Lipids are essential for neuron development and physiology1-3. Yet, the central hubs that coordinate lipid supply and demand in neurons remain unclear4. Here we show the presence and functional significance of neuronal lipid droplets (nLDs) in vivo using invertebrate and vertebrate models. We validate5 the presence of nLDs in vivo and demonstrate that triglyceride metabolism enzymes and LD-associated proteins control nLD formation through both canonical and recently discovered pathways6. Modulation of nLDs has conserved and male-biased effects on whole-body energy homeostasis across flies and mice, specifically in neurons that couple environmental cues with energy homeostasis. Mechanistically, nLD-derived lipids support neuron function by providing fatty acids and phospholipids to sustain mitochondrial and endoplasmic reticulum function and homeostasis. This identifies a conserved role for nLDs in coordinating lipid supply and demand in neurons, which has implications for maintenance of neuronal lipid homeostasis and function in health and disease.

Alzheimer’s disease (AD) is associated with cognitive deficits and sleep disturbances. Research suggests the involvement of dysfunctions in lipid metabolism in the brain of AD patients and animal models. The inhibition of stearoyl-CoA desaturase (SCD), a lipid-converting enzyme, was shown to restore memory in triple transgenic (3xTg)-AD mice. In the brain, astrocytes regulate the synthesis of specific lipids. This project tested whether the inhibition of SCD restores sleep in 3xTg-AD mice, and whether this associates with modifications in lipids, astrocytic function and the transcriptome. Wild-type (WT) and 3xTg-AD female mice received a SCD inhibitor (SCDi) or vehicle, which was followed by an electrocorticographic (ECoG) recording. Brain slices were stained for lipid droplets, astrocytic markers or processed for spatial transcriptomics. The reduced time spent awake (increased time spent in slow wave sleep) in 3xTg-AD mice was not restored by SCDi treatment. Rhythmic and scale-free ECoG activities were markedly altered in 3xTg-AD mice for all wake/sleep states, and SCDi changed these ECoG signatures differently in mutant in comparison to WT mice. GFAP-positive cell density and lipid droplet count were elevated in hippocampal CA1, and rescued by SCDi. The treatment also rescued the expression of several genes in a manner mainly overlapping between brain regions. The findings suggest that the multiple wake/sleep alterations in 3xTg-AD mice are not mitigated by SCD inhibition, but that this treatment can revert changes in hippocampal astrocytes, lipids and in the brain transcriptome. This work will benefit the understanding of the AD pathophysiology and associated sleep disturbances.

Pediatric cardiomyopathy is a major cause of diastolic heart failure; yet, its mechanisms remain unclear. Global myocardial transcriptomic and blood lipidomic profiling revealed a distinct metabolic signature of diastolic dysfunction marked by dysregulated lipid signaling. A machine learning model using these gene markers accurately classified diastolic dysfunction across cardiomyopathy subtypes. Lipidomic changes included excess saturated lipids and impaired oxidation that correlated with myocardial gene expression. Induced pluripotent stem cell cardiomyocytes from patients with diastolic dysfunction exhibited lipid accumulation, and mitochondrial dysfunction that was rescued by semaglutide. These findings define a new molecular phenotype of diastolic dysfunction and point to abnormal lipid signaling as a promising therapeutic target in childhood cardiomyopathy.

Lipid droplets (LD) are triglyceride storing organelles that have emerged as an important component of cellular inflammatory responses. LD lipolysis via adipose triglyceride lipase (ATGL), the enzyme that catalyses the rate-limiting step of triglyceride lipolysis, regulates inflammation in peripheral immune and non-immune cells. ATGL elicits both pro- and anti-inflammatory responses in the periphery in a cell-type dependent manner. The present study determined the impact of ATGL inhibition and microglia-specific ATGL genetic loss-of-function on acute inflammatory and behavioural responses to pro-inflammatory insult. First, we evaluated the impact of lipolysis inhibition on lipopolysaccharide (LPS)-induced expression and secretion of cytokines and phagocytosis in mouse primary microglia cultures. Lipase inhibitors (ORlistat and ATGListatin) and LPS led to LD accumulation in microglia. Pan-lipase inhibition with ORlistat alleviated LPS-induced expression of IL-1 beta and IL-6. Specific inhibition of ATGL had a similar action on CCL2, IL-1 beta and IL-6 expression in both neonatal and adult microglia cultures. CCL2 and IL-6 secretion were also reduced by ATGListatin or knockdown of ATGL. ATGListatin increased phagocytosis in neonatal cultures independently from LPS treatment. Second, targeted and untargeted lipid profiling revealed that ATGListatin reduced LPS-induced generation of pro-inflammatory prostanoids and modulated ceramide species in neonatal microglia. Finally, the role of microglial ATGL in neuroinflammation was assessed using a novel microglia-specific and inducible ATGL knockout mouse model. Loss of microglial ATGL in adult male mice dampened LPS-induced expression of IL-6 and IL-1 beta and microglial density. LPS-induced sickness- and anxiety-like behaviours were also reduced in male mice with loss of ATGL in microglia. Together, our results demonstrate potent anti-inflammatory effects produced by pharmacological or genetic inhibition of ATGL-mediated triglyceride lipolysis and thereby propose that supressing microglial LD lipolysis has beneficial actions in acute neuroinflammatory conditions.

Background: Platelets play a central role in hemostatic and inflammatory responses during septic shock, with lipids being essential for their function. However, the specific lipidomic alterations occurring in platelets during septic shock remain poorly understood. Objectives: This study aimed to characterize platelet lipidomic changes in septic shock and investigate their associations with disease severity. Methods: In this matched case-control study, platelets were isolated from 49 septic shock patients and 47 nonseptic controls (matched for age, gender, and comorbidities). Lipidomic profiling was performed using untargeted lipidomics to identify significant alterations in the platelet lipidome. Associations among lipid changes, clinical data, and plasma biomarkers of coagulopathy and inflammation were explored. Results: More than 60% of the annotated platelet lipids were significantly altered in septic shock. Cholesteryl esters, sphingomyelins, lysophosphatidylcholines, and ether-lipids were significantly reduced, while ceramide levels increased. Fatty acyl chain remodeling displayed distinct patterns, with polyunsaturated fatty acids increasing in triacylglycerols and decreasing in phospholipids. Lipid alterations were strongly associated with thrombocytopenia, and lysophosphatidylcholine levels inversely correlated with disease severity, as indicated by the Sequential Organ Failure Assessment score. Conclusions: Septic shock induces significant disruptions in the platelet lipidome, with the extent of these alterations correlating with sepsis-associated thrombocytopenia severity. The observed changes affect multiple lipid classes, surpassing those reported under physiological conditions or in other diseases. These findings highlight the impact of sepsis-driven dysregulated inflammation and coagulopathy on platelet lipid composition, providing new insights into sepsis pathophysiology.

OBJECTIVES:Oxidative stress, driven by iron imbalance from recurrent blood transfusions, is a major contributor to cardiometabolic complications in transfusion-dependent thalassemia (TDT). N-acetylcysteine (NAC), a glutathione precursor, is a well-known antioxidant with cardioprotective effects achieved by mitigating the impact of oxidative stress on cell metabolism. Current study aimed to evaluate the effect of a six-month NAC intervention by focusing on previously reported changes in the plasma lipidome in TDT patients. Design & Methods A randomized cohort of 62 Thai TDT patients was divided into two groups: both received six months of cocktail therapy involving standardized blood transfusions and iron chelator therapy, with the intervention group additionally receiving 600 mg oral NAC daily and the control group receiving a placebo. Plasma lipidomic profiling was performed using mass spectrometry to assess 339 previously annotated lipid features significantly altered in TDT patients. Clinical parameters, including heart rate variability (HRV), were measured before and after the intervention. RESULTS:NAC treatment significantly altered 152 plasma lipid features (P < 0.03), 78 of which were also altered in the placebo group. Importantly, 29 lipid features (26 unique lipids) were restored toward healthy control levels following NAC treatment. Within this subset, circulating diacylglycerophosphocholines PC(14:0_20:4) and cholesteryl ester CE 18:3 positively correlated with HRV, a clinical marker markedly improved in NAC-treated patients. CONCLUSIONS:Six-month oral NAC intervention modified the plasma lipidomic profile in TDT patients, partially restoring lipid species likely disrupted by chronic oxidative stress. The observed correlation between NAC-responsive lipids and improved HRV suggests a potential cardioprotective effect. These findings highlight the potential of NAC as an adjunctive therapy to mitigate cardiometabolic complications in TDT.

Understanding molecular traits through metabolomics offers an avenue to tailor cardiovascular prevention, diagnosis and treatment strategies more effectively. This study focuses on the application of machine learning (ML) and explainable artificial intelligence (XAI) algorithms to detect discriminant molecular signatures in heart failure (HF). We aim to uncover metabolites with significant predictive value by analyzing targeted metabolomics data through ML and XAI algorithms. After quality control, we analyzed 55 metabolites from 124 plasma samples, including 53 HF patients and 71 controls, comparing Ridge Logistic Regression, Support Vector Machine and eXtreme Gradient Boosting models. All achieved high accuracy in predicting group labels: 84.0% [95% CI: 75.3 - 92.7], 85.73 [95% CI: 78.6 - 92.9], and 84.8% [95% CI: 76.1 - 93.5], respectively. Permutation-based variable importance and Local Interpretable Model-agnostic Explanations (LIME) were used for group-level and individual-level explainability, respectively, complemented by H-Friedman statistics for variable interactions, yielding reliable, explainable insights of the ML models. Metabolites well-known for their association with HF, such as glucose and cholesterol, and more recently described, the C18:1 carnitine, were reaffirmed in our analysis. The novel discovery of lignoceric acid (C24:0 fatty acid) as a critical discriminator, was confirmed in a replication cohort, underscoring its potential as a metabolite marker. Furthermore, our study highlights the utility of 2-way variable interaction analysis in unveiling a network of metabolite interactions essential for accurate disease prediction. The results demonstrate our approach's efficacy in identifying key metabolites and their interactions, illustrating the power of ML and XAI in advancing personalized healthcare solutions.

Background: The major challenge in mitochondrial diseases relies in their great heterogeneity, in terms of genetic origin (i.e. nuclear or mitochondrial), mutation diversity and clinical manifestations. The underlying mechanisms are still unclear and often considered common to most mitochondrial diseases. Hypothesis: According to the genetic origin, the different lipidomic profiles will reveal common as well as mutation-dependent signatures. Aims: i) Establish the characteristic signatures of human fibroblasts from mitochondrial diseases of distinct genetic origins: either nuclear: LRPPRC and MTFMT, or mitochondrial: NDUFS4, UQCRC2 and ECHS1 compared with healthy fibroblasts and, based on the established signatures, ii) identify the underlying mechanisms. Methods: We used a mass spectrometry-based untargeted lipidomics approach to scan for thousands lipid signals. Based on the identified signatures, selected players were evaluated by quantitative PCR. Results: Principal component analysis shows that each cell line differs from the others. Our lipidomics analyses show that few lipids were modulated in a similar way irrespective of the genetic origin, and especially the increase in cholesterol esters. In contrast, ganglioside metabolism was differentially changed according to the genetic origin. More specifically, while GM3 forms were mostly and significantly increased for MTFMT, UQCRC2 and ECHS1 and moderately increased for NDUFS4, GM3 were decreased for LRPPRC. In addition, the GA1 and GM2 forms produced from GM3 were reduced in all cell lines except for LRPPRC and NDUFS4 where their signals remained unchanged. Since the formation of GA1 and GM2 involves the B4GALNT1 enzyme, we measured its mRNA expression and observed a drastic decrease up to 90% for MTFMT, while it remained unchanged for LRPPRC. Conclusion: The use of untargeted lipidomics is a relevant and powerful approach for the identification of specific signatures and underlying mechanisms of mitochondrial disorders, illustrated here by differentially affected ganglioside metabolism according to genetic origin, providing a starting point that will ultimately guide towards nutritional and therapeutic alternatives. Montreal Heart Institute Fundation, Lactic Acidosis Association and FRQS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Heart failure (HF) is a global health problem requiring more effective therapeutic alternatives. While HF involves disturbances in cardiac metabolism associated with lipidomic remodeling, there is a lack of approaches to specifically target cardiac metabolism. Recently, it has been shown in murine models of HF, that the use of nicotinamide riboside (vitamin B3) alone or the combination of B9 and B12 vitamins, before the development of HF, improved cardiac metabolism and function. Our hypothesis is that the combination of B3, B9 and B12 vitamins, used when HF is declared, will improve lipid metabolism, cardiac function and survival. The aim of this study is to evaluate the curative benefit of a synthetic diet enriched with the combination of these three vitamins (VitB) in a murine model of HF with reduced ejection fraction (EF). Method: Pressure overload was induced by constriction of the transverse aorta (TAC) in male and female mice. After 4 weeks, TAC mice reaching a pressure gradient of 60 mmHg, an increased left ventricle mass >30% and a reduced EF >10%, were randomized to a VitB-enriched diet or not. Results: In females, VitB improved survival and cardiac hypertrophy (-24%, p<0.05) as well as EF (+22%, p<0.01). We also observed a reduction of cardiac fibrosis emphasized by a significant decrease expression of collagen 1a and 3 (-30% and -19% respectively) and hydroxyproline concentration (-32%, p<0.01). No benefit was observed in males in terms of survival, cardiac function and fibrosis. Untargeted mass spectrometry (MS)-based lipidomics on plasmas at 8 weeks of treatment showed a decrease in several individual triglycerides (TG from 43 to 48 %; p<0.05) in TAC females that were normalized by VitB. In contrast, in TAC males, most of the annotated TGs were increased (from ̴35 to 210%; p<0.05), a profile exacerbated upon VitB treatment. MSMS identification of TG side chains revealed a differential profile in terms of fatty acid composition according to sex and treatment. In VitB females, longer and polyunsaturated TGs (53 to 60C; 5 to 12 double bonds) were significantly increased (1.6- to 2.1-fold) while, in VitB males, the increased TGs (1.2- to 5.2-fold) were shorter (24C to 48C) and saturated (no double bonds). Conclusion: Our study pointed out a sexual dimorphism in the response to VitB treatment in HF in favor of females and implying a lipidomic remodeling promoting cardioprotective polyunsaturated TGs. European Research Area Network on Cardiovascular Diseases, Canadian Institutes of Health Research, Fonds de recherche du Québec - Santé. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lipids are essential for neuron development and physiology. Yet, the central hubs that coordinate lipid supply and demand in neurons remain unclear. Here, we combine invertebrate and vertebrate models to establish the presence and functional significance of neuronal lipid droplets (LD) in vivo. We find that LD are normally present in neurons in a non-uniform distribution across the brain, and demonstrate triglyceride metabolism enzymes and lipid droplet-associated proteins control neuronal LD formation through both canonical and recently-discovered pathways. Appropriate LD regulation in neurons has conserved and male-biased effects on whole-body energy homeostasis across flies and mice, specifically neurons that couple environmental cues with energy homeostasis. Mechanistically, LD-derived lipids support neuron function by providing phospholipids to sustain mitochondrial and endoplasmic reticulum homeostasis. Together, our work identifies a conserved role for LD as the organelle that coordinates lipid management in neurons, with implications for our understanding of mechanisms that preserve neuronal lipid homeostasis and function in health and disease.

Lean patients with NAFLD may develop cardiac complications independently of pre-existent metabolic disruptions and comorbidities. To address the underlying mechanisms independent of the development of obesity, we used a murine model of hepatic mitochondrial deficiency. The liver-heart axis was studied as these mice develop microvesicular steatosis without obesity. Our results unveil a sex-dependent phenotypic remodeling beyond liver damage. Males, more than females, show fasting hypoglycemia and increased insulin sensitivity. They exhibit diastolic dysfunction, remodeling of the circulating lipoproteins and cardiac lipidome. Conversely, females do not manifest cardiac dysfunction but exhibit cardiometabolic impairments supported by impaired mitochondrial integrity and β-oxidation, remodeling of circulating lipoproteins and intracardiac accumulation of deleterious triglycerides. This study underscores metabolic defects in the liver resulting in significant sex-dependent cardiac abnormalities independent of obesity. This experimental model may prove useful to better understand the sex-related variability, notably in the heart, involved in the progression of lean-NAFLD.

The inflammatory bowel diseases (IBD) known as Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the gastrointestinal tract believed to arise because of an imbalance between the epithelial, immune and microbial systems. It has been shown that biological differences (genetic, epigenetic, microbial, environmental, etc.) exist between patients with IBD, with multiple risk factors been associated with disease susceptibility and IBD-related phenotypes (e.g. disease location). It is also known that there is heterogeneity in terms of response to therapy in patients with IBD, including to biological therapies that target very specific biological pathways (e.g. TNF-alpha signaling, IL-23R signaling, immune cell trafficking, etc.). It is hypothesized that the better the match between the biology targeted by these advanced therapies and the predominant disease-associated pathways at play in each patient will favor a beneficial response. The aim of this pilot study was to identify potential biological differences associated with differential treatment response to the anti α4β7 integrin therapy known as Vedolizumab. Our approach was to measure a broad range of analytes in the serum of patients prior to initiation of therapy and at the first clinical assessment visit, to identify potential markers of biological differences between patients at baseline and to see which biomarkers are most affected by treatment in responders. Our focus on early clinical response was to study the most proximal effects of therapy and to minimize confounders such as loss of response that occurs further distal to treatment initiation. Specifically, we performed targeted analyses of >150 proteins and metabolites, and untargeted analyses of >1100 lipid entities, in serum samples from 92 IBD patients (42 CD, 50 UC) immediately prior to initiation of therapy with vedolizumab (baseline samples) and at their first clinical assessment (14-week samples). We found lower levels of SDF-1a, but higher levels of PDGF-ββ, lactate, lysine, phenylalanine, branched chain amino acids, alanine, short/medium chain acylcarnitines, and triglycerides containing myristic acid in baseline serum samples of responders as compared to non-responders. We also observed an increase in serum levels of CXCL9 and citrate, as well as a decrease in IL-10, between baseline and week 14 samples. In addition, we observed that a group of metabolites and protein analytes was strongly associated with both treatment response and BMI status, although BMI status was not associated with treatment response.

Agrobacterium Tumefaciens is a natural genetic engineer that transfers DNA into plants and this is the most frequently applied process for the generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system localized in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2Å resolution and that of the related plasmid pKM101-determined N-pilus at 3Å resolution. Both pili contain a main pilus protein (VirB2 in A. Tumefaciens and TraM in pKM101) and phospholipids arranged in a 5-start helical assembly. They contain positively charged amino acids in the pilus lumen and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge to the lumen. Mutagenesis of the lumen-exposed Arg91 residue in VirB2 resulted in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion system pili and that the charge of the lumen is of functional importance.

Peroxisomes play a central role in tuning metabolic and signaling programs in a tissue- and cell-type-specific manner. However, the mechanisms by which the status of peroxisomes is communicated and integrated into cellular signaling pathways are not yet understood. Herein, we report the cellular responses to peroxisomal proteotoxic stress upon silencing the peroxisomal protease/chaperone LONP2. Depletion of LONP2 triggered the accumulation of its substrate TYSND1 protease, while the overall expression of peroxisomal proteins, as well as TYSND1-dependent ACOX1 processing appeared normal, reflecting early stages of peroxisomal proteotoxic stress. Consequently, the alteration of peroxisome size and numbers, and luminal protein import failure was coupled with induction of cell-specific cellular stress responses. Specific to COS-7 cells was a strong activation of the integrated stress response (ISR) and upregulation of ribosomal biogenesis gene expression levels. Common changes between COS-7 and U2OS cell lines included repression of the retinoic acid signaling pathway and upregulation of sphingolipids. Cholesterol accumulated in the endomembrane compartments in both cell lines, consistent with evidence that peroxisomes are required for cholesterol flux out of late endosomes. These unexpected consequences of peroxisomal stress provide an important insight into our understanding of the tissue-specific responses seen in peroxisomal disorders.

Very-long chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of mitochondrial long chain (LC) fatty acid β-oxidation (FAO). Inherited VLCAD deficiency (VLCADD) predisposes to neonatal arrhythmias whose pathophysiology is still not understood. We hypothesized that VLCADD results in global disruption of cardiac complex lipid homeostasis, which may set conditions predisposing to arrhythmia. To test this, we assessed the cardiac lipidome and related molecular markers in seven-month-old VLCAD−/− mice, which mimic to some extent the human cardiac phenotype. Mice were sacrificed in the fed or fasted state after receiving for two weeks a chow or a high-fat diet (HFD), the latter condition being known to worsen symptoms in human VLCADD. Compared to their littermate counterparts, HFD/fasted VLCAD−/− mouse hearts displayed the following lipid alterations: (1) Lower LC, but higher VLC-acylcarnitines accumulation, (2) higher levels of arachidonic acid (AA) and lower docosahexaenoic acid (DHA) contents in glycerophospholipids (GPLs), as well as (3) corresponding changes in pro-arrhythmogenic AA-derived isoprostanes and thromboxane B2 (higher), and anti-arrythmogenic DHA-derived neuroprostanes (lower). These changes were associated with remodeling in the expression of gene or protein markers of (1) GPLs remodeling: higher calcium-dependent phospholipase A2 and lysophosphatidylcholine-acyltransferase 2, (2) calcium handling perturbations, and (3) endoplasmic reticulum stress. Altogether, these results highlight global lipid dyshomeostasis beyond FAO in VLCAD−/− mouse hearts, which may set conditions predisposing the hearts to calcium mishandling and endoplasmic reticulum stress and thereby may contribute to the pathogenesis of arrhythmias in VLCADD in mice as well as in humans.

Adipose triglyceride lipase (ATGL), the enzyme that catalyses the rate-limiting step of triglyceride lipolysis, regulates inflammation in peripheral tissues. ATGL has been associated with both pro- and anti-inflammatory responses in different tissues suggesting its actions are dependent on cell type. Recent studies in microglia and macrophages suggest that lipid droplets (LD), a triglyceride storing organelle, and LD lipolysis via ATGL are important components of inflammatory responses. Here, we determined the impact of ATGL inhibition and microglia-specific ATGL loss-of-function on inflammatory and behavioural responses to acute pro-inflammatory insult. First, we evaluated the impact of lipolysis inhibition on lipopolysaccharide (LPS)-induced expression and secretion of cytokines in mouse primary microglia cultures. LPS led to LD accumulation in microglia and altered the expression of lipolysis regulators. The pan-lipase inhibitor ORlistat alleviated LPS-induced expression of IL-1β and IL-6. Specific inhibition of ATGL by ATGListatin had similar anti-inflammatory action on cytokines expression and secretion in both neonatal and adult microglia cultures. Second, targeted and untargeted lipidomic studies revealed that ATGL inhibition reduced LPS-induced generation of pro-inflammatory prostanoids and affected ceramide profile. Finally, the role of ATGL in neuroinflammation was assessed in a novel mouse model with inducible ATGL deletion specifically in microglia. Loss of microglial ATGL in adult male mice dampened LPS-induced expression of IL-6 and reduced LPS-induced sickness behaviour. Together, our results demonstrate that pharmacological inhibition or loss of ATGL-mediated triglyceride lipolysis reduces LPS-induced inflammation to suggest that inhibition of lipolysis plays a beneficial role in neuroinflammation.### Competing Interest StatementThe authors have declared no competing interest.

Age-related macular degeneration is a prevalent neuroinflammatory condition and a major cause of blindness driven by genetic and environmental factors such as obesity. In diseases of aging, modifiable factors can be compounded over the life span. We report that diet-induced obesity earlier in life triggers persistent reprogramming of the innate immune system, lasting long after normalization of metabolic abnormalities. Stearic acid, acting through Toll-like receptor 4 (TLR4), is sufficient to remodel chromatin landscapes and selectively enhance accessibility at binding sites for activator protein-1 (AP-1). Myeloid cells show less oxidative phosphorylation and shift to glycolysis, ultimately leading to proinflammatory cytokine transcription, aggravation of pathological retinal angiogenesis, and neuronal degeneration associated with loss of visual function. Thus, a past history of obesity reprograms mononuclear phagocytes and predisposes to neuroinflammation.

Collectively, these results indicate that metabolic defects in the liver can result in significant sex-dependent abnormalities that affect both the mitochondrial/metabolic phenotype and contractile function independent of obesity. This experimental model may prove useful to better understand the mechanisms underlying the sex-related variability in the progression of lean NAFLD in humans.

Plasmalogens belong to a subclass of phospholipids called ether lipids. The peroxisome synthesize these lipids and involve the limiting AGPS enzyme. In addition, mutations in the Agps gene are associated with cardiac manifestations. We hypothesized that decreased plasmalogens in cells negatively affect mitochondrial function and cell survival. We aimed to evaluate the metabolic and functional consequences of plasmalogens deficiency in a cardiomyoblastic cell line (H9c2 cells) modified using a CRISPR-Cas9 strategy targeting the Agps gene. We characterized this cell line using qPCR and immunoblotting to explore the influence on markers related to mitochondrial function [biogenesis, fatty acid metabolism (FA)], endoplasmic reticulum (ER) stress and apoptosis. AGPS reduction was validated at the gene (−58%) and protein (−78%) level, and is associated with decreased plasmalogens (−76%) that we analysed using mass spectrometry. This deficiency leaded to mitochondrial perturbations as revealed by changes on the expression of genes involved in biogenesis: Pgc-1α and ß (−27%; −61%), dynamics: Opa1 and Mff (−15%) as well as in the use of FA: Cd36 and Cpt1 (−52%; −51%) and in the oxidation of FA: Vlcad and Lcad (−22%; −13%). Consistently, using mass spectrometry, we observed decreased acylcarnitines (̴75%) used as markers of mitochondrial FA metabolism. This is accompanied with signs of increased ER stress and apoptosis as suggested by the increase in Chop gene expression (+31%) and the decreased uncleaved form of the Caspase-3 protein (−32%), respectively. Finally, we were able to normalize the levels of plasmalogen in our cells using alkylglycerols. Collectively, these results demonstrate that lowering plasmalogens affects mitochondrial function and cell stress in H9c2 cells. The normalisation of plasmalogens using alkylglycerols is encouraging to further characterize their metabolic and functional benefits.