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Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons leading to muscle paralysis and death. While a link between dysregulated lipid metabolism and ALS has been proposed, lipidome alterations involved in disease progression are still understudied. Using a rodent model of ALS overexpressing mutant human Cu/Zn-superoxide dismutase gene (SOD1-G93A), we performed a comparative lipidomic analysis in motor cortex and spinal cord tissues of SOD1-G93A and WT rats at asymptomatic (~70 days) and symptomatic stages (~120 days). Interestingly, lipidome alterations in motor cortex were mostly related to age than ALS. In contrast, drastic changes were observed in spinal cord of SOD1-G93A 120d group, including decreased levels of cardiolipin and a 6-fold increase in several cholesteryl esters linked to polyunsaturated fatty acids. Consistent with previous studies, our findings suggest abnormal mitochondria in motor neurons and lipid droplets accumulation in aberrant astrocytes. Although the mechanism leading to cholesteryl esters accumulation remains to be established, we postulate a hypothetical model based on neuroprotection of polyunsaturated fatty acids into lipid droplets in response to increased oxidative stress. Implicated in the pathology of other neurodegenerative diseases, cholesteryl esters appear as attractive targets for further investigations.

Dietary sugar is an important determinant of the development and progression of nonalcoholic fatty liver disease (NAFLD). However, the molecular mechanisms underlying the deleterious effects of sugar intake on NAFLD under energy-balanced conditions are still poorly understood. Here, we provide a comprehensive analysis of the liver lipidome and mechanistic insights into the pathogenesis of NAFLD induced by the chronic consumption of high-sugar diet (HSD). Newly weaned male Wistar rats were fed either a standard chow diet or an isocaloric HSD for 18 weeks. Livers were harvested for histological, oxidative stress, gene expression, and lipidomic analyses. Intake of HSD increased oxidative stress and induced severe liver injury, microvesicular steatosis, and ballooning degeneration of hepatocytes. Using untargeted lipidomics, we identified and quantified 362 lipid species in the liver. Rats fed with HSD displayed increased hepatic levels of triacylglycerol enriched in saturated and monounsaturated fatty acids, lipids related to mitochondrial function/structure (phosphatidylglycerol, cardiolipin, and ubiquinone), and acylcarnitine (an intermediate lipid of fatty acid beta-oxidation). HSD-fed animals also presented increased levels of some species of membrane lipids and a decreased content of phospholipids containing omega-6 fatty acids. These changes in the lipidome were associated with the downregulation of genes involved in fatty acid oxidation in the liver. In conclusion, our data suggest that the chronic intake of a HSD, even under isocaloric conditions, induces lipid overload, and inefficient/impaired fatty acid oxidation in the liver. Such events lead to marked disturbance in hepatic lipid metabolism and the development of NAFLD.

Mechanistic target of rapamycin complex (mTORC)1 activity is increased in adipose tissue of obese insulin-resistant mice, but its role in the regulation of tissue inflammation is unknown. Herein, we investigated the effects of adipocyte mTORC1 deficiency on adipose tissue inflammation and glucose homeostasis. For this, mice with adipocyte raptor deletion and controls fed a chow or a high-fat diet were evaluated for body mass, adiposity, glucose homeostasis, and adipose tissue inflammation. Despite reducing adiposity, adipocyte mTORC1 deficiency promoted hepatic steatosis, insulin resistance, and adipose tissue inflammation (increased infiltration of macrophages, neutrophils, and B lymphocytes; crown-like structure density; TNF-α, interleukin (IL)-6, and monocyte chemoattractant protein 1 expression; IL-1β protein content; lipid peroxidation; and de novo ceramide synthesis). The anti-oxidant, N-acetylcysteine, partially attenuated, whereas treatment with de novo ceramide synthesis inhibitor, myriocin, completely blocked adipose tissue inflammation and nucleotide oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3)-inflammasome activation, but not hepatic steatosis and insulin resistance induced by adipocyte raptor deletion. Rosiglitazone treatment, however, completely abrogated insulin resistance induced by adipocyte raptor deletion. In conclusion, adipocyte mTORC1 deficiency induces adipose tissue inflammation and NLRP3-inflammasome activation by promoting oxidative stress and de novo ceramide synthesis. Such adipose tissue inflammation, however, is not an underlying cause of the insulin resistance displayed by these mice.

Palmitoleic acid (POA, 16:1n-7) is a lipokine that has potential nutraceutical use to treat non-alcoholic fatty liver disease. We tested the effects of POA supplementation (daily oral gavage, 300 mg/Kg, 15 days) on murine liver inflammation induced by a high fat diet (HFD, 59% fat, 12 weeks). In HFD-fed mice, POA supplementation reduced serum insulin and improved insulin tolerance compared with oleic acid (OA, 300 mg/Kg). The livers of POA-treated mice exhibited less steatosis and inflammation than those of OA-treated mice with lower inflammatory cytokine levels and reduced toll-like receptor 4 protein content. The anti-inflammatory effects of POA in the liver were accompanied by a reduction in liver macrophages (LM, CD11c+; F4/80+; CD86+), an effect that could be triggered by peroxisome proliferator activated receptor (PPAR)-γ, a lipogenic transcription factor upregulated in livers of POA-treated mice. We also used HFD-fed mice with selective deletion of PPAR-γ in myeloid cells (PPAR-γ KOLyzCre+) to test whether the beneficial anti-inflammatory effects of POA are dependent on macrophages PPAR-γ. POA-mediated improvement of insulin tolerance was tightly dependent on myeloid PPAR-γ, while POA anti-inflammatory actions including the reduction in liver inflammatory cytokines were preserved in mice bearing myeloid cells deficient in PPAR-γ. This overlapped with increased CD206+ (M2a) cells and downregulation of CD86+ and CD11c+ liver macrophages. Moreover, POA supplementation increased hepatic AMPK activity and decreased expression of the fatty acid binding scavenger receptor, CD36. We conclude that POA controls liver inflammation triggered by fat accumulation through induction of M2a macrophages independently of myeloid cell PPAR-γ., Graphical abstract Unlabelled Image, Highlights • Palmitoleic acid (POA) supplementation reduced serum insulin and improved insulin tolerance; • Livers of POA-treated mice exhibited less steatosis and inflammation; • POA lowered the liver M1 macrophages population and the expression of inflammation-related immune-cell markers; • POA increased PPAR-γ, a transcription factor that regulates anti-inflammatory effects in macrophages; • However, POA reduced liver inflammation even in mice that lack PPAR-γ expression in myeloid cells; • POA controls liver inflammation through induction of M2a macrophages independently of PPAR-γ in myeloid cells.

Scope Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated. Methods and results Severe lipoatrophic mice induced by PPAR-γ deletion exclusively in adipocytes (A-PPARγ KO) and littermate controls (A-PPARγ WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis. Conclusion Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.

The nutrient sensors peroxisome proliferator-activated receptor γ (PPARγ) and mechanistic target of rapamycin complex 1 (mTORC1) closely interact in the regulation of adipocyte lipid storage. The precise mechanisms underlying this interaction and whether this extends to other metabolic processes and the endocrine function of adipocytes are still unknown. We investigated herein the involvement of mTORC1 as a mediator of the actions of the PPARγ ligand rosiglitazone in subcutaneous inguinal white adipose tissue (iWAT) mass, endocrine function, lipidome, transcriptome and branched-chain amino acid (BCAA) metabolism. Mice bearing regulatory associated protein of mTOR (Raptor) deletion and therefore mTORC1 deficiency exclusively in adipocytes and littermate controls were fed a high-fat diet supplemented or not with the PPARγ agonist rosiglitazone (30 mg/kg/day) for 8 weeks and evaluated for iWAT mass, lipidome, transcriptome (Rnaseq), respiration and BCAA metabolism. Adipocyte mTORC1 deficiency not only impaired iWAT adiponectin transcription, synthesis and secretion, PEPCK mRNA levels, triacylglycerol synthesis and BCAA oxidation and mRNA levels of related proteins but also completely blocked the upregulation in these processes induced by pharmacological PPARγ activation with rosiglitazone. Mechanistically, adipocyte mTORC1 deficiency impairs PPARγ transcriptional activity by reducing PPARγ protein content, as well as by downregulating C/EBPα, a co-partner and facilitator of PPARγ. In conclusion, mTORC1 and PPARγ are essential partners involved in the regulation of subcutaneous adipose tissue adiponectin production and secretion and BCAA oxidative metabolism.

Cytochrome c (cytc) is a heme protein of 12 kDa that transfers electrons in the mitochondrial respiratory chain. Increased cytc peroxidase activity leads to cardiolipin (CL) oxidation, a hallmark of early apoptosis stage. Here, we aimed to investigate the interaction between cytc with cardiolipin hydroperoxide (CLOOH) in a mimetic mitochondrial membrane. Cytc-CL peroxidase reaction occurred at faster rates with CLOOH than with H2O2. Moreover, liposomes containing CLOOH promoted increased protein aggregation with minor or no release of cytc from the membrane. Dimeric and trimeric cytc species were observed in the first 15 min, followed by increased formation of high-molecular-weight aggregates afterwards. nLC-MS/MS analysis identified several Lys and His residues covalently modified by lipid aldehydes that showed mass increments corresponding to 4-hydroxynonenal (HNE), 4-oxononenal (ONE), hexanoyl, heptenal and octenal addition. Noteworthy, most modifications were observed at Lys and His residues located at A-site (K73, K87, K88), L-site (H26, H33, and K27) membrane binding sites. Further, dityrosine cross-linked peptides were also characterized at residues Y48-Y74, Y48-Y97 and Y74-Y97. Collectively, our findings show that CLOOH causes irreversible protein damage and crosslinking of cytc in the membrane.

Microglial activation by oleate and palmitate differentially modulates brain inflammatory status. However, the metabolic reprogramming supporting these reactive phenotypes remains unknown. Employing real-time metabolic measurements and lipidomic analysis, we show that both fatty acids promote microglial oxidative metabolism, while lipopolysaccharide (LPS) enhances glycolytic rates. Interestingly, oleate treatment was followed by enrichment in storage lipids bound to polyunsaturated fatty acids (PUFA), in parallel with protection against oxidative imbalance. Palmitate, in turn, induced a distinct lipid distribution defined by PUFA linked to membrane phospholipids, which are more susceptible to lipid peroxidation and inflammatory signaling cascades. This distribution was mirrored by LPS treatment, which led to a strong pro-inflammatory phenotype in microglia. Thus, although both oleate and palmitate preserve mitochondrial function, a contrasting lipid distribution supports differences in fatty acid-induced neuroinflammation. These data reinforce the concept that reactive microglial profiles are achieved by stimulus-evoked remodeling in cell metabolism.

Scope The mechanisms and involvement of uncoupling protein 1 (UCP1) in the protection from obesity and insulin resistance induced by intake of a high-fat diet rich in omega-3 (n-3) fatty acids are investigated. Methods and results C57BL/6J mice are fed either a low-fat (control group) or one of two isocaloric high-fat diets containing either lard (HFD) or fish oil (HFN3) as fat source and evaluated for body weight, adiposity, energy expenditure, glucose homeostasis, and inguinal white and interscapular brown adipose tissue (iWAT and iBAT, respectively) gene expression, lipidome, and mitochondrial bioenergetics. HFN3 intake protected from obesity, glucose and insulin intolerances, and hyperinsulinemia. This is associated with increased energy expenditure, iWAT UCP1 expression, and incorporation of n-3 eicosapentaenoic and docosahexaenoic fatty acids in iWAT and iBAT triacylglycerol. Importantly, HFN3 is equally effective in reducing body weight gain, adiposity, and glucose intolerance and increasing energy expenditure in wild-type and UCP1-deficient mice without recruiting other thermogenic processes in iWAT and iBAT, such as mitochondrial uncoupling and SERCA-mediated calcium and creatine-driven substrate cyclings. Conclusion Intake of a high-fat diet rich in omega-3 fatty acids protects both wild-type and UCP1-deficient mice from obesity and insulin resistance by increasing energy expenditure through unknown mechanisms.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by degeneration of upper and lower motor neurons. While the fundamental causes of the disease are still unclear, the accumulation of Cu,Zn-superoxide dismutase (SOD1) immunoreactive aggregates is associated with familial ALS cases. Cholesterol 5,6-secosterol aldehydes (Seco A and Seco B) are reported to contribute to neurodegenerative disease pathology by inducing protein modification and aggregation. Here we have investigated the presence of secosterol aldehydes in ALS SOD1-G93A rats and their capacity to induce SOD1 aggregation. Secosterol aldehydes were analyzed in blood plasma, spinal cord and motor cortex of ALS rats at the pre-symptomatic and symptomatic stages. Seco B was significantly increased in plasma of symptomatic ALS rats compared to pre-symptomatic animals, suggesting an association with disease progression. In vitro experiments showed that both Seco A and Seco B induce the formation of high molecular weight (HMW) SOD1 aggregates with amorphous morphology. SOD1 adduction to ω-alkynyl-secosterols analyzed by click assay showed that modified proteins are only detected in the HMW region, indicating that secosterol adduction generates species highly prone to aggregate. Of note, SOD1-secosterol adducts containing up to five secosterol molecules were confirmed by MALDI-TOF analysis. Interestingly, mass spectrometry sequencing of SOD1 aggregates revealed preferential secosterol adduction to Lys residues located at the electrostatic loop (Lys 122, 128 and 136) and nearby the dimer interface (Lys 3 and 9). Altogether, our results show that secosterol aldehydes are increased in plasma of symptomatic ALS rats and represent a class of aldehydes that can potentially modify SOD1 enhancing its propensity to aggregate., Graphical abstract fx1, Highlights • Secosterol aldehydes are ubiquitously present in motor cortex and spinal cord; • These aldehydes modify SOD1 in vitro, producing high molecular weight aggregates; • Lys residues at electrostatic loop and nearby the dimer interface are mainly modified; • Secosterol adduction increases protein hydrophobicity and its propensity to aggregate.

Uric acid is the final product of purine metabolism in humans and is considered to be quantitatively the main antioxidant in plasma. In vitro studies showed that the oxidation of uric acid by peroxidases, in presence of superoxide, generates urate free radical and urate hydroperoxide. Urate hydroperoxide is a strong oxidant and might be a relevant intermediate in inflammatory conditions. However, the identification of urate hydroperoxide in cells and biological samples has been a challenge due to its high reactivity. By using mass spectrometry, we undoubtedly demonstrated the formation of urate hydroperoxide and its corresponding alcohol, hydroxyisourate during the respiratory burst in peripheral blood neutrophils and in human leukemic cells differentiated in neutrophils (dHL-60). The respiratory burst was induced by phorbol myristate acetate (PMA) and greatly increased oxygen consumption and superoxide production. Both oxygen consumption and superoxide production were further augmented by incubation with uric acid. Conversely, uric acid significantly decreased the levels of HOCl, probably because of the competition with chloride by the catalysis of myeloperoxidase. In spite of the decrease in HOCl, the overall oxidative status, measured by GSH/GSSG ratio, was augmented in the presence of uric acid. In summary, the present results support the formation of urate hydroperoxide, a novel oxidant in neutrophils oxidative burst. Urate hydroperoxide is a strong oxidant and alters the redox balance toward a pro-oxidative environment. The production of urate hydroperoxide in inflammatory conditions could explain, at least in part, the harmful effect associated to uric acid.

The 2015–2016 Zika virus (ZIKV) outbreak in Brazil was remarkably linked to the incidence of microcephaly and other deleterious clinical manifestations, including eye abnormalities, in newborns. It is known that ZIKV targets the placenta, triggering an inflammatory profile that may cause placental insufficiency. Transplacental lipid transport is delicately regulated during pregnancy and deficiency on the delivery of lipids such as arachidonic and docosahexaenoic acids may lead to deficits in both brain and retina during fetal development. Here, plasma lipidome profiles of ZIKV exposed microcephalic and normocephalic newborns were compared to non-infected controls. Our results reveal major alterations in circulating lipids from both ZIKV exposed newborns with and without microcephaly relative to controls. In newborns with microcephaly, the plasma concentrations of hydroxyoctadecadienoic acid (HODE), primarily as 13-HODE isomer, derived from linoleic acid were higher as compared to normocephalic ZIKV exposed newborns and controls. Total HODE concentrations were also positively associated with levels of other oxidized lipids and several circulating free fatty acids in newborns, indicating a possible plasma lipidome signature of microcephaly. Moreover, higher concentrations of lysophosphatidylcholine in ZIKV exposed normocephalic newborns relative to controls suggest a potential disruption of polyunsaturated fatty acids transport across the blood-brain barrier of fetuses. The latter data is particularly important given the neurocognitive and neurodevelopmental abnormalities observed in follow-up studies involving children with antenatal ZIKV exposure, but normocephalic at birth. Taken together, our data reveal that plasma lipidome alterations associated with antenatal exposure to ZIKV could contribute to identification and monitoring of the wide spectrum of clinical phenotypes at birth and further, during childhood., Author summary Antenatal exposure to Zika virus (ZIKV) is linked to a wide range of clinical presentations at birth, from asymptomatic cases to microcephaly, and other neurocognitive and neurodevelopmental abnormalities manifested in the early childhood. Stratification of these clinical phenotypes in newborns with suspected antenatal ZIKV exposure is challenging, but critical to improve early assessment of rehabilitative interventions. In this study, plasma lipidome profiling of 274 lipid species was performed in both normocephalic and microcephalic newborns with antenatal ZIKV exposure and compared to non-infected controls. Multiple lipid species were independent predictors of antenatal ZIKV exposure. More specifically, microcephaly was strongly associated with an oxidized free fatty acid and ZIKV exposed normocephalic newborns exhibited higher plasma concentrations of lysophosphatidylcholine relative to controls. These findings emphasize the need for studies focused on the role of individual lipids in neuropathogenesis of ZIKV and raise the potential of plasma lipidome profiling for early diagnosis of newborns with suspected antenatal ZIKV exposure. To validate the predictive ability of this approach, prospective studies with a larger cohort of newborns are now required.