Effect of Total SMS Activity on LDL Catabolism in Mice.

BACKGROUND: Sphingomyelin (SM) and cholesterol are 2 key lipid partners on cell membranes and on lipoproteins. Many studies have indicated the influence of cholesterol on SM metabolism. This study examined the influence of SM biosynthesis on cholesterol metabolism. METHODS: Inducible global Sms1 KO (knockout)/global Sms2 KO mice were prepared to evaluate the effect of whole-body SM biosynthesis deficiency on lipoprotein metabolism. Tissue cholesterol, SM, ceramide, and glucosylceramide levels were measured. Triglyceride production rate and LDL (low-density lipoprotein) catabolism were measured. Lipid rafts were isolated and LDL receptor mass and function were evaluated. Also, the effects of exogenous sphingolipids on hepatocytes were investigated. RESULTS: We found that total SMS (SM synthase) depletion significantly reduced plasma SM levels. Also, the total deficiency significantly induced plasma cholesterol, apoB (apolipoprotein B), and apoE (apolipoprotein E) levels. Importantly, total SMS deficiency, but not SMS2 deficiency, dramatically decreased LDL receptors in the liver and attenuated LDL uptake through the receptor. Further, we found that total SMS deficiency greatly reduced LDL receptors in the lipid rafts, which contained significantly lower SM and significantly higher glucosylceramide, as well as cholesterol. Furthermore, we treated primary hepatocytes and Huh7 cells (a human hepatoma cell line) with SM, ceramide, or glucosylceramide, and we found that only SM could upregulate LDL receptor levels in a dose-dependent fashion. CONCLUSIONS: Whole-body SM biosynthesis plays an important role in LDL cholesterol catabolism. The total SMS deficiency, but not SMS2 deficiency, reduces LDL uptake and causes LDL cholesterol accumulation in the circulation. Given the fact that serum SM level is a risk factor for cardiovascular diseases, inhibiting SMS2 but not SMS1 should be the desirable approach.

Effect of Total Sphingomyelin Synthase Activity on Low Density Lipoprotein Catabolism in Mice.

Background: Sphingomyelin (SM) and cholesterol are two key lipid partners on cell membranes and on lipoproteins. Many studies have indicated the influence of cholesterol on SM metabolism. This study examined the influence of SM biosynthesis on cholesterol metabolism. Methods: Inducible global Sms1 KO/global Sms2 KO mice were prepared to evaluate the effect of whole-body SM biosynthesis deficiency on lipoprotein metabolism. Tissue cholesterol, SM, ceramide, and glucosylceramide levels were measured. TG production rate and LDL catabolism were measured. Lipid rafts were isolated and LDL receptor mass and function were evaluated. Also, the effects of exogenous sphingolipids on hepatocytes were investigated. Results: We found that total SMS depletion significantly reduced plasma SM levels. Also, the total deficiency significantly induced plasma cholesterol, apoB, and apoE levels. Importantly, total SMS deficiency, but not SMS2 deficiency, dramatically decreased LDL receptors in the liver and attenuated LDL uptake through the receptor. Further, we found that total SMS deficiency greatly reduced LDL receptors in the lipid rafts which contained significantly lower SM and significantly higher glucosylceramide as well as cholesterol. Furthermore, we treated primary hepatocytes and Huh7 cells (a human hepatoma cell line) with SM, ceramide, or glucosylceramide, and we found that only SM could up-regulate LDL receptor levels in a dose-dependent fashion. Conclusions: Whole-body SM biosynthesis plays an important role in LDL-cholesterol catabolism. The total SMS deficiency, but not SMS2 deficiency, reduces LDL uptake and causes LDL-cholesterol accumulation in the circulation. Given the fact that serum SM level is a risk factor for cardiovascular diseases, inhibiting SMS2 but not SMS1 should be the desirable approach.

Microfluidic isolation of aptamers with affinity towards multiple myeloma monoclonal immunoglobulins.

Multiple myeloma (MM) is a bone marrow cancer of resident plasma cells that affects 125,000 patients in the U.S. with about 30,000 new cases per year. Its signature is the clonal proliferation of a single plasma cell that secretes a patient specific monoclonal immunoglobulin (M-Ig). Targeting the M-Ig in patient serum could allow sensitive and noninvasive identification of minimal residual disease in multiple myeloma. Aptamers, which are single-stranded oligonucleotides with affinity and specificity to a target molecule, have recently been introduced as affinity reagents for recognition of MM M-Igs. Here we exploit microfluidic SELEX technology to enable rapid and efficient generation of aptamers against M-Ig proteins from MM patients. We first characterize the technology by isolating aptamers with affinity towards the monoclonal antibody rituximab as a model M-Ig and then apply the technology to isolating aptamers specifically targeting M-Igs obtained from serum samples of MM patients. We demonstrate that high-affinity DNA aptamers (KD < 50 nM) for M-Ig proteins from a patient sample could be isolated via microfluidic SELEX within approximately 12 h and using less than 100 micrograms of patient M-Ig. Such aptamers can potentially be used in personalized monitoring of minimal residual disease in MM patients.

Liver sphingomyelin synthase 1 deficiency causes steatosis, steatohepatitis, fibrosis, and tumorigenesis: An effect of glucosylceramide accumulation.

Glucosylceramide (GluCer) was accumulated in sphingomyelin synthase 1 (SMS1) but not SMS2 deficient mouse tissues. In current study, we studied GluCer accumulation-mediated metabolic consequences. Livers from liver-specific Sms1/global Sms2 double-knockout (dKO) exhibited severe steatosis under a high-fat diet. Moreover, chow diet-fed ≥6-month-old dKO mice had liver impairment, inflammation, and fibrosis, compared with wild type and Sms2 KO mice. RNA sequencing showed 3- to 12-fold increases in various genes which are involved in lipogenesis, inflammation, and fibrosis. Further, we found that direct GluCer treatment (in vitro and in vivo) promoted hepatocyte to secrete more activated TGFß1, which stimulated more collagen 1α1 production in hepatic stellate cells. Additionally, GluCer promoted more ß-catenin translocation into the nucleus, thus promoting tumorigenesis. Importantly, human NASH patients had higher liver GluCer synthase and higher plasma GluCer. These findings implicated that GluCer accumulation is one of triggers promoting the development of NAFLD into NASH, then, fibrosis, and tumorigenesis.

The microbiome affects liver sphingolipids and plasma fatty acids in a murine model of the Western diet based on soybean oil.

Studies in mice using germfree animals as controls for microbial colonization have shown that the gut microbiome mediates diet-induced obesity. Such studies use diets rich in saturated fat, however, Western diets in the United States America are enriched in soybean oil, composed of unsaturated fatty acids, either linoleic or oleic acid. Here, we addressed whether the microbiome is a variable in fat metabolism in mice on a soybean oil diet. We used conventionally-raised, low-germ, and germfree mice fed for 10 weeks diets either high or low in high-linoleic-acid soybean oil as the sole source of fat. Conventional and germfree mice gained relative fat weight and all mice consumed more calories on the high fat vs. low fat soybean oil diet. Plasma fatty acid levels were generally dependent on diet, with microbial colonization status affecting iso-C18:0, C20:3n-6, C14:0, and C15:0 levels. Colonization status, but not diet, impacted levels of liver sphingolipids including ceramides, sphingomyelins, and sphinganine. Our results confirm that absorbed fatty acids are mainly a reflection of the diet and that microbial colonization influences liver sphingolipid pools regardless of diet.

Personalized immunoglobulin aptamers for detection of multiple myeloma minimal residual disease in serum.

Multiple myeloma (MM) is a neoplasm of plasma cells that secrete patient specific monoclonal immunoglobulins. A recognized problem in MM treatment is the early recognition of minimal residual disease (MRD), the major cause of relapse. Current MRD detection methods (multiparameter flow cytometry and next generation sequencing) are based on the analysis of bone marrow plasma cells. Both methods cannot detect extramedullary disease and are unsuitable for serial measurements. We describe the methodology to generate high affinity DNA aptamers that are specific to a patient's monoclonal Fab region. Such aptamers are 2000-fold more sensitive than immunofixation electrophoresis and enabled detection and quantification of MRD in serum when conventional MRD methods assessed complete remission. The aptamer isolation process that requires small volumes of serum is automatable, and Fab specific aptamers are adaptable to multiple diagnostic formats including point-of-care devices.

Sphingolipids produced by gut bacteria enter host metabolic pathways impacting ceramide levels.

Gut microbes are linked to host metabolism, but specific mechanisms remain to be uncovered. Ceramides, a type of sphingolipid (SL), have been implicated in the development of a range of metabolic disorders from insulin resistance (IR) to hepatic steatosis. SLs are obtained from the diet and generated by de novo synthesis in mammalian tissues. Another potential, but unexplored, source of mammalian SLs is production by Bacteroidetes, the dominant phylum of the gut microbiome. Genomes of Bacteroides spp. and their relatives encode serine palmitoyltransfease (SPT), allowing them to produce SLs. Here, we explore the contribution of SL-production by gut Bacteroides to host SL homeostasis. In human cell culture, bacterial SLs are processed by host SL-metabolic pathways. In mouse models, Bacteroides-derived lipids transfer to host epithelial tissue and the hepatic portal vein. Administration of B. thetaiotaomicron to mice, but not an SPT-deficient strain, reduces de novo SL production and increases liver ceramides. These results indicate that gut-derived bacterial SLs affect host lipid metabolism.

Liver serine palmitoyltransferase activity deficiency in early life impairs adherens junctions and promotes tumorigenesis.

Serine palmitoyltransferase is the key enzyme in sphingolipid biosynthesis. Mice lacking serine palmitoyltransferase are embryonic lethal. We prepared liver-specific mice deficient in the serine palmitoyltransferase long chain base subunit 2 gene using an albumin-cyclization recombination approach and found that the deficient mice have severe jaundice. Moreover, the deficiency impairs hepatocyte polarity, attenuates liver regeneration after hepatectomy, and promotes tumorigenesis. Importantly, we show that the deficiency significantly reduces sphingomyelin but not other sphingolipids in hepatocyte plasma membrane; greatly reduces cadherin, the major protein in adherens junctions, on the membrane; and greatly induces cadherin phosphorylation, an indication of its degradation. The deficiency affects cellular distribution of ß-catenin, the central component of the canonical Wnt pathway. Furthermore, such a defect can be partially corrected by sphingomyelin supplementation in vivo and in vitro. CONCLUSION: The plasma membrane sphingomyelin level is one of the key factors in regulating hepatocyte polarity and tumorigenesis. (Hepatology 2016;64:2089-2102).

Airway Epithelial Cell Release of GABA is Regulated by Protein Kinase A.

INTRODUCTION: γ-amino butyric acid (GABA) is not only the major inhibitory neurotransmitter in the central nervous system (CNS), but it also plays an important role in the lung, mediating airway smooth muscle relaxation and mucus production. As kinases such as protein kinase A (PKA) are known to regulate the release and reuptake of GABA in the CNS by GABA transporters, we hypothesized that ß-agonists would affect GABA release from airway epithelial cells through activation of PKA. METHODS: C57/BL6 mice received a pretreatment of a ß-agonist or vehicle (PBS), followed by methacholine or PBS. Bronchoalveolar lavage (BAL) was collected and the amount of GABA was quantified using HPLC mass spectrometry. For in vitro studies, cultured BEAS-2B human airway epithelial cells were loaded with (3)H-GABA. (3)H-GABA released was measured during activation and inhibition of PKA and tyrosine kinase signaling pathways. RESULTS: ß-agonist pretreatment prior to methacholine challenge attenuated in vivo GABA release in mouse BAL and (3)H-GABA release from depolarized BEAS-2B cells. GABA release was also decreased in BEAS-2B cells by increases in cAMP but not by Epac or tyrosine kinase activation. CONCLUSION: ß-agonists decrease GABA release from airway epithelium through the activation of cAMP and PKA. This has important therapeutic implications as ß-agonists and GABA are important mediators of both mucus production and airway smooth muscle tone.

Low sphingosine-1-phosphate impairs lung dendritic cells in cystic fibrosis.

Dysfunction of the cystic fibrosis transmembrane regulator (CFTR) leads to chronic inflammation and infection of the respiratory tract. The role of CFTR for cells of the pulmonary immune system is only partly understood. The present study analyzes the phenotype and immune stimulatory capacity of lung dendritic cells (DCs) from CFTR knockout (CF) mice. Total numbers of conventional DCs, plasmacytoid DCs, and CD103-positive DCs were lower in CF mice compared with wild-type (WT) control mice, as was the expression of major histocompatibility complex class II molecules (MHCII), CD40, and CD86. After pulmonary infection with respiratory syncytial virus, DC numbers increased in WT mice but not in CF mice, and the T cell-stimulatory capacity of CF DCs was impaired. The culture of CF lung DCs with bronchoalveolar lavage fluid (BALF) from WT mice increased the expression of MHCII, CD40, and CD86. The supplementation of CF BALF with sphingosine-1-phosphate (S1P), a mediator of immune cell migration and activation that is decreased in CF BALF, rescued the reduced expression of MHCII and CD40 in WT lung DCs and human blood DCs. These findings suggest that DCs are impaired in the CF lung, and that altered S1P affects lung DC function. These findings provide a novel link between defective CFTR and pulmonary innate immune dysfunction in CF.

Fatty acids regulate endothelial lipase and inflammatory markers in macrophages and in mouse aorta: a role for PPARγ.

OBJECTIVE: Macrophage endothelial lipase (EL) is associated with increased atherosclerosis and inflammation. Because of their anti-inflammatory properties we hypothesized that n-3 fatty acids, in contrast to saturated fatty acids, would lower macrophages and arterial EL and inflammatory markers. METHODS AND RESULTS: Murine J774 and peritoneal macrophages were incubated with eicosapentaenoic acid or palmitic acid in the presence or absence of lipopolysaccaride (LPS). LPS increased EL mRNA and protein. Palmitic acid alone or with LPS dose-dependently increased EL mRNA and protein. In contrast, eicosapentaenoic acid dose-dependently abrogated effects of LPS or palmitic acid on increasing EL expression. EL expression closely linked to peroxisome proliferator activated receptor (PPAR)γ expression. Eicosapentaenoic acid blocked rosiglitazone (a PPARγ agonist)-mediated EL activation and GW9662 (a PPARγ antagonist)-blocked palmitic acid-mediated EL stimulation. Eicosapentaenoic acid alone or with LPS blunted LPS-mediated stimulation of macrophage proinflammatory interleukin-6, interleukin-12p40, and toll-like receptor-4 mRNA and increased anti-inflammatory interleukin-10 and mannose receptor mRNA. In vivo studies in low density lipoprotein receptor knockout mice showed that high saturated fat rich diets, but not n-3 diets, increased arterial EL, PPARγ, and proinflammatory cytokine mRNA. CONCLUSIONS: n-3 fatty acids, in contrast to saturated fatty acids, decrease EL in parallel with modulating pro- and anti-inflammatory markers, and these effects on EL link to PPARγ.

Proinflammatory phenotype and increased caveolin-1 in alveolar macrophages with silenced CFTR mRNA.

The inflammatory milieu in the respiratory tract in cystic fibrosis (CF) has been linked to the defective expression of the cystic transmembrane regulator (CFTR) in epithelial cells. Alveolar macrophages (AM), important contibutors to inflammatory responses in the lung, also express CFTR. The present study analyzes the phenotype of human AM with silenced CFTR. Expression of CFTR mRNA and the immature form of the CFTR protein decreased 100-fold and 5.2-fold, respectively, in AM transfected with a CFTR specific siRNA (CFTR-siRNA) compared to controls. Reduction of CFTR expression in AM resulted in increased secretion of IL-8, increased phosphorylation of NF-kappaB, a positive regulator of IL-8 expression, and decreased expression of IkappaB-alpha, the inhibitory protein of NF-kappaB activation. AM with silenced CFTR expression also showed increased apoptosis. We hypothesized that caveolin-1 (Cav1), a membrane protein that is co-localized with CFTR in lipid rafts and that is related to inflammation and apoptosis in macrophages, may be affected by decreased CFTR expression. Messenger RNA and protein levels of Cav1 were increased in AM with silenced CFTR. Expression and transcriptional activity of sterol regulatory element binding protein (SREBP), a negative transcriptional regulator of Cav1, was decreased in AM with silenced CFTR, but total and free cholesterol mass did not change. These findings indicate that silencing of CFTR in human AM results in an inflammatory phenotype and apoptosis, which is associated to SREBP-mediated regulation of Cav1.

Lipid metabolism in cystic fibrosis.

PURPOSE OF REVIEW: Expression of defective cystic fibrosis transmembrane conductance regulator (CFTR), the cause for cystic fibrosis, affects fatty acid, cholesterol and sphingolipid metabolism. This review summarizes recent observations and evaluates current understanding of mechanisms. RECENT FINDINGS: Recent observations implicate CFTR, in addition to known effects on fatty acid and cholesterol metabolism, in the regulation of sphingolipid metabolism and suggest that this pathway is relevant to inflammation and infection. A common mechanism on how CFTR affects such a wide spectrum of lipid classes is currently not known. One mechanism for low linoleic acid, amenable to inhibition by docosahexaenoic acid, is increased metabolism in the n-6 fatty acid pathway. Accumulation of free cholesterol in distinct perinuclear compartments, reversible by overexpression of rab9, suggests that cystic fibrosis and the lysosomal storage disease Niemann-Pick-C could share similar cell signaling defects, in addition to increased cAMP signaling and sterol-regulatory element binding protein (SREBP) expression that affect cholesterol metabolism. Novel is the recognition that CFTR modulates ceramide mass and uptake of sphingosine-1- phosphate. Experiments in different cystic fibrosis-mouse models, although not able to establish whether ceramide mass is increased or decreased, suggest that normalization of ceramide decreases infection and selected parameters of inflammation, of relevance to the complex phenotype that characterizes cystic fibrosis. SUMMARY: Expression of defective CFTR has profound effects on fatty acid, cholesterol and sphingolipid metabolism, for which mechanisms are currently poorly understood. Recent studies in different cystic fibrosis models suggest a causal relationship between altered ceramide mass and increased inflammation and susceptibility to infection. Studies in cystic fibrosis knockout mouse models suggest that normalization of ceramide decreases infection and inflammation. Studies that evaluate the diagnostic and clinical relevance of sphingolipids in patients with cystic fibrosis are needed.

Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signaling.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that affect protein structure and channel function. CFTR, localized in the apical membrane within cholesterol and sphingomyelin rich regions, is an ABC transporter that functions as a chloride channel. Here, we report that expression of defective CFTR (DeltaF508CFTR or decreased CFTR) in human lung epithelial cell lines increases sphingolipid synthesis and mass of sphinganine, sphingosine, four long-chain saturated ceramide species, C16 dihydroceramide, C22, C24, C26-ceramide, and sphingomyelin, and decreases mass of C18 and unsaturated C18:1 ceramide species. Decreased expression of CFTR is associated with increased expression of long-chain base subunit 1 of serine-palmitoyl CoA, the rate-limiting enzyme of de novo sphingolipid synthesis and increased sphingolipid synthesis. Overexpression of DeltaF508CFTR in bronchoalveolar cells that do not express CFTR increases sphingolipid synthesis and mass, whereas overexpression of wild-type CFTR, but not of an unrelated ABC transporter, ABCA7, decreases sphingolipid synthesis and mass. The data are consistent with a model in which CFTR functions within a feedback system that affects sphingolipid synthesis and in which increased sphingolipid synthesis could reflect a physiological response to sequestration of sphingolipids or altered membrane structure.

n-3 fatty acids and gene expression.

Accumulating evidence in both humans and animal models clearly indicates that a group of very-long-chain polyunsaturated fatty acids, the n-3 fatty acids (or omega-3), have distinct and important bioactive properties compared with other groups of fatty acids. n-3 Fatty acids are known to reduce many risk factors associated with several diseases, such as cardiovascular diseases, diabetes, and cancer. The mechanisms whereby n-3 fatty acids affect gene expression are complex and involve multiple processes. As examples, n-3 fatty acids regulate 2 groups of transcription factors, such as sterol-regulatory-element binding proteins and peroxisome proliferator-activated receptors, that are critical for modulating the expression of genes controlling both systemic and tissue-specific lipid homeostasis. Modulation of specific genes by n-3 fatty acids and cross-talk between these genes are responsible for many effects of n-3 fatty acids.

Sterol and fatty acid regulatory pathways in a Giardia lamblia-derived promoter: evidence for SREBP as an ancient transcription factor.

The sterol regulatory element binding-proteins (SREBPs) are transcription factors that regulate the genes of lipid metabolism. Cholesterol and unsaturated fatty acids regulate SREBPs. Giardia lamblia (GL) is an intestinal parasite and one of the earliest derived members within the eukaryotic lineage. GLs exist as trophozoites and cysts. Growth in cholesterol depletion induces transcription of cyst-wall protein (CWP) genes that are upregulated during encystation. The hypothesis was investigated that SREBP-like pathways have a role in cwp gene transcription. Chinese hamster ovary cells were transfected with a cwp-2 promoter reporter construct. Incubation with cholesterol or oleate reduced cwp-2 mediated gene transcription to about half of the control. Incubation in sterol-depleted media, or in the presence of either an inhibitor of intracellular cholesterol movement or inhibitor of cholesterol synthesis, increased gene expression up to 3-fold. Overexpression of SREBPs increased reporter gene activity 2.5-fold. In the absence of functional SREBPs, cwp-2 was not regulated by cholesterol. Footprint analysis of cwp-2 reveals three novel binding sites for mammalian SREBPs with no homologies in other species or humans. The data show that SREBP binds to and can modulate transcription of a regulatory element from an ancient eukaryote and suggest the existence of an SREBP homolog in GL.

Selective uptake from LDL is stimulated by unsaturated fatty acids and modulated by cholesterol content in the plasma membrane: role of plasma membrane composition in regulating non-SR-BI-mediated selective lipid transfer.

We previously reported that unsaturated fatty acids stimulated low-density lipoprotein (LDL) particle uptake in J774 macrophages by increasing LDL receptor activity. Since free fatty acids (FFA) also change plasma membrane properties, a putative cholesteryl ester (CE) acceptor for selective uptake (SU), we questioned the ability of FFA to modulate SU from LDL. Using [(3)H]cholesteryl ether/(125)I-LDL to trace CE core and whole particle uptake, we found that oleic acid and eicosapentaenoic acid, but not saturated stearic acid, increased SU by 30% over control levels. An ACAT inhibitor, Dup128, abolished FFA effects on SU, indicating that increased SU by FFA was secondary to changes in cell-free cholesterol (FC). Consistent with these observations, ACAT inhibition increased cell FC and reduced LDL SU by half. The important role of plasma membrane composition was further demonstrated in that beta-cyclodextrin- (beta-CD-) mediated FC removal from the plasma membrane increased SU from LDL and was further stimulated by U18666A, a compound that inhibits FC transport between lysosomes and the plasma membrane. In contrast, cholesterol-saturated beta-CD markedly reduced LDL SU. In contrast to LDL SU, oleic acid, ACAT inhibition, U18666A, or beta-CD had no effects on HDL SU. Moreover, HDL SU was inhibited by antimouse SR-BI antibody by more than 50% but had little effect on LDL SU. In C57BL/6 mice fed a high fat diet, plasma FFA levels increased, and SU accounted for an almost 4-fold increased proportion of total cholesterol delivery to the arterial wall. Taken together, these data suggest that LDL SU is mediated by pathways independent of SR-BI and is influenced by plasma membrane FC content. Moreover, in conditions where elevated plasma FFA occur, SU from LDL can be an important mechanism for cholesterol delivery in vivo.