Tingbo Ding

Deficiency in Lysophosphatidylcholine Acyltransferase 3 Reduces Plasma Levels of Lipids by Reducing Lipid Absorption in Mice

BACKGROUND & AIMS Phosphatidylcholines (PCs) are structural and functional constituents of cell membranes. The activity of acyltransferase (lysophosphatidylcholine acyltransferase [LPCAT]) is required for addition of polyunsaturated fatty acids to the sn-2 position of PCs and is therefore required to maintain cell membrane structure and function. LPCAT3 is the most abundant isoform of LPCAT in the small intestine and liver, which are important sites of plasma lipoprotein metabolism. We investigated the effects of Lpcat3 disruption on lipid metabolism in mice. METHODS We disrupted the gene Lpcat3 in C57BL/6J mice to create LPCAT3 knockout (KO) mice. Livers and small intestinal tissues were collected from LPCAT3 KO and C57BL/6J parental strain (controls), and levels of LPCAT messenger RNAs and protein were measured. Levels of lipids and lipoproteins were measured in plasma samples. We isolated enterocytes from mice and measured levels of RNAs and proteins involved in lipid uptake by real-time polymerase chain reaction and immunoblot assays, respectively. We assessed lipid absorption and PC subspecies in the enterocyte plasma membrane using liquid chromatography with tandem mass spectometry. RESULTS LPCAT3 KO mice survived only 3 weeks after birth. Oil Red O staining showed that the control but not LPCAT3 KO mice accumulated lipids in the small intestine; levels of Niemann-Pick C1-like 1 (NPC1L1) and fatty acid transporter protein 4 (FATP4), which regulate lipid uptake, were greatly reduced in the small intestines of LPCAT3 KO mice. Oral administration of PC and olive oil allowed the LPCAT3 KO mice to survive with the same body weights as controls, but the KO mice had shorter and wider small-intestinal villi and longer and bigger small intestines. Plasma membranes of enterocytes from LPCAT3 KO mice also had significant reductions in the composition of polyunsaturated PCs and reduced levels of NPC1L1, CD36, and FATP4 proteins. These reductions were associated with reduced intestinal uptake of lipid by the small intestine and reduced plasma levels of cholesterol, phospholipid, and triglyceride. CONCLUSIONS LPCAT3 KO mice have longer and larger small intestines than control mice, with shorter wide villi, reduced lipid absorption, and lower levels NPC1L1, CD36, and FATP4 proteins. Inhibition of LPCAT3 in the small intestine could be developed as an approach to treat hyperlipidemia.

Sphingomyelin Synthase 2 Activity and Liver Steatosis An Effect of Ceramide-Mediated Peroxisome Proliferator-Activated Receptor γ2 Suppression

Objective—Sphingolipid de novo biosynthesis is related to nonalcoholic fatty liver disease or hepatic steatosis. However, the mechanism is still unclear. Sphingomyelin synthase (SMS), using ceramide as one of the substrates to produce sphingomyelin, sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. SMS2 is the major isoform in liver. Approach and Results—To investigate the relationship between liver SMS2 activity–mediated sphingolipid changes and hepatic steatosis, we used 2 mouse models: Sms2 liver-specific transgenic and Sms2 knockout mice. We found that Sms2 liver-specific transgenic livers have lower ceramide and higher sphingomyelin, whereas Sms2 knockout livers have higher ceramide and lower sphingomyelin. We also found that liver Sms2 overexpression promoted fatty acid uptake and liver steatosis, whereas Sms2 deficiency had an opposite effect in comparison with their respective controls. Importantly, the exogenous ceramide supplementation to Huh7 cells, a human hepatoma cell line, reduced the expression of peroxisome proliferator-activated receptor &ggr;2 and its target genes, Cd36 and Fsp27. Peroxisome proliferator-activated receptor &ggr; reporter analysis confirmed this phenomenon. Furthermore, peroxisome proliferator-activated receptor &ggr; antagonist treatment significantly decreased triglyceride accumulation in Sms2 liver-specific transgenic liver. Conclusions—We attributed these effects to ceramide that can suppress peroxisome proliferator-activated receptor &ggr;2, thus reducing the expression of Cd36 and Fsp27 and reducing liver steatosis. After all, SMS2 inhibition in the liver could diminish liver steatosis.Objective— Sphingolipid de novo biosynthesis is related to nonalcoholic fatty liver disease or hepatic steatosis. However, the mechanism is still unclear. Sphingomyelin synthase (SMS), using ceramide as one of the substrates to produce sphingomyelin, sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. SMS2 is the major isoform in liver. Approach and Results— To investigate the relationship between liver SMS2 activity–mediated sphingolipid changes and hepatic steatosis, we used 2 mouse models: Sms2 liver-specific transgenic and Sms2 knockout mice. We found that Sms2 liver-specific transgenic livers have lower ceramide and higher sphingomyelin, whereas Sms2 knockout livers have higher ceramide and lower sphingomyelin. We also found that liver Sms2 overexpression promoted fatty acid uptake and liver steatosis, whereas Sms2 deficiency had an opposite effect in comparison with their respective controls. Importantly, the exogenous ceramide supplementation to Huh7 cells, a human hepatoma cell line, reduced the expression of peroxisome proliferator-activated receptor γ2 and its target genes, Cd36 and Fsp27 . Peroxisome proliferator-activated receptor γ reporter analysis confirmed this phenomenon. Furthermore, peroxisome proliferator-activated receptor γ antagonist treatment significantly decreased triglyceride accumulation in Sms2 liver-specific transgenic liver. Conclusions— We attributed these effects to ceramide that can suppress peroxisome proliferator-activated receptor γ2, thus reducing the expression of Cd36 and Fsp27 and reducing liver steatosis. After all, SMS2 inhibition in the liver could diminish liver steatosis. # Significance {#article-title-42}

Sphingomyelin Synthase 2 Activity and Liver Steatosis

Objective—Sphingolipid de novo biosynthesis is related to nonalcoholic fatty liver disease or hepatic steatosis. However, the mechanism is still unclear. Sphingomyelin synthase (SMS), using ceramide as one of the substrates to produce sphingomyelin, sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. SMS2 is the major isoform in liver. Approach and Results—To investigate the relationship between liver SMS2 activity–mediated sphingolipid changes and hepatic steatosis, we used 2 mouse models: Sms2 liver-specific transgenic and Sms2 knockout mice. We found that Sms2 liver-specific transgenic livers have lower ceramide and higher sphingomyelin, whereas Sms2 knockout livers have higher ceramide and lower sphingomyelin. We also found that liver Sms2 overexpression promoted fatty acid uptake and liver steatosis, whereas Sms2 deficiency had an opposite effect in comparison with their respective controls. Importantly, the exogenous ceramide supplementation to Huh7 cells, a human hepatoma cell line, reduced the expression of peroxisome proliferator-activated receptor &ggr;2 and its target genes, Cd36 and Fsp27. Peroxisome proliferator-activated receptor &ggr; reporter analysis confirmed this phenomenon. Furthermore, peroxisome proliferator-activated receptor &ggr; antagonist treatment significantly decreased triglyceride accumulation in Sms2 liver-specific transgenic liver. Conclusions—We attributed these effects to ceramide that can suppress peroxisome proliferator-activated receptor &ggr;2, thus reducing the expression of Cd36 and Fsp27 and reducing liver steatosis. After all, SMS2 inhibition in the liver could diminish liver steatosis.Objective— Sphingolipid de novo biosynthesis is related to nonalcoholic fatty liver disease or hepatic steatosis. However, the mechanism is still unclear. Sphingomyelin synthase (SMS), using ceramide as one of the substrates to produce sphingomyelin, sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. SMS2 is the major isoform in liver. Approach and Results— To investigate the relationship between liver SMS2 activity–mediated sphingolipid changes and hepatic steatosis, we used 2 mouse models: Sms2 liver-specific transgenic and Sms2 knockout mice. We found that Sms2 liver-specific transgenic livers have lower ceramide and higher sphingomyelin, whereas Sms2 knockout livers have higher ceramide and lower sphingomyelin. We also found that liver Sms2 overexpression promoted fatty acid uptake and liver steatosis, whereas Sms2 deficiency had an opposite effect in comparison with their respective controls. Importantly, the exogenous ceramide supplementation to Huh7 cells, a human hepatoma cell line, reduced the expression of peroxisome proliferator-activated receptor γ2 and its target genes, Cd36 and Fsp27 . Peroxisome proliferator-activated receptor γ reporter analysis confirmed this phenomenon. Furthermore, peroxisome proliferator-activated receptor γ antagonist treatment significantly decreased triglyceride accumulation in Sms2 liver-specific transgenic liver. Conclusions— We attributed these effects to ceramide that can suppress peroxisome proliferator-activated receptor γ2, thus reducing the expression of Cd36 and Fsp27 and reducing liver steatosis. After all, SMS2 inhibition in the liver could diminish liver steatosis. # Significance {#article-title-42}

All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity

Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase Km and Vmax for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo.

Pharmacologic inhibition of sphingomyelin synthase (SMS) activity reduces apolipoprotein-B secretion from hepatocytes and attenuates endotoxin-mediated macrophage inflammation.

Sphingomyelin synthase (SMS) plays an important role in plasma atherogenic lipoprotein metabolism, inflammation, and the development of atherosclerosis. To understand whether the impaired apoB secretion and inflammation response is a direct result from lack of SMS activity, in this study, we prepared a series of compounds that inhibit SMS activity. Further, we characterized Dy105, the most potent inhibitor. We found that Dy105 treatment significantly reduces SM levels in SM-rich microdomain on cell membranes. Moreover, we found that SMS inhibition reduces apoB secretion in a human hepatoma cell line and reduces the activation of NFκB and p38, a MAP kinase, in bone marrow derived macrophages. These studies provided further evidence that SMS activity regulates atherogenic lipoprotein metabolism and inflammatory responses. Pharmacologic inhibition of SMS may be a new therapy for atherosclerosis by reducing apoB secretion, and reducing inflammation.

Discovery, synthesis and biological evaluation of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) as novel sphingomyelin synthase 1 inhibitors

Sphingomyelin synthase (SMS) has been proved to be a potential drug target for the treatment of atherosclerosis. However, few SMS inhibitors have been reported. In this paper, structure-based virtual screening was performed on hSMS1. SAPA 1a was discovered as a novel SMS1 inhibitor with an IC50 value of 5.2 μM in enzymatic assay. A series of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) were synthesized and their biological activities toward SMS1 were evaluated. Among them, SAPA 1j was found to be the most potent SMS1 inhibitor with an IC50 value of 2.1 μM in in vitro assay. The molecular docking studies suggested the interaction modes of SMS1 inhibitors and PC with the active site of SMS1. Site-directed mutagenesis validated the involvement of residues Arg342 and Tyr338 in enzymatic sphingomyelin production. The discovery of SAPA derivatives as a novel class of SMS1 inhibitors would advance the development of more effective SMS1 inhibitors.

Sphingomyelin Synthase 2 Activity and Liver SteatosisSignificance

Objective—Sphingolipid de novo biosynthesis is related to nonalcoholic fatty liver disease or hepatic steatosis. However, the mechanism is still unclear. Sphingomyelin synthase (SMS), using ceramide as one of the substrates to produce sphingomyelin, sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. SMS2 is the major isoform in liver. Approach and Results—To investigate the relationship between liver SMS2 activity–mediated sphingolipid changes and hepatic steatosis, we used 2 mouse models: Sms2 liver-specific transgenic and Sms2 knockout mice. We found that Sms2 liver-specific transgenic livers have lower ceramide and higher sphingomyelin, whereas Sms2 knockout livers have higher ceramide and lower sphingomyelin. We also found that liver Sms2 overexpression promoted fatty acid uptake and liver steatosis, whereas Sms2 deficiency had an opposite effect in comparison with their respective controls. Importantly, the exogenous ceramide supplementation to Huh7 cells, a human hepatoma cell line, reduced the expression of peroxisome proliferator-activated receptor &ggr;2 and its target genes, Cd36 and Fsp27. Peroxisome proliferator-activated receptor &ggr; reporter analysis confirmed this phenomenon. Furthermore, peroxisome proliferator-activated receptor &ggr; antagonist treatment significantly decreased triglyceride accumulation in Sms2 liver-specific transgenic liver. Conclusions—We attributed these effects to ceramide that can suppress peroxisome proliferator-activated receptor &ggr;2, thus reducing the expression of Cd36 and Fsp27 and reducing liver steatosis. After all, SMS2 inhibition in the liver could diminish liver steatosis.Objective— Sphingolipid de novo biosynthesis is related to nonalcoholic fatty liver disease or hepatic steatosis. However, the mechanism is still unclear. Sphingomyelin synthase (SMS), using ceramide as one of the substrates to produce sphingomyelin, sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. SMS2 is the major isoform in liver. Approach and Results— To investigate the relationship between liver SMS2 activity–mediated sphingolipid changes and hepatic steatosis, we used 2 mouse models: Sms2 liver-specific transgenic and Sms2 knockout mice. We found that Sms2 liver-specific transgenic livers have lower ceramide and higher sphingomyelin, whereas Sms2 knockout livers have higher ceramide and lower sphingomyelin. We also found that liver Sms2 overexpression promoted fatty acid uptake and liver steatosis, whereas Sms2 deficiency had an opposite effect in comparison with their respective controls. Importantly, the exogenous ceramide supplementation to Huh7 cells, a human hepatoma cell line, reduced the expression of peroxisome proliferator-activated receptor γ2 and its target genes, Cd36 and Fsp27 . Peroxisome proliferator-activated receptor γ reporter analysis confirmed this phenomenon. Furthermore, peroxisome proliferator-activated receptor γ antagonist treatment significantly decreased triglyceride accumulation in Sms2 liver-specific transgenic liver. Conclusions— We attributed these effects to ceramide that can suppress peroxisome proliferator-activated receptor γ2, thus reducing the expression of Cd36 and Fsp27 and reducing liver steatosis. After all, SMS2 inhibition in the liver could diminish liver steatosis. # Significance {#article-title-42}

Sphingomyelin synthase 2 activity and liver steatosis: an effect of ceramide-mediated PPARγ2 suppression

Objective—Sphingolipid de novo biosynthesis is related to nonalcoholic fatty liver disease or hepatic steatosis. However, the mechanism is still unclear. Sphingomyelin synthase (SMS), using ceramide as one of the substrates to produce sphingomyelin, sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. SMS2 is the major isoform in liver. Approach and Results—To investigate the relationship between liver SMS2 activity–mediated sphingolipid changes and hepatic steatosis, we used 2 mouse models: Sms2 liver-specific transgenic and Sms2 knockout mice. We found that Sms2 liver-specific transgenic livers have lower ceramide and higher sphingomyelin, whereas Sms2 knockout livers have higher ceramide and lower sphingomyelin. We also found that liver Sms2 overexpression promoted fatty acid uptake and liver steatosis, whereas Sms2 deficiency had an opposite effect in comparison with their respective controls. Importantly, the exogenous ceramide supplementation to Huh7 cells, a human hepatoma cell line, reduced the expression of peroxisome proliferator-activated receptor &ggr;2 and its target genes, Cd36 and Fsp27. Peroxisome proliferator-activated receptor &ggr; reporter analysis confirmed this phenomenon. Furthermore, peroxisome proliferator-activated receptor &ggr; antagonist treatment significantly decreased triglyceride accumulation in Sms2 liver-specific transgenic liver. Conclusions—We attributed these effects to ceramide that can suppress peroxisome proliferator-activated receptor &ggr;2, thus reducing the expression of Cd36 and Fsp27 and reducing liver steatosis. After all, SMS2 inhibition in the liver could diminish liver steatosis.Objective— Sphingolipid de novo biosynthesis is related to nonalcoholic fatty liver disease or hepatic steatosis. However, the mechanism is still unclear. Sphingomyelin synthase (SMS), using ceramide as one of the substrates to produce sphingomyelin, sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. SMS2 is the major isoform in liver. Approach and Results— To investigate the relationship between liver SMS2 activity–mediated sphingolipid changes and hepatic steatosis, we used 2 mouse models: Sms2 liver-specific transgenic and Sms2 knockout mice. We found that Sms2 liver-specific transgenic livers have lower ceramide and higher sphingomyelin, whereas Sms2 knockout livers have higher ceramide and lower sphingomyelin. We also found that liver Sms2 overexpression promoted fatty acid uptake and liver steatosis, whereas Sms2 deficiency had an opposite effect in comparison with their respective controls. Importantly, the exogenous ceramide supplementation to Huh7 cells, a human hepatoma cell line, reduced the expression of peroxisome proliferator-activated receptor γ2 and its target genes, Cd36 and Fsp27 . Peroxisome proliferator-activated receptor γ reporter analysis confirmed this phenomenon. Furthermore, peroxisome proliferator-activated receptor γ antagonist treatment significantly decreased triglyceride accumulation in Sms2 liver-specific transgenic liver. Conclusions— We attributed these effects to ceramide that can suppress peroxisome proliferator-activated receptor γ2, thus reducing the expression of Cd36 and Fsp27 and reducing liver steatosis. After all, SMS2 inhibition in the liver could diminish liver steatosis. # Significance {#article-title-42}