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.

Bile acids (BAs) have been established as ubiquitous regulatory molecules implicated in a large variety of healthy and pathological processes. However, the scope of BA heterogeneity is often underrepresented in current literature. This is due in part to inadequate detection methods, which fail to distinguish the individual constituents of the BA pool. Thus, the primary aim of this study was to develop a method that would allow the simultaneous analysis of specific C24 BA species, and to apply that method to biological systems of interest. Herein, we describe the generation and validation of an LC-MS/MS assay for quantification of numerous BAs in a variety of cell systems and relevant biofluids and tissue. These studies included the first baseline level assessment for planar BAs, including allocholic acid, in cell lines, biofluids, and tissue in a nonhuman primate (NHP) laboratory animal, Macaca mulatta, in healthy conditions. These results indicate that immortalized cell lines make poor models for the study of BA synthesis and metabolism, whereas human primary hepatocytes represent a promising alternative model system. We also characterized the BA pool of M. mulatta in detail. Our results support the use of NHP models for the study of BA metabolism and pathology in lieu of murine models. Moreover, the method developed here can be applied to the study of common and planar C24 BA species in other systems.

The analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for bottom-up lipid raftomics based on suspension-trapping (S-Trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection, and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in flotillin-1, LAT, and cholesterol were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2,680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from nonstimulated versus anti-CD3/CD28 T cell receptor (TCR)-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an S-Trap-based approach for sample preparation increases the specificity and sensitivity of lipid raftomics.

Accompanied with nutrition transition, non-HDL-C levels of individuals in Asian countries has increased rapidly, which has caused the global epicenter of nonoptimal cholesterol to shift from Western countries to Asian countries. Thus, it is critical to underline major genetic and dietary determinants. In the current study of 2,330 Chinese individuals, genetic risk scores (GRSs) were calculated for total cholesterol (TC; GRS_TC, 57 SNPs), LDL-C (GRS_LDL-C, 45 SNPs), and HDL-C (GRS_HDL-C, 65 SNPs) based on SNPs from the Global Lipid Genetics Consortium study. Cholesterol intake was estimated by a 74-item food-frequency questionnaire. Associations of dietary cholesterol intake with plasma TC and LDL-C strengthened across quartiles of the GRS_TC (effect sizes: –0.29, 0.34, 2.45, and 6.47; P_interaction = 0.002) and GRS_LDL-C (effect sizes: –1.35, 0.17, 5.45, and 6.07; P_interaction = 0.001), respectively. Similar interactions with non-HDL-C were observed between dietary cholesterol and GRS_TC (P_interaction = 0.001) and GRS_LDL-C (P_interaction = 0.004). The adverse effects of GRS_TC on TC (effect sizes across dietary cholesterol quartiles: 0.51, 0.82, 1.21, and 1.31; P_interaction = 0.023) and GRS_LDL-C on LDL-C (effect sizes across dietary cholesterol quartiles: 0.66, 0.52, 1.12, and 1.56; P_interaction = 0.020) were more profound in those having higher cholesterol intake compared with those with lower intake. Our findings suggest significant interactions between genetic susceptibility and dietary cholesterol intake on plasma cholesterol profiles in a Chinese population.

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.

Some cases of chylomicronemia are caused by autoantibodies against glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1), an endothelial cell protein that shuttles LPL to the capillary lumen. GPIHBP1 autoantibodies prevent binding and transport of LPL by GPIHBP1, thereby disrupting the lipolytic processing of triglyceride-rich lipoproteins. Here, we review the "GPIHBP1 autoantibody syndrome" and summarize clinical and laboratory findings in 22 patients. All patients had GPIHBP1 autoantibodies and chylomicronemia, but we did not find a correlation between triglyceride levels and autoantibody levels. Many of the patients had a history of pancreatitis, and most had clinical and/or serological evidence of autoimmune disease. IgA autoantibodies were present in all patients, and IgG4 autoantibodies were present in 19 of 22 patients. Patients with GPIHBP1 autoantibodies had low plasma LPL levels, consistent with impaired delivery of LPL into capillaries. Plasma levels of GPIHBP1, measured with a monoclonal antibody–based ELISA, were very low in 17 patients, reflecting the inability of the ELISA to detect GPIHBP1 in the presence of autoantibodies (immunoassay interference). However, GPIHBP1 levels were very high in five patients, indicating little capacity of their autoantibodies to interfere with the ELISA. Recently, several GPIHBP1 autoantibody syndrome patients were treated successfully with rituximab, resulting in the disappearance of GPIHBP1 autoantibodies and normalization of both plasma triglyceride and LPL levels. The GPIHBP1 autoantibody syndrome should be considered in any patient with newly acquired and unexplained chylomicronemia.

Familial LCAT deficiency (FLD) is a rare genetic disorder of HDL metabolism, caused by loss-of-function mutations in the LCAT gene and characterized by a variety of symptoms including corneal opacities and kidney failure. Renal disease represents the leading cause of morbidity and mortality in FLD cases. However, the prognosis is not known and the rate of deterioration of kidney function is variable and unpredictable from patient to patient. In this article, we present data from a follow-up of the large Italian cohort of FLD patients, who have been followed for an average of 12 years. We show that renal failure occurs at the median age of 46 years, with a median time to a second recurrence of 10 years. Additionally, we identify high plasma unesterified cholesterol level as a predicting factor for rapid deterioration of kidney function. In conclusion, this study highlights the severe consequences of FLD, underlines the need of correct early diagnosis and referral of patients to specialized centers, and highlights the urgency for effective treatments to prevent or slow renal disease in patients with LCAT deficiency.

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.

NAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin’s anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

ABCB4/MDR3 is located in the canalicular membrane of hepatocytes and translocates PC-lipids from the cytoplasmic to the extracellular leaflet. ABCB4 is an ATP-dependent transporter that reduces the harsh detergent effect of the bile salts by counteracting self-digestion. To do so, ABCB4 provides PC lipids for extraction into bile. PC lipids account for 40% of the entire pool of lipids in the canalicular membrane with an unknown distribution over both leaflets. Extracted PC lipids end up in so-called mixed micelles. Mixed micelles are composed of phospholipids, bile salts, and cholesterol. Ninety to ninety-five percent of the phospholipids are members of the PC family, but only a subset of mainly 16.0-18:1 PC and 16:0-18:2 PC variants are present. To elucidate whether ABCB4 is the key discriminator in this enrichment of specific PC lipids, we used in vitro studies to identify crucial determinants in substrate selection. We demonstrate that PC-lipid moieties alone are insufficient for stimulating ABCB4 ATPase activity, and that at least two acyl chains and the backbone itself are required for a productive interaction. The nature of the fatty acids, like length or saturation has a quantitative impact on the ATPase activity. Our data demonstrate a two-step enrichment and protective function of ABCB4 to mitigate the harsh detergent effect of the bile salts, because ABCB4 can translocate more than just the PC-lipid variants found in bile.

Beiging of white adipose tissue (WAT) has beneficial effects on metabolism. Although it is known that beige adipocytes are active in lipid catabolism and thermogenesis, how they are regulated deserves more explorations. In this study, we demonstrate that stearoyl-CoA desaturase 1 (SCD1) in subcutaneous WAT (scWAT) responded to cold stimulation and was able to promote mobilization of triacylglycerol [TAG (triglyceride)]. In vitro studies showed that SCD1 promoted lipolysis in C3H10T1/2 white adipocytes. The lipolytic effect was contributed by one of SCD1’s products, oleic acid (OA). OA upregulated adipose TAG lipase and hormone-sensitive lipase expression. When SCD1 was overexpressed in the scWAT of mice, lipolysis was enhanced, and oxygen consumption and heat generation were increased. These effects were also demonstrated by the SCD1 knockdown experiments in mice. In conclusion, our study suggests that SCD1, known as an enzyme for lipid synthesis, plays a role in upregulating lipid mobilization through its desaturation product, OA.

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^–/– mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp^–/–) mice and age-weight matched wild-type control mice. Young (10–12-week-old) L-Mttp^–/– mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp^–/– mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKC activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp^–/– mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKC activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp^–/– mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKC activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp^–/– mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp^–/– mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp^–/– mice.

Phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and regulates the synthesis of membrane phospholipids. There is much interest in this enzyme because it controls the cellular levels of its substrate, phosphatidate (PA), and product, DAG; defects in the metabolism of these lipid intermediates are the basis for lipid-based diseases such as obesity, lipodystrophy, and inflammation. The measurement of PAP activity is required for studies aimed at understanding its mechanisms of action, how it is regulated, and for screening its activators and/or inhibitors. Enzyme activity is determined through the use of radioactive and nonradioactive assays that measure the product, DAG, or P_i. However, sensitivity and ease of use are variable across these methods. This review summarizes approaches to synthesize radioactive PA, to analyze radioactive and nonradioactive products, DAG and P_i, and discusses the advantages and disadvantages of each PAP assay.

A comprehensive and standardized system to report lipid structures analyzed by MS is essential for the communication and storage of lipidomics data. Herein, an update on both the LIPID MAPS classification system and shorthand notation of lipid structures is presented for lipid categories Fatty Acyls (FA), Glycerolipids (GL), Glycerophospholipids (GP), Sphingolipids (SP), and Sterols (ST). With its major changes, i.e., annotation of ring double bond equivalents and number of oxygens, the updated shorthand notation facilitates reporting of newly delineated oxygenated lipid species as well. For standardized reporting in lipidomics, the hierarchical architecture of shorthand notation reflects the diverse structural resolution powers provided by mass spectrometric assays. Moreover, shorthand notation is expanded beyond mammalian phyla to lipids from plant and yeast phyla. Finally, annotation of atoms is included for the use of stable isotope-labeled compounds in metabolic labeling experiments or as internal standards. This update on lipid classification, nomenclature, and shorthand annotation for lipid mass spectra is considered a standard for lipid data presentation.

S-Acylation, a reversible post-translational lipid modification of proteins, controls the properties and function of various proteins, including ion channels. Large conductance Ca2+-activated potassium (BK) channels are S-acylated at two sites that impart distinct functional effects. Whereas the enzymes that attach lipid groups are known, the enzymes mediating lipid removal (i.e. deacylation) are largely unknown. Here, McClafferty et al. identify two enzymes, ABHD17a and ABHD17c, that excise BK channel lipid groups with remarkable precision. These findings lend insights into mechanisms that orchestrate the (de)acylation that fine-tunes ion channel function in physiology and disease.

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0–S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

The organic anion transporters (OATs) and organic anion–transporting polypeptides (OATPs) belong to the solute carrier (SLC) transporter superfamily and play important roles in handling various endogenous and exogenous compounds of anionic charge. The OATs and OATPs are often implicated in drug therapy by impacting the pharmacokinetics of clinically important drugs and, thereby, drug exposure in the target organs or cells. Various mechanisms (e.g. genetic, environmental, and disease-related factors, drug-drug interactions, and food-drug interactions) can lead to variations in the expression and activity of the anion drug-transporting proteins of OATs and OATPs, possibly impacting the therapeutic outcomes. Previous investigations mainly focused on the regulation at the transcriptional level and drug-drug interactions as competing substrates or inhibitors. Recently, evidence has accumulated that cellular trafficking, post-translational modification, and degradation mechanisms serve as another important layer for the mechanisms underlying the variations in the OATs and OATPs. This review will provide a brief overview of the major OATs and OATPs implicated in drug therapy and summarize recent progress in our understanding of the post-translational modifications, in particular ubiquitination and degradation pathways of the individual OATs and OATPs implicated in drug therapy.

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming α1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ−1−4) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.4+ex47 and Cav1.4Δex47, respectively), and the auxiliary subunits, β2X13 and α2δ-4. We find that IT caused both Cav1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits β2X13 and α2δ-4 but not in those with β2A and α2δ-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Cav1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of β2X13 and α2δ-4. Our results reveal complex actions of IT in modifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.

SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.

The assembly of the postsynaptic transmitter sensing machinery at inhibitory nerve cell synapses requires the intimate interplay between cell adhesion proteins, scaffold and adaptor proteins, and γ-aminobutyric acid (GABA) or glycine receptors. We developed an in vitro membrane system to reconstitute this process, to identify the essential protein components, and to define their mechanism of action, with a specific focus on the mechanism by which the cytosolic C terminus of the synaptic cell adhesion protein Neuroligin-2 alters the conformation of the adaptor protein Collybistin-2 and thereby controls Collybistin-2-interactions with phosphoinositides (PtdInsPs) in the plasma membrane. Supported hybrid membranes doped with different PtdInsPs and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) to allow for the specific adsorption of the His6-tagged intracellular domain of Neuroligin-2 (His-cytNL2) were prepared on hydrophobically functionalized silicon dioxide substrates via vesicle spreading. Two different collybistin variants, the WT protein (CB2SH3) and a mutant that adopts an intrinsically 'open' and activated conformation (CB2SH3/W24A-E262A), were bound to supported membranes in the absence or presence of His-cytNL2. The corresponding binding data, obtained by reflectometric interference spectroscopy, show that the interaction of the C terminus of Neuroligin-2 with Collybistin-2 induces a conformational change in Collybistin-2 that promotes its interaction with distinct membrane PtdInsPs.

The dominant role of CaV2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of CaV2 and CaV1 channels, and less so CaV3 channels, it is unclear why there have not been major shifts toward dependence on other CaV channels for synaptic transmission. Here, we provide a structural and functional profile of the CaV2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possesses single gene homologues for CaV1–CaV3 channels. Remarkably, the highly divergent channel possesses similar features as human CaV2.1 and other CaV2 channels, including high voltage–activated currents that are larger in external Ba2+ than in Ca2+; voltage-dependent kinetics of activation, inactivation, and deactivation; and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax CaV2 suggests that the core features of presynaptic CaV2 channels were established early during animal evolution, after CaV1 and CaV2 channels emerged via proposed gene duplication from an ancestral CaV1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian CaV2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA and to metal cation blockers Cd2+ and Ni2+. Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gβγ subunits, which nevertheless inhibited the human CaV2.1 channel, suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G proteins. Last, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior and other likely secretory cells, suggesting contributions to regulated exocytosis.