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Driven by the energy of a photon, the visual pigments in rod and cone photoreceptor cells isomerize 11-cis-retinal to the all-trans configuration. This photochemical reaction initiates the signal transduction pathway that eventually leads to the transmission of a visual signal to the brain and leaves the opsins insensitive to further light stimulation. For the eye to restore light sensitivity, opsins require recharging with 11-cis-retinal. This trans–cis back conversion is achieved through a series of enzymatic reactions composing the retinoid (visual) cycle. Although it is evident that the classical retinoid cycle is critical for vision, the existence of an adjunct pathway for 11-cis-retinal regeneration has been debated for many years. Retinal pigment epithelium (RPE)–retinal G protein-coupled receptor (RGR) has been identified previously as a mammalian retinaldehyde photoisomerase homologous to retinochrome found in invertebrates. Using pharmacological, genetic, and biochemical approaches, researchers have now established the physiological relevance of the RGR in 11-cis-retinal regeneration. The photoisomerase activity of RGR in the RPE and Müller glia explains how the eye can remain responsive in daylight. In this review, we will focus on retinoid metabolism in the eye and visual chromophore regeneration mediated by RGR.  

Functions of the gut microbiome have a growing number of implications for host metabolic health, with diet being one of the most significant influences on microbiome composition. Compelling links between diet and the gut microbiome suggest key roles for various macronutrients, including lipids, yet how individual classes of dietary lipids interact with the microbiome remains largely unknown. Sphingolipids are bioactive components of most foods and are also produced by prominent gut microbes. This makes sphingolipids intriguing candidates for shaping diet–microbiome interactions. Here, we used a click chemistry–based approach to track the incorporation of bioorthogonal dietary omega-alkynyl sphinganine (sphinganine alkyne [SAA]) into the murine gut microbial community (Bioorthogonal labeling). We identified microbial and SAA-specific metabolic products through fluorescence-based sorting of SAA-containing microbes (Sort), 16S rRNA gene sequencing to identify the sphingolipid-interacting microbes (Seq), and comparative metabolomics to identify products of SAA assimilation by the microbiome (Spec). Together, this approach, termed Bioorthogonal labeling-Sort-Seq-Spec (BOSSS), revealed that SAA assimilation is nearly exclusively performed by gut Bacteroides, indicating that sphingolipid-producing bacteria play a major role in processing dietary sphinganine. Comparative metabolomics of cecal microbiota from SAA-treated mice revealed conversion of SAA to a suite of dihydroceramides, consistent with metabolic activities of Bacteroides and Bifidobacterium. Additionally, other sphingolipid-interacting microbes were identified with a focus on an uncharacterized ability of Bacteroides and Bifidobacterium to metabolize dietary sphingolipids. We conclude that BOSSS provides a platform to study the flux of virtually any alkyne-labeled metabolite in diet–microbiome interactions.

Genetic variants that increase the risk of fatty liver disease (FLD) and cirrhosis have recently been identified in the proximity of membrane bound O-acyltransferase domain-containing 7 (MBOAT7).  To elucidate the link between these variants and FLD we characterized Mboat7 liver-specific knock-out mice (Mboat7-LSKO).  Chow-fed Mboat7-LSKO mice developed fatty livers and associated liver injury.  Lipidomic analysis of liver using mass spectrometry revealed a pronounced reduction in 20-carbon polyunsaturated fatty acid content in phosphatidylinositols (PIs), but not in other phospholipids. The change in fatty acid composition of PIs in these mice was associated with a marked increase in de novo lipogenesis due to activation of SREBP-1c, a transcription factor that coordinates the activation of genes encoding enzymes in the fatty acid biosynthesis pathway. Hepatic removal of both SREBP cleavage activating protein (Scap) and Mboat7 normalized hepatic triglycerides relative to Scap only hepatic knock-out showing increased SREBP-1c processing is required for Mboat7 induced steatosis.  This study reveals a clear relationship between PI fatty acid composition and regulation of hepatic fat synthesis and delineates the mechanism by which mutations in MBOAT7 cause hepatic steatosis.

Roux-en-Y gastric bypass (RYGB) is one of the most commonly performed weight-loss procedures, but how severe obesity and RYGB affects circulating HDL-associated microRNAs (miRNAs) remains unclear. Here, we aim to investigate how HDL-associated miRNAs are regulated in severe obesity and how weight loss after RYGB surgery affects HDL-miRNAs. Plasma HDL were isolated from patients with severe obesity (n=53) before, 6 and 12 months after RYGB by immunoprecipitation using goat anti-human apoA-I microbeads. HDL were also isolated from 18 healthy participants. miRNAs were extracted from isolated HDL and levels of miR-24, miR-126, miR-222 and miR-223 were determined by TaqMan miRNA assays. We found that HDL-associated miR-126, miR-222 and miR-223 levels, but not miR-24 levels, were significantly higher in patients with severe obesity when compared with healthy controls. There were significant increases in HDL-associated miR-24, miR-222 and miR-223 at 12 months after RYGB. Additionally, cholesterol efflux capacity and paraoxonase (PON1) activity were increased and intracellular adhesion molecule-1 (ICAM-1) levels decreased. The increases in HDL-associated miR-24 and miR-223 were positively correlated with increase in cholesterol efflux capacity (r=0.326, P=0.027 and r=0.349, P=0.017 respectively). An inverse correlation was observed between HDL-associated miR-223 and ICAM-1 at baseline. Together, these findings show that HDL-associated miRNAs are differentially regulated in healthy versus patients with severe obesity and are altered after RYGB. These findings provide insights into how miRNAs are regulated in obesity before and after weight reduction, and may lead to the development of novel treatment strategies for obesity and related metabolic disorders.

Adiponectin, an adipocyte-derived protein, has anti-atherogenic and anti-diabetic effects, but how it confers the anti-atherogenic effects is not well understood. To study the anti-atherogenic mechanisms of adiponectin, we examined whether it interacts with atherogenic low-density lipoprotein (LDL) to attenuate LDL’s atherogenicity. L5, the most electronegative subfraction of LDL, induces atherogenic responses similarly to copper-oxidized LDL (oxLDL). Unlike native LDL endocytosed via the LDL receptor, L5 and oxLDL are internalized by cells via the lectin-like oxidized LDL receptor-1 (LOX-1). Using enzyme-linked immunosorbent assays (ELISAs), we showed that adiponectin preferentially bound oxLDL but not native LDL. In Chinese hamster ovary (CHO) cells transfected with LOX-1 or LDL receptor, adiponectin selectively inhibited the uptake of oxLDL but not of native LDL, respectively. Furthermore, adiponectin suppressed the internalization of oxLDL in human coronary artery endothelial cells (HCAECs) and THP-1–derived macrophages. Western blot analysis of human plasma showed that adiponectin was abundant in L5 but not in L1, the least electronegative subfraction of LDL. Sandwich ELISAs with anti-adiponectin and anti–apolipoprotein B antibodies confirmed the binding of adiponectin to L5 and oxLDL. In LOX-1–expressing CHO cells, adiponectin inhibited cellular responses to oxLDL and L5, including nuclear factor-B activation and ERK phosphorylation. In HCAECs, adiponectin inhibited oxLDL-induced endothelin-1 secretion and ERK phosphorylation. Conversely, oxLDL suppressed the adiponectin-induced activation of adenosine monophosphate–activated protein kinase in COS-7 cells expressing adiponectin receptor AdipoR1. Our findings suggest that adiponectin binds and inactivates atherogenic LDL, providing novel insight into the anti-atherogenic mechanisms of adiponectin.

Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) is essential for the transportation of cholesterol between peripheral tissues and the liver. However, specific mutations in Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) are responsible for a late-onset systemic amyloidosis, the pathological accumulation of protein fibrils in tissues and organs. Carriers of these mutations do not exhibit increased cardiovascular disease risk despite displaying reduced levels of ApoA-I/ HDL-cholesterol. To explain this paradox, we show that the HDL particle profile of patients carrying either L75P or L174S ApoA-I amyloidogenic variants a higher relative abundance of the 8.4 nm vs 9.6 nm particles, and that serum from patients, as well as reconstituted 8.4 and 9.6 nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogen-deuterium exchange revealed that the variants in 8.4 nm rHDL have altered secondary structure composition and display a more flexible binding to lipids compared to their native counterpart. The reduced HDL-cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles and better cholesterol efflux due to altered, region-specific protein structure dynamics.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism by inducing the degradation of hepatic low-density lipoprotein receptor (LDLR). Plasma PCSK9 has two main molecular forms: a 62-kDa mature form (PCSK9_62) and a 55-kDa, furin-cleaved form (PCSK9_55). PCSK9_55 is considered less active than PCSK9_62 in degrading LDLR. We aimed to identify the site of PCSK9_55 formation (intra- vs. extracellular) and to further characterize the LDLR-degradative function of PCSK9_55 relative to PCSK9_62. Co-expressing PCSK9_62 with furin in cell culture induced formation of PCSK9_55, most of which was found in the extracellular space. Under the same conditions we found that: i) adding a cell-permeable furin inhibitor preferentially decreased the formation of PCSK9_55 extracellularly; ii) using pulse-chase, we observed the formation of PCSK9_55 exclusively extracellularly in a time-dependent manner. A recombinant form of PCSK9_55 was efficiently produced but displayed impaired secretion that resulted in its intracellular trapping. However, the non-secreted PCSK9_55 was able to induce degradation of LDLR, though with 50% lower efficiency compared with PCSK9_62. Collectively, our data show that PCSK9_55 is generated in the extracellular space, and that intracellular PCSK9_55 is not secreted but retains the ability to degrade the LDLR through an intracellular pathway.

Smith-Lemli-Opitz Syndrome (SLOS) is a developmental disorder (OMIM #270400) caused by autosomal recessive mutations in the Dhcr7 gene, which encodes the enzyme 3β-hydroxysterol-7 reductase. SLOS patients present clinically with dysmorphology and neurological, behavioral and cognitive defects, with characteristically elevated levels of 7-dehydrocholesterol (7-DHC) in all bodily tissues and fluids. Previous mouse models of SLOS have been hampered by postnatal lethality when Dhcr7 is knocked out globally, while a hypomorphic mouse model showed improvement in the biochemical phenotype with ageing, and did not manifest most other characteristic features of SLOS. We report the generation of a conditional knockout of Dhcr7 (Dhcr7flx/flx), validated by generating a mouse with a liver-specific deletion (Dhcr7L-KO). Phenotypic characterization of liver-specific knockout mice revealed no significant changes in viability, fertility, growth curves, liver architecture, hepatic triglyceride secretion, or parameters of systemic glucose homeostasis. Furthermore, qPCR and RNA-Seq analyses of livers revealed no perturbations in pathways responsible for cholesterol synthesis, either in male or female Dhcr7L-KO mice, suggesting hepatic disruption of post-squalene cholesterol synthesis leads to minimal impact on sterol metabolism in the liver. This validated conditional Dhcr7 knockout model may now allow us to systematically explore the pathophysiology of SLOS, by allowing for temporal, cell and tissue-specific loss of DHCR7.

Lecithin:cholesterol-acyl-transferase (LCAT) plays a major role in cholesterol metabolism as it is the only extracellular enzyme able to esterify cholesterol. LCAT activity is required for lipoprotein remodelling and, most specifically, for the growth and maturation of HDLs. In fact, genetic alterations affecting LCAT func- tionality may cause a severe reduction in plasma levels of HDL-cholesterol with important clinical consequences. Although several hypotheses were formulated, the exact molecular recognition mechanism between LCAT and HDLs is still unknown. We employed a combination of structural bioinformatics procedures to deepen the insights into the HDL-LCAT interplay that promotes LCAT activation and cholesterol esterification. We have generated a data-driven model of reconstituted HDL (rHDL) and studied the dynamics of an assembled rHDL::LCAT supramolecular complex, pinpointing the conformational changes originating from the interaction between LCAT and apolipoprotein A-I (apoA-I) that are necessary for LCAT activation. Specifically, we propose a mechanism in which the anchoring of LCAT lid to apoA-I helices allows the formation of a hydrophobic hood that expands LCAT active site and shields it from the solvent, allowing the enzyme to process large hydrophobic substrates.

Bacterial lipopolysaccharides (LPSs or endotoxins) can bind most proteins of the lipid transfer/LPS-binding protein (LT/LBP) family in host organisms. The LPS-bound LT/LBP proteins then trigger either an LPS-induced proinflammatory cascade or LPS binding to lipoproteins that are involved in endotoxin inactivation and detoxification. Cholesteryl ester transfer protein (CETP) is an LT/LBP member, but its impact on LPS metabolism and sepsis outcome is unclear. Here, we performed fluorescent LPS transfer assays to assess the ability of CETP to bind and transfer LPS. The effects of intravenous (iv) infusion of purified LPS or polymicrobial infection (cecal ligation and puncture [CLP]) were compared in transgenic mice expressing human CETP and wild-type mice naturally having no CETP activity. CETP displayed no LPS transfer activity in vitro, but it tended to reduce biliary excretion of LPS in vivo. The CETP expression in mice was associated with significantly lower basal plasma lipid levels and with higher mortality rates in both models of endotoxemia and sepsis. Furthermore, CETPTg plasma modified cytokine production of macrophages in vitro. In conclusion, despite having no direct LPS binding and transfer property, human CETP worsens sepsis outcomes in mice by altering the protective effects of plasma lipoproteins against endotoxemia, inflammation, and infection.

The LDL receptor-related protein-1 (LRP1) is highly expressed in numerous cell types, and its impairment is associated with obesity, diabetes, and fatty liver disease. However, the mechanisms linking LRP1 to metabolic disease are not completely understood. Here, we compared the metabolic phenotype of C57BL/6J wild type and LRP1 knock-in mice carrying an inactivating mutation in the distal NPxY motif after feeding a low fat (LF) diet or high fat diets with (HFHC) or without (HF) cholesterol supplementation. In response to HF feeding, both groups developed hyperglycemia, hyperinsulinemia, and hyperlipidemia, as well as increased adiposity with adipose tissue inflammation and liver steatosis. However, when animals were fed the HF diet supplemented with cholesterol, the LRP1 NPxY mutation prevents hypercholesterolemia, reduces adipose tissue and brain inflammation, and limits liver progression to steatohepatitis. Nevertheless, insulin signaling is impaired in LRP1 NPxY mutant hepatocytes and this mutation does not protect against HFHC-induced insulin resistance. The selective metabolic improvement observed in HFHC-fed LRP1 NPxY mutant mice is due to an apparent increase of hepatic LDL receptor levels, leading to an elevated rate of plasma lipoprotein clearance and lowering of plasma and hepatic cholesterol levels. The unique metabolic phenotypes displayed by LRP1 NPxY mutant mice in response to HF or HFHC diet feeding indicate an LRP1-cholesterol axis in modulating tissue inflammation. The LRP1 NPxY mutant mouse phenotype differs from phenotypes observed in mice with tissue-specific LRP1 inactivation, thus highlighting the importance of an integrative approach to evaluate how global LRP1 dysfunction contributes to metabolic disease development.

Apolipoproteins C-I, C-II and C-III interact with ApoE to regulate lipoprotein metabolism and contribute to Alzheimer’s disease pathophysiology. In plasma, apoC-I and C-II exist as truncated isoforms, while apoC-III exhibits multiple glycoforms. This study aimed to 1. delineate apoC-I, C-II and C-III isoform profiles in CSF and plasma in a cohort of non-demented older individuals (n = 61), and 2. examine the effect of APOE4 on these isoforms and their correlation with CSF Aβ42, a surrogate of brain amyloid accumulation. The isoforms of the apoCs were immunoaffinity enriched and measured with MALDI-TOF mass spectrometry, revealing a significantly higher percentage of truncated apoC-I and apoC-II in CSF compared to matched plasma, with positive correlation between CSF and plasma. A greater percentage of monosialylated and disialylated apoC-III isoforms was detected in CSF, accompanied by a lower percentage of the two non-sialylated apoC-III isoforms, with significant linear correlations between CSF and plasma. Furthermore, a greater percentage of truncated apoC-I in CSF, and apoC-II in plasma and CSF, was observed in individuals carrying at least one apoE E4 allele. Increased apoC-I and apoC-II truncations were  associated with lower CSF Aβ42. Finally, monosialylated apoC-III was lower, and disialylated apoC-III greater in the CSF of E4 carriers. Together, these results reveal distinct patterns of the apoCs isoforms in CSF, implying CSF-specific apoCs processing. These patterns were accentuated in APOE E4 allele carriers, suggesting an association between APOE4 genotype and Alzheimer’s disease pathology with apoCs processing and function in the brain.

Recent studies have highlighted an important role for lysophosphatidylcholine acyltransferase 3 (LPCAT3) in controlling the PUFA composition of cell membranes in the liver and intestine. In these organs, LPCAT3 critically supports cell membrane-associated processes such as lipid absorption or lipoprotein secretion. However, the role of LPCAT3 in macrophages remains controversial. Here, we investigated LPCAT3’s role in macrophages both in vitro and in vivo in mice with atherosclerosis and obesity. To accomplish this, we used the LysMCre strategy to develop a mouse model with conditional Lpcat3 deficiency in myeloid cells (Lpcat3KOMac). We observed that partial Lpcat3 deficiency (approx. 75% reduction) in macrophages alters the PUFA composition of all phospholipid (PL) subclasses, including phosphatidylinositols and phosphatidylserines. A reduced incorporation of C20 PUFAs (mainly arachidonic acid [AA]) into PLs was associated with a redistribution of these FAs toward other cellular lipids such as cholesteryl esters. Lpcat3 deficiency had no obvious impact on macrophage inflammatory response or endoplasmic reticulum (ER) stress; however, Lpcat3KOMac macrophages exhibited a reduction in cholesterol efflux in vitro. In vivo, myeloid Lpcat3 deficiency did not affect atherosclerosis development in LDL receptor deficient mouse (Ldlr-/-) mice. Lpcat3KOMac mice on a high-fat diet displayed a mild increase in hepatic steatosis associated with alterations in several liver metabolic pathways and in liver eicosanoid composition. We conclude that alterations in AA metabolism along with myeloid Lpcat3 deficiency may secondarily affect AA homeostasis in the whole liver, leading to metabolic disorders and triglyceride accumulation.

Perilipin (PLIN) 5 is a lipid droplet-associated protein that coordinates intracellular lipolysis in highly oxidative tissues and is thought to regulate lipid metabolism in response to phosphorylation by protein kinase A (PKA). We sought to identify PKA phosphorylation sites in PLIN5 and assess their functional relevance in cultured cells and the livers of mice. We detected phosphorylation on S155, S161 and S163 of recombinant PLIN5 by PKA in vitro and identified S155 as a functionally important site for lipid metabolism. Expression of phosphorylation-defective PLIN5 S155A in Plin5 null cells resulted in decreased rates of lipolysis and triglyceride-derived fatty acid oxidation compared with cells expressing wildtype PLIN5. These differences in lipid metabolism were not associated with differences in the cellular distribution of PLIN5. Rather, FLIM-FRET analysis of protein-protein interactions showed that PLIN5 S155 phosphorylation regulates PLIN5 interaction with adipose triglyceride lipase (ATGL) at the lipid droplet, but not with the co-activator of ATGL, α-β hydrolase domain-containing 5 (ABHD5). Re-expression of PLIN5 S155A in the liver of Plin5 liver-specific null mice reduced lipolysis when compared to mice with wildtype PLIN5 re-expression, but was not associated with other changes in hepatic lipid metabolism, such as fatty acid oxidation, de novo lipogenesis and triglyceride secretion. Furthermore, glycemic control was impaired in mice with expression of PLIN5 S155A compared with mice expressing PLIN5. Together, these studies demonstrate that PLIN5 S155 is required for PKA-mediated lipolysis and builds on the body of evidence demonstrating a critical role for PLIN5 in coordinating lipid and glucose metabolism

Carotid atherosclerosis is a risk factor for ischemic stroke, one of the main causes of mortality and disability worldwide. The disease is characterized by plaques, heterogeneous deposits of lipids and necrotic debris in the vascular wall, which grow gradually and may remain asymptomatic for decades. However, at some point a plaque can evolve to a high-risk plaque phenotype, which may trigger a cerebrovascular event. Lipids play a key role in the development and progression of atherosclerosis, but the nature of their involvement is not fully understood. Using matrix-assisted laser desorption/ionization mass spectrometry imaging, we visualized the distribution of approximately 200 different lipid signals, originating of > 90 uniquely assigned species, in 106 tissue sections of 12 human carotid atherosclerotic plaques. We performed unsupervised classification of the mass spectrometry dataset, as well as a histology-directed multivariate analysis. These data allowed us to extract the spatial lipid patterns associated with morphological plaque features in advanced plaques from a symptomatic population, revealing spatial lipid patterns in atherosclerosis and their relation to histological tissue type. The abundances of sphingomyelin and oxidized cholesteryl ester species were elevated specifically in necrotic intima areas, while diacylglycerols and triacylglycerols were spatially correlated to areas containing the coagulation protein fibrin. These results demonstrate a clear co-localization between plaque features and specific lipid classes, as well as individual lipid species in high-risk atherosclerotic plaques.

Genome-wide association studies (GWAS) have implicated ~380 genetic loci for plasma lipid regulation. However, these loci only explain 17-27% of the trait variance and a comprehensive understanding of the molecular mechanisms has not been achieved. In this study, we utilized an integrative genomics approach leveraging diverse genomic data from human populations to investigate whether genetic variants associated with various plasma lipid traits, namely total cholesterol (TC), high and low density lipoprotein cholesterol (HDL and LDL), and triglycerides (TG), from GWAS were concentrated on specific parts of tissue-specific gene regulatory networks. In addition to the expected lipid metabolism pathways, gene subnetworks involved in ‘interferon signaling’, ‘autoimmune/immune activation’, ‘visual transduction’, and ‘protein catabolism’ were significantly associated with all lipid traits. Additionally, we detected trait-specific subnetworks, including cadherin-associated subnetworks for LDL, glutathione metabolism for HDL, valine, leucine and isoleucine biosynthesis for TC, and insulin signaling and complement pathways for TG. Finally, utilizing gene-gene relations revealed by tissue-specific gene regulatory networks, we detected both known (e.g. APOH, APOA4, and ABCA1) and novel (e.g. F2 in adipose tissue) key regulator genes in these lipid-associated subnetworks. Knockdown of the F2 gene (Coagulation Factor II, Thrombin) in 3T3-L1 and C3H10T1/2 adipocytes reduced gene expression of Abcb11, Apoa5, Apof, Fabp1, Lipc, and Cd36, reduced intracellular adipocyte lipid content, and increased extracellular lipid content, supporting a link between adipose thrombin and lipid regulation. Our results shed light on the complex mechanisms underlying lipid metabolism and highlight potential novel targets for lipid regulation and lipid-associated diseases.

Deficiency of glucocerebrosidase (GBA), a lysosomal β-glucosidase, causes Gaucher disease. The enzyme hydrolyzes β-glucosidic substrates and transglucosylates cholesterol to cholesterol-β-glucoside. Here we show that recombinant human GBA also cleaves β-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as acceptor for subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced β-glucosidase activity were similarly impaired in β-xylosidase, transglucosidase and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from Gaucher disease patients. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous β-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing β-glucosidase GBA2. We later sought an endogenous β-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyse formation of XylCer. Thus, food-derived β-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids.

Microtubules are polymers composed of αβ-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human iPS cell-derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in C. reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry-mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms (CRPs) in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that CRPs mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function. 

Angiopoietin-like protein (ANGPTL)3 regulates plasma lipids by inhibiting LPL and endothelial lipase (EL). ANGPTL3 inactivation lowers LDL-C independently of the classical LDLR-mediated pathway and represents a promising therapeutic approach for individuals with homozygous familial hypercholesterolemia due to LDLR mutations. Yet, how ANGPTL3 regulates LDL-C levels is unknown. Here, we demonstrate in hyperlipidemic humans and mice that ANGPTL3 controls VLDL catabolism upstream of LDL. Using kinetic, lipidomic, and biophysical studies, we show that ANGPTL3 inhibition reduces VLDL-lipid content and size, generating remnant particles that are efficiently removed from the circulation. This suggests that ANGPTL3 inhibition lowers LDL-C by limiting LDL particle production. Mechanistically, we discovered that EL is a key mediator of ANGPTL3’s novel pathway. Our experiments revealed that, although dispensable in the presence of LDLR, EL-mediated processing of VLDL becomes critical for LDLR-independent particle clearance. In the absence of EL and LDLR, ANGPTL3 inhibition perturbed VLDL catabolism, promoted accumulation of atypical remnants, and failed to reduce LDL-C. Taken together, we uncover ANGPTL3 at the helm of a novel EL-dependent pathway that lowers LDL-C in the absence of LDLR.

Lipoprotein (a) [Lp(a)] is a risk factor for CVD and a target of therapy, but Lp(a) measurements are not globally standardized. Commercially available assays generally use polyclonal antibodies that detect multiple sites within the kringle (K)IV₂ repeat region of Lp(a) and may lead to inaccurate assessments of plasma levels. With increasing awareness of Lp(a) as a cardiovascular risk factor and the active clinical development of new potential therapeutic approaches, the broad availability of reagents capable of providing isoform independence of Lp(a) measurements is paramount. To address this issue, we generated a murine monoclonal antibody that binds to only one site on apo(a). A BALB/C mouse was immunized with a truncated version of apo(a) that contained eight total KIV repeats, including only one copy of KIV₂. We generated hybridomas, screened them, and successfully produced a KIV₂-independent monoclonal antibody, named LPA-KIV9. Using a variety of truncated apo(a) constructs to map its binding site, we found that LPA-KIV9 binds to KIV₉ without binding to plasminogen. Fine peptide mapping revealed that LPA-KIV9 bound to the sequence ⁴⁰⁷⁶LETPTVV⁴⁰⁸² on KIV₉. In conclusion, the generation of monoclonal antibody LPA-KIV9 may be a useful reagent in basic research studies and in the clinical application of Lp(a) measurements.

TG-rich lipoprotein (TRL)-related biomarkers, including TRL-cholesterol (TRL-C), remnant-like lipoprotein particle-cholesterol (RLP-C), and apoC-III have been associated with atherosclerosis. However, their prognostic values have not been fully determined, especially in patients with previous CAD. This study aimed to examine the associations of TRL-C, RLP-C, and apoC-III with incident cardiovascular events (CVEs) in the setting of secondary prevention of CAD. Plasma TRL-C, RLP-C, and total apoC-III were directly measured. A total of 4,355 participants with angiographically confirmed CAD were followed up for the occurrence of CVEs. During a median follow-up period of 5.1 years (interquartile range: 3.9–6.4 years), 543 (12.5%) events occurred. Patients with incident CVEs had significantly higher levels of TRL-C, RLP-C, and apoC-III than those without events. Multivariable Cox analysis indicated that a log unit increase in TRL-C, RLP-C, and apoC-III increased the risk of CVEs by 49% (95% CI: 1.16–1.93), 21% (95% CI: 1.09–1.35), and 40% (95% CI: 1.11–1.77), respectively. High TRL-C, RLP-C, and apoC-III were also independent predictors of CVEs in individuals with LDL-C levels ≤1.8 mmol/l (n = 1,068). The addition of RLP-C level to a prediction model resulted in a significant increase in discrimination, and all three TRL biomarkers improved risk reclassification. Thus, TRL-C, RLP-C, and apoC-III levels were independently associated with incident CVEs in Chinese CAD patients undergoing statin therapy.

For three decades, the LPL–specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the half-life, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than long-chain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions.

Sphingolipids have become established participants in the pathogenesis of obesity and its associated maladies. Sphingosine kinase 1 (SPHK1), which generates S1P, has been shown to increase in liver and adipose of obese humans and mice and to regulate inflammation in hepatocytes and adipose tissue, insulin resistance, and systemic inflammation in mouse models of obesity. Previous studies by us and others have demonstrated that global sphingosine kinase 1 KO mice are protected from diet-induced obesity, insulin resistance, systemic inflammation, and NAFLD, suggesting that SPHK1 may mediate pathological outcomes of obesity. As adipose tissue dysfunction has gained recognition as a central instigator of obesity-induced metabolic disease, we hypothesized that SPHK1 intrinsic to adipocytes may contribute to HFD-induced metabolic pathology. To test this, we depleted Sphk1 from adipocytes in mice (SK1^fatKO) and placed them on a HFD. In contrast to our initial hypothesis, SK1^fatKO mice displayed greater weight gain on HFD and exacerbated impairment in glucose clearance. Pro-inflammatory cytokines and neutrophil content of adipose tissue were similar, as were levels of circulating leptin and adiponectin. However, SPHK1-null adipocytes were hypertrophied and had lower basal lipolytic activity. Interestingly, hepatocyte triacylglycerol accumulation and expression of pro-inflammatory cytokines and collagen 1a1 were exacerbated in SK1^fatKO mice on a HFD, implicating a specific role for adipocyte SPHK1 in adipocyte function and inter-organ cross-talk that maintains overall metabolic homeostasis in obesity. Thus, SPHK1 serves a previously unidentified essential homeostatic role in adipocytes that protects from obesity-associated pathology. These findings may have implications for pharmacological targeting of the SPHK1/S1P signaling axis.

Lipoprotein (a) [Lp(a)] is a well-known risk factor for cardiovascular disease, but analysis on Lp(a) and renal dysfunction is scarce. We aimed to investigate prospectively the association of serum Lp(a) with the risk of reduced renal function, and further investigated whether diabetic or hypertensive status modified such association. Six thousand two hundred and fifty-seven Chinese adults aged ≤40 years and free of reduced renal function at baseline were included in the study. Reduced renal function was defined as estimated glomerular filtration rate <60 ml/min/1.73 m². During a mean follow-up of 4.4 years, 158 participants developed reduced renal function. Each one-unit increase in log₁₀-Lp(a) (milligrams per deciliter) was associated with a 1.99-fold (95% CI 1.15–3.43) increased risk of incident reduced renal function; the multivariable-adjusted odds ratio (OR) for the highest tertile of Lp(a) was 1.61 (95% CI 1.03–2.52) compared with the lowest tertile (P for trend = 0.03). The stratified analysis showed the association of serum Lp(a) and incident reduced renal function was more prominent in participants with prevalent diabetes [OR 4.04, 95% CI (1.42–11.54)] or hypertension [OR 2.18, 95% CI (1.22–3.89)]. A stronger association was observed in the group with diabetes and high Lp(a) (>25 mg/dl), indicating a combined effect of diabetes and high Lp(a) on the reduced renal function risk. An elevated Lp(a) level was independently associated with risk of incident reduced renal function, especially in diabetic or hypertensive patients.

Cognitive decline with age is a harmful process that can reduce quality of life. Multiple factors have been established to contribute to cognitive decline, but the overall etiology remains unknown. Here, we hypothesized that cognitive dysfunction is mediated, in part, by increased levels of inflammatory cytokines that alter allopregnanolone (AlloP) levels, an important neurosteroid in the brain. We assessed the levels and regulation of AlloP and the effects of AlloP supplementation on cognitive function in 4-month-old and 24-month-old male C57BL/6 mice. With age, the expression of enzymes involved in the AlloP synthetic pathway was decreased and corticosterone (CORT) synthesis increased. Supplementation of AlloP improved cognitive function. Interestingly, interleukin 6 (IL-6) infusion in young animals significantly reduced the production of AlloP compared with controls. It is notable that inhibition of IL-6 with its natural inhibitor, soluble membrane glycoprotein 130, significantly improved spatial memory in aged mice. These findings were supported by in vitro experiments in primary murine astrocyte cultures, indicating that IL-6 decreases production of AlloP and increases CORT levels. Our results indicate that age-related increases in IL-6 levels reduce progesterone substrate availability, resulting in a decline in AlloP levels and an increase in CORT. Furthermore, our results indicate that AlloP is a critical link between inflammatory cytokines and the age-related decline in cognitive function.

Atherosclerosis is characterized by the pathological accumulation of cholesterol-laden macrophages in the arterial wall. Atherosclerosis is also the main underlying cause of CVDs, and its development is largely driven by elevated plasma cholesterol. Strong epidemiological data find an inverse association between plasma β-carotene with atherosclerosis, and we recently showed that β-carotene oxygenase 1 (BCO1) activity, responsible for β-carotene cleavage to vitamin A, is associated with reduced plasma cholesterol in humans and mice. In this study, we explore whether intact β-carotene or vitamin A affects atherosclerosis progression in the atheroprone LDLR-deficient mice. Compared with control-fed Ldlr^–/– mice, β-carotene-supplemented mice showed reduced atherosclerotic lesion size at the level of the aortic root and reduced plasma cholesterol levels. These changes were absent in Ldlr^–/–/Bco1^–/– mice despite accumulating β-carotene in plasma and atherosclerotic lesions. We discarded the implication of myeloid BCO1 in the development of atherosclerosis by performing bone marrow transplant experiments. Lipid production assays found that retinoic acid, the active form of vitamin A, reduced the secretion of newly synthetized triglyceride and cholesteryl ester in cell culture and mice. Overall, our findings provide insights into the role of BCO1 activity and vitamin A in atherosclerosis progression through the regulation of hepatic lipid metabolism.

N-acylethanolamines (NAEs) are endogenous lipid-signaling molecules derived from fatty acids that regulate numerous biological functions, including in the brain. Interestingly, NAEs are elevated in the absence of fatty acid amide hydrolase (FAAH) and following CO₂-induced ischemia/hypercapnia, suggesting a neuroprotective response. Tetracosahexaenoic acid (THA) is a product and precursor to DHA; however, the NAE product, tetracosahexaenoylethanolamide (THEA), has never been reported. Presently, THEA was chemically synthesized as an authentic standard to confirm THEA presence in biological tissues. Whole brains were collected and analyzed for unesterified THA, total THA, and THEA in wild-type and FAAH-KO mice that were euthanized by either head-focused microwave fixation, CO₂ + microwave, or CO₂ only. PPAR activity by transient transfection assay and ex vivo neuronal output in medium spiny neurons (MSNs) of the nucleus accumbens by patch clamp electrophysiology were determined following THEA exposure. THEA in the wild-type mice was nearly doubled (P < 0.05) following ischemia/hypercapnia (CO₂ euthanization) and up to 12 times higher (P < 0.001) in the FAAH-KO compared with wild-type. THEA did not increase (P > 0.05) transcriptional activity of PPARs relative to control, but 100 nM of THEA increased (P < 0.001) neuronal output in MSNs of the nucleus accumbens. Here were identify a novel NAE, THEA, in the brain that is elevated upon ischemia/hypercapnia and by KO of the FAAH enzyme. While THEA did not activate PPAR, it augmented the excitability of MSNs in the nucleus accumbens. Overall, our results suggest that THEA is a novel NAE that is produced in the brain upon ischemia/hypercapnia and regulates neuronal excitation.

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

Bile acids, which are synthesized from cholesterol by the liver, are chemically transformed along the intestinal tract by the gut microbiota, and the products of these transformations signal through host receptors, affecting overall host health. These transformations include bile acid deconjugation, oxidation, and 7α-dehydroxylation. An understanding of the biogeography of bile acid transformations in the gut is critical because deconjugation is a prerequisite for 7α-dehydroxylation and because most gut microorganisms harbor bile acid transformation capacity. Here, we used a coupled metabolomic and metaproteomic approach to probe in vivo activity of the gut microbial community in a gnotobiotic mouse model. Results revealed the involvement of Clostridium scindens in 7α-dehydroxylation, of the genera Muribaculum and Bacteroides in deconjugation, and of six additional organisms in oxidation (the genera Clostridium, Muribaculum, Bacteroides, Bifidobacterium, Acutalibacter, and Akkermansia). Furthermore, the bile acid profile in mice with a more complex microbiota, a dysbiosed microbiota, or no microbiota was considered. For instance, conventional mice harbor a large diversity of bile acids, but treatment with an antibiotic such as clindamycin results in the complete inhibition of 7α-dehydroxylation, underscoring the strong inhibition of organisms that are capable of carrying out this process by this compound. Finally, a comparison of the hepatic bile acid pool size as a function of microbiota revealed that a reduced microbiota affects host signaling but not necessarily bile acid synthesis. In this study, bile acid transformations were mapped to the associated active microorganisms, offering a systematic characterization of the relationship between microbiota and bile acid composition.

Among the virulence factors in Neisseria infections, a major inducer of inflammatory cytokines is the lipooligosaccharide (LOS). The activation of NF-B via extracellular binding of LOS or lipopolysaccharide (LPS) to the toll-like receptor 4 and its coreceptor, MD-2, results in production of pro-inflammatory cytokines that initiate adaptive immune responses. LOS can also be absorbed by cells and activate intracellular inflammasomes, causing the release of inflammatory cytokines and pyroptosis. Studies of LOS and LPS have shown that their inflammatory potential is highly dependent on lipid A phosphorylation and acylation, but little is known on the location and pattern of these posttranslational modifications. Herein, we report on the localization of phosphoryl groups on phosphorylated meningococcal lipid A, which has two to three phosphate and zero to two phosphoethanolamine substituents. Intact LOS with symmetrical hexa-acylated and asymmetrical penta-acylated lipid A moieties was subjected to high-resolution ion mobility spectrometry MALDI-TOF MS. LOS molecular ions readily underwent in-source decay to give fragments of the oligosaccharide and lipid A formed by cleavage of the ketosidic linkage, which enabled performing MS/MS (pseudo-MS³). The resulting spectra revealed several patterns of phosphoryl substitution on lipid A, with certain species predominating. The extent of phosphoryl substitution, particularly phosphoethanolaminylation, on the 4'-hydroxyl was greater than that on the 1-hydroxyl. The heretofore unrecognized phosphorylation patterns of lipid A of meningococcal LOS that we detected are likely determinants of both pathogenicity and the ability of the bacteria to evade the innate immune system.

Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, results generated by independent groups are rarely comparable, which increases the need for the implementation of internationally agreed upon protocols. We performed an interlaboratory comparison for the MS-based quantitative analysis of total oxylipins. Five independent laboratories assessed the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e., seven quality control plasmas), standard calibration series, and analytical methods. The quantitative analysis was based on a standard calibration series with isotopically labeled internal standards. Using the standardized protocol, the technical variance was within ±15% for 73% of oxylipins; however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of concentrations determined by the laboratories was examined using consensus value estimates and unsupervised/supervised multivariate analysis (i.e., principal component analysis and partial least squares discriminant analysis). Interlaboratory variability was limited and did not interfere with our ability to distinguish the different plasmas. Moreover, all laboratories were able to identify similar differences between plasmas. In summary, we show that by using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all oxylipin extraction and analysis steps led to reliable, reproducible, and comparable oxylipin concentrations in independent laboratories, allowing the generation of biologically meaningful oxylipin patterns.

Fabry disease is caused by deficient activity of α-galactosidase A, an enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins, and subsequent accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb₃), globotriaosylsphingosine (lyso-Gb₃), and galabiosylceramide. However, there is no known link between these compounds and disease severity. In this study, we compared Gb₃ isoforms (various fatty acids) and lyso-Gb₃ analogs (various sphingosine modifications) in two strains of Fabry disease mouse models: a pure C57BL/6 (B6) background or a B6/129 mixed background, with the latter exhibiting more prominent cardiac and renal hypertrophy and thermosensation deficits. Total Gb₃ and lyso-Gb₃ levels in the heart, kidney, and dorsal root ganglion (DRG) were similar in the two strains. However, levels of the C20-fatty acid isoform of Gb₃ and particular lyso-Gb₃ analogs (+18, +34) were significantly higher in Fabry-B6/129 heart tissue when compared with Fabry-B6. By contrast, there was no difference in Gb₃ and lyso-Gb₃ isoforms/analogs in the kidneys and DRG between the two strains. Furthermore, using immunohistochemistry, we found that Gb₃ massively accumulated in DRG mechanoreceptors, a sensory neuron subpopulation with preserved function in Fabry disease. However, Gb₃ accumulation was not observed in nonpeptidergic nociceptors, the disease-relevant subpopulation that has remarkably increased isolectin-B4 (the marker of nonpeptidergic nociceptors) binding and enlarged cell size. These findings suggest that specific species of Gb₃ or lyso-Gb₃ may play major roles in the pathogenesis of Fabry disease, and that Gb₃ and lyso-Gb₃ are not responsible for the pathology in all tissues or cell types.

Human genetic studies recently identified an association of SNPs in the 17-β hydroxysteroid dehydrogenase 13 (HSD17B13) gene with alcoholic and nonalcoholic fatty liver disease development. Mutant HSD17B13 variants devoid of enzymatic function have been demonstrated to be protective from cirrhosis and liver cancer, supporting the development of HSD17B13 as a promising therapeutic target. Previous studies have demonstrated that HSD17B13 is a lipid droplet (LD)-associated protein. However, the critical domains that drive LD targeting or determine the enzymatic activity have yet to be defined. Here we used mutagenesis to generate multiple truncated and point-mutated proteins and were able to demonstrate in vitro that the N-terminal hydrophobic domain, PAT-like domain, and a putative α-helix/β-sheet/α-helix domain in HSD17B13 are all critical for LD targeting. Similarly, we characterized the predicted catalytic, substrate-binding, and homodimer interaction sites and found them to be essential for the enzymatic activity of HSD17B13, in addition to our previous identification of amino acid P260 and cofactor binding site. In conclusion, we identified critical domains and amino acid sites that are essential for the LD localization and protein function of HSD17B13, which may facilitate understanding of its function and targeting of this protein to treat chronic liver diseases.

Nonsmall cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. While mutations in Kras and overexpression of Myc are commonly found in patients, the role of altered lipid metabolism in lung cancer and its interplay with oncogenic Myc is poorly understood. Here we use a transgenic mouse model of Kras-driven lung adenocarcinoma with reversible activation of Myc combined with surface analysis lipid profiling of lung tumors and transcriptomics to study the effect of Myc activity on cholesterol homeostasis. Our findings reveal that the activation of Myc leads to the accumulation of cholesteryl esters (CEs) stored in lipid droplets. Subsequent Myc deactivation leads to further increases in CEs, in contrast to tumors in which Myc was never activated. Gene expression analysis linked cholesterol transport and storage pathways to Myc activity. Our results suggest that increased Myc activity is associated with increased cholesterol influx, reduced efflux, and accumulation of CE-rich lipid droplets in lung tumors. Targeting cholesterol homeostasis is proposed as a promising avenue to explore for novel treatments of lung cancer, with diagnostic and stratification potential in human NSCLC.

Adaptive thermogenesis is highly dependent on uncoupling protein 1 (UCP1), a protein expressed by thermogenic adipocytes present in brown adipose tissue (BAT) and white adipose tissue (WAT). Thermogenic capacity of human and mouse BAT can be measured by positron emission tomography-computed tomography quantifying the uptake of ¹⁸F-fluodeoxyglucose or lipid tracers. BAT activation is typically studied in response to cold exposure or treatment with β-3-adrenergic receptor agonists such as CL316,243 (CL). Currently, it is unknown whether cold-stimulated uptake of glucose or lipid tracers is a good surrogate marker of UCP1-mediated thermogenesis. In metabolic studies using radiolabeled tracers, we found that glucose uptake is increased in mildly cold-activated BAT of Ucp1^–/– versus WT mice kept at subthermoneutral temperature. Conversely, lower glucose disposal was detected after full thermogenic activation achieved by sustained cold exposure or CL treatment. In contrast, uptake of lipoprotein-derived fatty acids into chronically activated thermogenic adipose tissues was substantially increased in UCP1-deficient mice. This effect is linked to higher sympathetic tone in adipose tissues of Ucp1^–/– mice, as indicated by elevated levels of thermogenic genes in BAT and WAT. Thus, glucose and lipoprotein handling does not necessarily reflect UCP1-dependent thermogenic activity, but especially lipid uptake rather mirrors sympathetic activation of adipose tissues.

Lipopolysaccharide (LPS) is a key player for innate immunity activation. It is therefore a prime target for sepsis treatment, as antibiotics are not sufficient to improve outcome during septic shock. An extracorporeal removal method by polymyxin (PMX) B direct hemoperfusion (PMX-DHP) is used in Japan, but recent trials failed to show a significant lowering of circulating LPS levels after PMX-DHP therapy. PMX-DHP has a direct effect on LPS molecules. However, LPS is not present in a free form in the circulation, as it is mainly carried by lipoproteins, including LDLs. Lipoproteins are critical for physiological LPS clearance, as LPSs are carried by LDLs to the liver for elimination. We hypothesized that LDL apheresis could be an alternate method for LPS removal. First, we demonstrated in vitro that LDL apheresis microbeads are almost as efficient as PMX beads to reduce LPS concentration in LPS-spiked human plasma, whereas it is not active in PBS. We found that PMX was also adsorbing lipoproteins, although less specifically. Then, we found that endogenous LPS of patients treated by LDL apheresis for familial hypercholesterolemia is also removed during their LDL apheresis sessions, with both electrostatic-based devices and filtration devices. Finally, LPS circulating in the plasma of septic shock and severe sepsis patients with gram-negative bacteremia was also removed in vitro by LDL adsorption. Overall, these results underline the importance of lipoproteins for LPS clearance, making them a prime target to study and treat endotoxemia-related conditions.

Of the known regulators of atherosclerosis, miRNAs have been demonstrated to play critical roles in lipoprotein homeostasis and plaque formation. Here, we generated a novel animal model of atherosclerosis by knocking in LDLR^W483X in C57BL/6 mice, as the W483X mutation in LDLR is considered the most common newly identified pathogenic mutation in Chinese familial hypercholesterolemia (FH) individuals. Using the new in vivo mouse model combined with a well-established atherosclerotic in vitro human cell model, we identified a novel atherosclerosis-related miRNA, miR-23a-3p, by microarray analysis of mouse aortic tissue specimens and human aortic endothelial cells (HAECs). miR-23a-3p was consistently downregulated in both models, which was confirmed by qPCR. Bioinformatics analysis and further validation experiments revealed that the TNFα-induced protein 3 (TNFAIP3) gene was the key target of miR-23a-3p. The miR-23a-3p-related functional pathways were then analyzed in HAECs. Collectively, the present results suggest that miR-23a-3p regulates inflammatory and apoptotic pathways in atherogenesis by targeting TNFAIP3 through the NF-B and p38/MAPK signaling pathways.

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.

Spatial changes of FAs in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids (PLs), EPA-containing TGs, DHA-containing PLs, or DHA-containing TGs. Qualitative changes in FA composition of plasma, erythrocytes, and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of PC and PE species in retina were determined by LC coupled to MS/MS and MALDI coupled to MS imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that PLs as well as TGs are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain PUFAs (more than 28 carbons) following PL-EPA and TG-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of FAs in the rat retina.

Lipins are eukaryotic proteins with functions in lipid synthesis and the homeostatic control of energy balance. They execute these functions by acting as phosphatidate phosphatase enzymes in the cytoplasm and by changing gene expression after translocation into the cell nucleus, in particular under fasting conditions. Here, we asked whether nuclear translocation and the enzymatic activity of Drosophila Lipin serve essential functions and how gene expression changes, under both fed and fasting conditions, when nuclear translocation is impaired. To address these questions, we created a Lipin null mutant, a mutant expressing Lipin lacking a nuclear localization signal (Lipin^NLS), and a mutant expressing enzymatically dead Lipin. Our data support the conclusion that the enzymatic but not nuclear gene regulatory activity of Lipin is essential for survival. Notably, adult Lipin^NLS flies were not only viable but also exhibited improved life expectancy. In contrast, they were highly susceptible to starvation. Both the improved life expectancy in the fed state and the decreased survival in the fasting state correlated with changes in metabolic gene expression. Moreover, increased life expectancy of fed flies was associated with a decreased metabolic rate. Interestingly, in addition to metabolic genes, genes involved in feeding behavior and the immune response were misregulated in Lipin^NLS flies. Altogether, our data suggest that the nuclear activity of Lipin influences the genomic response to nutrient availability with effects on life expectancy and starvation resistance. Thus, nutritional or therapeutic approaches that aim at lowering nuclear translocation of lipins in humans may be worth exploring.

Phospholipids, including ether phospholipids, are composed of numerous isomeric and isobaric species that have the same backbone and acyl chains. This structural resemblance results in similar fragmentation patterns by collision-induced dissociation of phospholipids regardless of class, yielding complicated MS/MS spectra when isobaric species are analyzed together. Furthermore, the presence of isobaric species can lead to misassignment of species when made solely based on their molecular weights. In this study, we used normal-phase HPLC for ESI-MS/MS analysis of phospholipids from bovine heart mitochondria. Class separation by HPLC eliminates chances for misidentification of isobaric species from different classes of phospholipids. Chromatography yields simple MS/MS spectra without interference from isobaric species, allowing clear identification of peaks corresponding to fragmented ions containing monoacylglycerol backbone derived from losing one acyl chain. Using these fragmented ions, we characterized individual and isomeric species in each class of mitochondrial phospholipids, including unusual species, such as PS, containing an ether linkage and species containing odd-numbered acyl chains in cardiolipin, PS, PI, and PG. We also characterized monolysocardiolipin and dilysocardiolipin, the least abundant but nevertheless important mitochondrial phospholipids. The results clearly show the power of HPLC-MS/MS for identification and characterization of phospholipids, including minor species.

The dysregulation of myeloid-derived cell metabolism can drive atherosclerosis. AMP-activated protein kinase (AMPK) controls various aspects of macrophage dynamics and lipid homeostasis, which are important during atherogenesis. Using LysM-Cre to drive the deletion of both the α1 and α2 catalytic subunits (MacKO), we aimed to clarify the role of myeloid-specific AMPK signaling in male and female mice made acutely atherosclerotic by injection of AAV vector encoding a gain-of-function mutant PCSK9 (PCSK9-AAV) and WD feeding. After 6 weeks of WD feeding, mice received a daily injection of either the AMPK activator A-769662 or a vehicle control for an additional 6 weeks. Following this (12 weeks total), we assessed myeloid cell populations and differences between genotype or sex were not observed. Similarly, aortic sinus plaque size, lipid staining, and necrotic area did not differ in male and female MacKO mice compared with their littermate floxed controls. Moreover, therapeutic intervention with A-769662 showed no treatment effect. There were also no observable differences in the amount of circulating total cholesterol or triglyceride, and only minor differences in the levels of inflammatory cytokines between groups. Finally, CD68+ area and markers of autophagy showed no effect of either lacking AMPK signaling or AMPK activation. Our data suggest that while defined roles for each catalytic AMPK subunit have been identified, complete deletion of myeloid AMPK signaling does not significantly impact atherosclerosis. Additionally, these findings suggest that intervention with the first-generation AMPK activator A-769662 is not able to stem the progression of atherosclerosis.

Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apo(a). The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analog, -aminocaproic acid. Interestingly, KIV-2 presents a lysine binding site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to be negligible in surface plasmon resonance experiments; and with the LBS being nonfunctional, we propose to rename it "pseudo-LBS". Further investigation of the protein by computational small-molecule docking allowed us to identify a possible heparin-binding site away from the LBS, which was confirmed by specific reverse charge mutations abolishing heparin binding. This study opens new possibilities to define the pathogenesis of Lp(a)-related diseases and to facilitate the design of specific therapeutic drugs.

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.

Xanthophyllomyces dendrorhous is a basidiomycete yeast that produces carotenoids, mainly astaxanthin. Astaxanthin is an organic pigment of commercial interest due to its antioxidant and coloring properties. X. dendrorhous has a functional SREBP pathway, and the Sre1 protein is the SREBP homolog in this yeast. However, how sterol regulatory element (Sre)1 promotes the biosynthesis of sterols and carotenoids in X. dendrorhous is unknown. In this work, comparative RNA-sequencing analysis between modified X. dendrorhous strains that have an active Sre1 protein and the WT was performed to identify Sre1-dependent genes. In addition, Sre1 direct target genes were identified through ChIP combined with lambda exonuclease digestion (ChIP-exo) assays. SRE motifs were detected in the promoter regions of several Sre1 direct target genes and were consistent with the SREs described in other yeast species. Sre1 directly regulates genes related to ergosterol biosynthesis as well as genes related to the mevalonate (MVA) pathway, which synthesizes the building blocks of isoprenoids, including carotenoids. Two carotenogenic genes, crtE and crtR, were also identified as Sre1 direct target genes. Thus, carotenogenesis in X. dendrorhous is regulated by Sre1 through the regulation of the MVA pathway and the regulation of the crtE and crtR genes. As the crtR gene encodes a cytochrome P450 reductase, Sre1 regulates pathways that include cytochrome P450 enzymes, such as the biosynthesis of carotenoids and sterols. These results demonstrate that Sre1 is a sterol master regulator that is conserved in X. dendrorhous.

Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue-destructive processes in human periodontitis. Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require the engagement of Toll-like receptor 2 (TLR2). Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2, but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6. The novel lipid class, termed Lipid 1256, consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide previously termed Lipid 654. Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes. Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms. Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans.