laying or locomotion.

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.

Pacific salmon (Oncorhynchus spp.) undergo intricate physiological changes during maturation as they migrate to spawning beds and breed before succumbing to a programmed senescence (semelparous life cycle). Research into the physiological mechanisms of semelparity in salmon has identified a clear and progressive rise in sex and stress hormone levels throughout their migration, which correlates with the emergence of morphological traits, as well as changes in behavioral patterns. We examined transcriptional changes in three critical tissues (gonads, head kidney, and skeletal muscle) across the spawning migration in male and female Pink salmon (Oncorhynchus gorbuscha) to capture the molecular changes occurring in these tissues during maturation and senescence. Major transcriptional changes occurred around the time of spawning, while only modest transcriptional changes were found as the fish migrated between saltwater and freshwater. Examination of enriched biological pathways identified the signatures of semelparous catabolic processes in all tissues and a strong immune response in somatic tissues. Evidence of shifts in lipid energy mobilization were also seen in somatic tissues. A closer investigation of the expression patterns of endocrine hormone receptors showed that many endocrine pathways prioritized expression of specific dominant ohnologs to orchestrate much of the hormone response in the analyzed tissues. Our characterization of the transcriptional profiles in migrating pink salmon adds critical context to link the molecular changes occurring in tissues to the physiological transitions that define semelparous maturation in Pacific salmon. NEW & NOTEWORTHYLarge transcriptional changes occurred in the gonads, head kidney, and skeletal muscle of pink salmon during the final stages of their spawning migration. Across the tissues and sexes, spawning was marked by coordinated activation of catabolic programs (autophagy, proteolysis, cell death), and a strong immune response in somatic tissues, alongside lipid mobilization. Endocrine receptor expression analyses revealed that the response to hormones was primarily mediated by a limited number of dominant ohnologs.

Coordination of metabolism, cell growth and cell division is essential to life. Recent single-cell measurements in S. cerevisiae have shown that metabolic processes and the cellular redox state are dynamic along the cell cycle. However, it is unknown whether similar metabolic oscillations also occur in other organisms. Until now, the dynamics of metabolism in other eukaryotes have predominantly been studied in cell cycle synchronised populations. Since cell cycle synchronisation methods can perturb metabolism, they may also introduce artefacts in the recorded dynamics. Here, we performed time-lapse microscopy analyses of exponentially growing single cells of the budding yeast S. cerevisiae, the fission yeast S. pombe and murine leukaemia L1210 cells. Measuring the NAD(P)H autofluorescence and the cell surface area growth rate in unsynchronised cells, we discovered oscillations along the cell cycle of the cellular redox state and lipid metabolism, respectively. Thus, our work shows that metabolism is dynamic along the cell cycle of these three evolutionarily distant eukaryotic organisms. This finding suggests that such metabolic oscillations could be a conserved characteristic among eukaryotes.

Sugar-induced cell death (SICD) remains an intriguing but poorly studied phenomenon in the physiology of Saccharomyces cerevisiae. Recently, it was shown that SICD development largely depends on the redirection of glucose fluxes between glycolysis and the pentose phosphate pathway (PPP). In particular, inhibition of glycolysis by iodoacetamide (IAA) was observed to reduce SICD levels. This study is devoted to further investigation of the relationship between SICD and the functionality of the two PPP branches. It was shown that deletion of the ZWF1 gene does not affect the decrease in SICD levels in IAA-treated cells. This allows us to conclude that the oxidative branch of the PPP is not involved in the suppression of SICD/ROS. Deletion of the GLR1 gene and attenuation of the TRR1 gene also did not restore SICD levels in cells after IAA treatment. The obtained results indicate that the level of reduced glutathione or thioredoxin does not affect SICD genesis. The addition of 5 mM ribose-5-phosphate (R5P) to the incubation medium led to suppression of SICD by 79%. At the same time, the addition of 5 mM ribose + 5 mM Pi suppressed SICD by only 20%. Suppression of SICD by 5 mM R5P in the{Delta} pho3 strain (83%) excludes the mechanism of extracellular dephosphorylation of R5P to ribose, its subsequent transport into the cell, and re-phosphorylation inside the cell. Furthermore, more than 70% suppression of SICD in the{Delta} end3 strain with 5 mM R5P excludes endocytosis as a mechanism of R5P import into the cell. The observed effect of R5P can be explained by the moonlighting function of some unknown protein. Thus, SICD development in S. cerevisiae cells depends on the final product of the non-oxidative PPP--R5P.

Atherosclerosis is a condition characterized by plaque growths in arteries, consisting of oxidized LDL (low-density lipoprotein) and localized cell cumulation. By the time of diagnosis for patients with atherosclerosis, the disease has often progressed into advanced stages. Statins are commonly prescribed; however, while these drugs can lower blood cholesterol levels, they cannot regress or stop the plaque growth. Currently, there are no treatments available to prevent the formation of new plaques. Such treatment options would require the identification of proteins that act during disease onset, initiating molecular mechanisms that promote plaque formation. Histone deacetylases (HDACs) and Ten Eleven Translocation (TET) demethylases are two important classes of epigenetic mediators. Some isoforms of these two classes of proteins have been found to transcriptionally regulate cellular inflammation, which may favor plaque formation. These transcriptional regulators seem to function early in the molecular mechanisms that are involved in disease progression. In the present work, we identified a clear role of these epigenetic proteins in foam cell formation. Foam cells have been implicated as part of the early steps which ultimately lead to atherosclerosis. Here we showed that in the presence of OxLDL (oxidized LDL), the protein isoform TET1 has a direct role in foam cell formation, while HDAC2 adopts a more indirect role. Using specific inhibitors of TET1 and HDAC2, we showed the inter-regulated molecular mechanisms between these proteins and how they regulate foam cell formation in vitro. In this study, we found that upon inhibition of TET1 in U937-derived macrophages, and subsequent foam cell formation via OxLDL treatment, a lower percentage of foam cells was observed. However, TET2 inhibition under the same treatment conditions had no effect on the inhibition of foam cell formation.

Insulin resistance, excessive and ectopic fat accumulation, chronic low-grade inflammation, and pancreatic beta-cell failure are pathological features of type 2 diabetes mellitus.MiR-130a has been demonstrated to suppress the mRNA levels of PPAR{gamma}, NF-{kappa}B, and TNF- in vitro. PPAR{gamma} is a master regulator of systemic fat and glucose metabolism. NF-{kappa}B and TNF- are pivotal modulators of inflammation. Therefore, we aimed to examine the systemic effect of miR130a on fat metabolism, glucose/insulin homeostasis, and inflammation in mice. We found that mirR130a-deficient mice exhibited larger white fat mass with hypertrophic adipocytes, increased lipogenic gene expression in fat, and elevated serum leptin levels than controls. The white fat pads of mirR130a-deficient mice showed significant macrophage infiltration with enhanced expression of pro-inflammatory genes. In addition, mirR130a-deficient mice had more severe hepatic steatosis and higher hepatic triglycerides content than controls. Similarly, mirR130a-deficient mice had increased macrophage infiltration and lipogenic and inflammatory gene expression in the liver. Consistently, we found that Lepob/ob mice expressed markedly decreased miR130a expression in the liver and white fat compared to controls. Importantly, mirR130a-deficient mice displayed impaired glucose tolerance and worsened insulin resistance, accompanied with reduced serum adiponectin levels. Furthermore, insulin secretion is reduced in mirR130a-deficient mice compared to controls. In conclusion, knockout of miR130a in mice results in fat hypertrophy, hepatic steatosis, increased macrophage infiltration in liver and fat, glucose intolerance, and insulin resistance. These data indicate miR130a exert systemic anti-diabetic effects.