De Novo Sphingolipid Biosynthesis in Atherosclerosis.

Atherosclerosis is the formation of fibrofatty lesions in the arterial wall, and this inflammatory state of the artery is the main cause of advanced pathological processes, including myocardial infarction and stroke. Dyslipidemic conditions with excess cholesterol accumulate within the arterial vessel wall and initiate atherogenic processes. Following vascular reaction and lipid accumulation, the vascular wall gradually thickens. Together with the occurrence of local inflammation, early atherosclerotic lesions lead to advanced pathophysiological events, plaque rupture, and thrombosis. Ceramide and sphingomyelin have emerged as major risk factors for atherosclerosis and coronary artery disease. Currently, the clinical association between de novo sphingolipid biosynthesis and coronary artery disease has been established. Furthermore, therapeutic strategies to modulate this pathway, especially those involving serine palmitoyltransferase and sphingomyelin synthase, against atherosclerosis, cancer, type 2 diabetes, and non-alcoholic fatty liver disease are actively under development. In this chapter, we focus on the relationship between de novo sphingolipid biosynthesis and coronary artery disease.

Upregulation of the serine palmitoyltransferase subunit SPTLC2 by endoplasmic reticulum stress inhibits the hepatic insulin response.

Endoplasmic reticulum (ER) stress is induced by various conditions, such as inflammation and the presence of excess nutrients. Abnormal accumulation of unfolded proteins leads to the activation of a collective signaling cascade, termed the unfolded protein response (UPR). ER stress is reported to perturb hepatic insulin response metabolism while promoting insulin resistance. Here, we report that ER stress regulates the de novo biosynthesis of sphingolipids via the activation of serine palmitoyltransferase (SPT), a rate-limiting enzyme involved in the de novo biosynthesis of ceramides. We found that the expression levels of Sptlc1 and Sptlc2, the major SPT subunits, were upregulated and that the cellular concentrations of ceramide and dihydroceramide were elevated by acute ER stress inducers in primary hepatocytes and HepG2 cells. Sptlc2 was upregulated and ceramide levels were elevated by tunicamycin in the livers of C57BL/6J wild-type mice. Analysis of the Sptlc2 promoter demonstrated that the transcriptional activation of Sptlc2 was mediated by the spliced form of X-box binding protein 1 (sXBP1). Liver-specific Sptlc2 transgenic mice exhibited increased ceramide levels in the liver and elevated fasting glucose levels. The insulin response was reduced by the inhibition of the phosphorylation of insulin receptor ß (IRß). Collectively, these results demonstrate that ER stress induces activation of the de novo biosynthesis of ceramide and contributes to the progression of hepatic insulin resistance via the reduced phosphorylation of IRß in hepatocytes.

Annona muricata leaf extract attenuates hepatic lipogenesis and adipogenesis.

Annona muricata (graviola) is a medicinal plant that can be used to alleviate chronic human diseases by providing antioxidants and inducing immunomodulation. In this study, we found that treatment of AML12 hepatocytes with steam (SGE) and ethanol (EGE) extracts of graviola leaf downregulated the expression of fatty acid (FA) oxidation genes, including ACOX1, CPT1, and PPARα, with no change in the expression of FA synthesis genes. However, whereas EGE inhibited the differentiation and lipid accumulation of 3T3-L1 adipocytes and downregulated FA synthesis genes, no similar changes were observed in response to treatment with SGE. In an in vivo experiment using mice fed a high-fat diet (HFD), body weight was reduced in response to treatment with EGE, which also dose-dependently alleviated liver hepatocyte ballooning induced by the consumption of a HFD. However, genes involved in FA oxidation and the secretion of very low density lipoprotein (VLDL) were downregulated. We also found that the size of adipocytes was reduced in response to EGE treatment, and that there was a downregulated expression of genes involved in adipogenesis and FA synthesis. Furthermore, we detected increases in the levels of cholesterol in the plasma, whereas ALT activity was reduced. Collectively, these results indicates that EGE inhibits lipid influx into the liver and adipogenesis in adipose tissues. These bioactive properties of EGE indicate its potential as a natural ingredient that can be used to prevent obesity.

Bioactive Sphingolipids as Major Regulators of Coronary Artery Disease.

Atherosclerosis is the deposition of plaque in the main arteries. It is an inflammatory condition involving the accumulation of macrophages and various lipids (low-density lipoprotein [LDL] cholesterol, ceramide, S1P). Moreover, endothelial cells, macrophages, leukocytes, and smooth muscle cells are the major players in the atherogenic process. Sphingolipids are now emerging as important regulators in various pathophysiological processes, including the atherogenic process. Various sphingolipids exist, such as the ceramides, ceramide-1-phosphate, sphingosine, sphinganine, sphingosine-1-phosphate (S1P), sphingomyelin, and hundreds of glycosphingolipids. Among these, ceramides, glycosphingolipids, and S1P play important roles in the atherogenic processes. The atherosclerotic plaque consists of higher amounts of ceramide, glycosphingolipids, and sphingomyelin. The inhibition of the de novo ceramide biosynthesis reduces the development of atherosclerosis. S1P regulates atherogenesis via binding to the S1P receptor (S1PR). Among the five S1PRs (S1PR1-5), S1PR1 and S1PR3 mainly exert anti-atherosclerotic properties. This review mainly focuses on the effects of ceramide and S1P via the S1PR in the development of atherosclerosis. Moreover, it discusses the recent findings and potential therapeutic implications in atherosclerosis.

Near-infrared-emitting nanoparticles activate collagen synthesis via TGFß signaling.

Research efforts towards developing near-infrared (NIR) therapeutics to activate the proliferation of human keratinocytes and collagen synthesis in the skin microenvironment have been minimal, and the subject has not been fully explored. Herein, we describe the novel synthesis Ag2S nanoparticles (NPs) by using a sonochemical method and reveal the effects of NIR irradiation on the enhancement of the production of collagen through NIR-emitting Ag2S NPs. We also synthesized Li-doped Ag2S NPs that exhibited significantly increased emission intensity because of their enhanced absorption ability in the UV-NIR region. Both Ag2S and Li-doped Ag2S NPs activated the proliferation of HaCaT (human keratinocyte) and HDF (human dermal fibroblast) cells with no effect on cell morphology. While Ag2S NPs upregulated TIMP1 by only twofold in HaCaT cells and TGF-ß1 by only fourfold in HDF cells, Li-doped Ag2S NPs upregulated TGF-ß1 by tenfold, TIMP1 by 26-fold, and COL1A1 by 18-fold in HaCaT cells and upregulated TGF-ß1 by fivefold and COL1A1 by fourfold in HDF cells. Furthermore, Ag2S NPs activated TGF-ß1 signaling by increasing the phosphorylation of Smad2 and Smad3. The degree of activation was notably higher in cells treated with Li-doped Ag2S NPs, mainly caused by the higher PL intensity from Li-doped Ag2S NPs. Ag2S NPs NIR activates cell proliferation and collagen synthesis in skin keratinocytes and HDF cells, which can be applied to clinical light therapy and the development of anti-wrinkle agents for cosmetics.

Hepatic Expression of the Serine Palmitoyltransferase Subunit Sptlc2 Reduces Lipid Droplets in the Liver by Activating VLDL Secretion.

OBJECTIVE: Ceramide is a signaling molecule that contributes to insulin resistance and hepatosteatosis. In the present study, we activated de novo ceramide synthesis by inducing the hepatic expression of Sptlc2 to investigate the role of ceramide in glucose and lipid metabolism. METHODS: We first constructed an adenovirus containing Sptlc2 (AdSptlc2), which encodes a major catalytic subunit of serine palmitoyltransferase (SPT). We then infected hepatocytes and mice fed a regular diet with AdSptlc2 to activate de novo ceramide biosynthesis. The liver-specific effects of ceramide biosynthesis on glucose and lipid metabolism were investigated by measuring changes in insulin signaling, lipid droplet formation, and very low-density lipoprotein (VLDL) secretion. RESULTS: In HepG2 hepatocytes, adenoviral Sptlc2 expression inhibited insulin signaling and increased ceramide levels via activation of c-Jun N-terminal kinase and serine phosphorylation of insulin receptor substrate 1. In contrast, in mice, AdSptlc2 infection decreased plasma glucose levels by downregulating gluconeogenic genes and increased plasma triglyceride levels by increasing VLDL secretion. In mice infected with AdSptlc2, glucose intolerance and insulin sensitivity improved, while pyruvate utilization via gluconeogenesis decreased. CONCLUSION: Hepatic ceramide was found to modulate hepatosteatosis and the insulin response via increased VLDL secretion and inhibition of gluconeogenesis in vivo. Although inhibition of the insulin response was observed in vitro, the compensatory mechanism of relieving ceramide-induced stress and reducing ceramide levels resulted in improvements of glucose and lipid metabolic profiles in vivo. This discrepancy between in vitro and in vivo regulation mechanisms suggests that ceramide plays a role in non-alcoholic fatty liver disease and insulin resistance.

Ceramide and Sphingosine 1-Phosphate in Liver Diseases.

The liver is an important organ in the regulation of glucose and lipid metabolism. It is responsible for systemic energy homeostasis. When energy need exceeds the storage capacity in the liver, fatty acids are shunted into nonoxidative sphingolipid biosynthesis, which increases the level of cellular ceramides. Accumulation of ceramides alters substrate utilization from glucose to lipids, activates triglyceride storage, and results in the development of both insulin resistance and hepatosteatosis, increasing the likelihood of major metabolic diseases. Another sphingolipid metabolite, sphingosine 1-phosphate (S1P) is a bioactive signaling molecule that acts via S1P-specific G protein coupled receptors. It regulates many cellular and physiological events. Since an increase in plasma S1P is associated with obesity, it seems reasonable that recent studies have provided evidence that S1P is linked to lipid pathophysiology, including hepatosteatosis and fibrosis. Herein, we review recent findings on ceramides and S1P in obesity-mediated liver diseases and the therapeutic potential of these sphingolipid metabolites.

Inhibition of sphingosine 1-phosphate lyase activates human keratinocyte differentiation and attenuates psoriasis in mice.

Sphingosine 1-phosphate (S1P) lyase is an intracellular enzyme that catalyzes the irreversible degradation of S1P and has been suggested as a therapeutic target for the treatment of psoriasis vulgaris. Because S1P induces differentiation of keratinocytes, we examined whether modulation of S1P lyase and altered intracellular S1P levels regulate proliferation and differentiation of human neonatal epidermal keratinocyte (HEKn) cells. To identify the physiological functions of S1P lyase in skin, we inhibited S1P lyase in HEKn cells with an S1P lyase-specific inhibitor (SLI) and with S1P lyase 1 (SGPL1)-specific siRNA (siSGPL1). In HEKn cells, pharmacological treatment with the SLI caused G1 arrest by upregulation of p21 and p27 and induced keratin 1, an early differentiation marker. Similarly, genetic suppression by siSGPL1 arrested the cell cycle at the G1 phase and activated differentiation. In addition, enzyme suppression by siSGPL1 upregulated keratin 1 and differentiation markers including involucrin and loricrin. When hyperproliferation of HEKn cells was induced by interleukin (IL)-17 and IL-22, pharmacologic inhibition of S1P lyase by SLI decreased proliferation and activated differentiation of HEKn cells simultaneously. In addition, SLI administration ameliorated imiquimod-induced psoriatic symptoms including erythema, scaling, and epidermal thickness in vivo. We thus demonstrated that S1P lyase inhibition reduces cell proliferation and induces keratinocyte differentiation, and that inhibition may attenuate psoriasiform changes. Collectively, these findings suggest that S1P lyase is a modulating factor for proliferation and differentiation, and support its potential as a therapeutic target for psoriasis in human keratinocytes.

Addition of an N-Terminal Poly-Glutamate Fusion Tag Improves Solubility and Production of Recombinant TAT-Cre Recombinase in Escherichia coli.

Cre recombinase is widely used to manipulate DNA sequences for both in vitro and in vivo research. Attachment of a trans-activator of transcription (TAT) sequence to Cre allows TATCre to penetrate the cell membrane, and the addition of a nuclear localization signal (NLS) helps the enzyme to translocate into the nucleus. Since the yield of recombinant TAT-Cre is limited by formation of inclusion bodies, we hypothesized that the positively charged arginine-rich TAT sequence causes the inclusion body formation, whereas its neutralization by the addition of a negatively charged sequence improves solubility of the protein. To prove this, we neutralized the positively charged TAT sequence by proximally attaching a negatively charged poly-glutamate (E12) sequence. We found that the E12 tag improved the solubility and yield of E12-TAT-NLS-Cre (E12-TAT-Cre) compared with those of TAT-NLS-Cre (TATCre) when expressed in E. coli. Furthermore, the growth of cells expressing E12-TAT-Cre was increased compared with that of the cells expressing TAT-Cre. Efficacy of the purified TATCre was confirmed by a recombination test on a floxed plasmid in a cell-free system and 293 FT cells. Taken together, our results suggest that attachment of the E12 sequence to TAT-Cre improves its solubility during expression in E. coli (possibly by neutralizing the ionic-charge effects of the TAT sequence) and consequently increases the yield. This method can be applied to the production of transducible proteins for research and therapeutic purposes.

Increased Intestinal Absorption of Vitamin U in Steamed Graviola Leaf Extract Activates Nicotine Detoxification.

Graviola leaves contain much vitamin U (vit U), but their sensory quality is not good enough for them to be developed as food ingredients. Addition of excipient natural ingredients formulated alongside vit U as active ingredients could enhance not only its sensory quality but also its bioavailability. The objectives of this study were to measure the bioaccessibility and intestinal cellular uptake of bioactive components, including rutin, kaempferol-rutinoside, and vit U, from steamed extract of graviola leaves (SGV) and SGV enriched with kale extract (SGK), and to examine how much they can detoxify nicotine in HepG2 cells. The bioaccessibility of vit U from SGV and SGK was 82.40% and 68.03%, respectively. The cellular uptake of vit U in SGK by Caco-2 cells was higher than that in SGV. Cotinine content converted from nicotine in HepG2 cells for 120 min was 0.22 and 0.25 µg/mg protein in 50 µg/mL of SGV and SGK, respectively, which were 2.86 and 3.57 times higher than the no-treatment control. SGK treatment of HepG2 cells upregulated CYP2A6 three times as much as did that of SGV. Our results suggest that graviola leaf extract enriched with excipient ingredients such as kale could improve vit U absorption and provide a natural therapy for detoxifying nicotine.

Fermented Cordyceps militaris Extract Prevents Hepatosteatosis and Adipocyte Hypertrophy in High Fat Diet-Fed Mice.

Nonalcoholic fatty liver diseases (NAFLD) is characterized by accumulation of lipid droplets in the liver. The objective of this study was to evaluate protective effects of fermented Cordyceps militaris extract by Pediococcus pentosaceus ON188 (ONE) against hepatosteatosis and obesity in mice fed a high-fat diet (HFD). Eight-week-old male C57BL/6J mice were fed HFD mixed with ONE for four weeks and its effects on hepatosteatosis and obesity were examined. Although ONE did not change food intake, it reduced body weights of mice at administration dose of 200 mg/kg/day. Activities of lactate dehydrogenase (LDH), aspartate transaminase (AST), and alanine transaminase (ALT) as plasma parameters were reduced by ONE in a dose-dependent manner. Hepatic lipid droplets and triglyceride (TG) levels were also reduced by ONE due to upregulation of fatty acid oxidizing genes such as carnithine palmitoyltransferase (CPT1) and peroxisomal proliferator activated receptor α(PPARα) mediated by induction of sphingosine kinase 2 (SPHK2). In epididymal fat tissue, sizes of adipocytes were significantly reduced by ONE in a dose-dependent manner. This is mainly due to suppression of lipogenesis and upregulation of adipocyte browning genes. Collectively, these results suggest that fermented ONE can activate fatty acid oxidation via SPHK2 in the liver. It can also suppress lipogenesis and activate browning in adipose tissue. Thus, ONE might have potential to be used for the development of functional foods against liver dysfunction and obesity.

Fermented Cordyceps militaris Extract Ameliorates Hepatosteatosis via Activation of Fatty Acid Oxidation.

Nonalcoholic fatty liver disease is a progressive disease involving the accumulation of lipid droplets in the liver. In this study, we investigated the anti-hepatosteatosis effects of fermented Cordyceps militaris extract (CME) in AML-12 hepatocytes. Although the levels of adenosine and cordycepin were reduced in the extracts of CM grown on germinated soybean (GSCE) and fermented CM grown on germinated soybean (GSC) by Pediococcus pentosaceus ON188 (ON188E), the expression of fatty acid oxidation (FAO) genes were upregulated only by GSC-ON188E treatment in a dose-dependent manner. In contrast, a lipogenic gene, stearoyl Coenzyme A desaturase 1, was downregulated by ON188E. Formation of intracellular lipid droplets by the addition of oleic acid was reduced by ON188E to levels observed in WY14643-treated cells. When cells were treated with ON188E, sphingosine kinase 2 mainly responsible for hepatic sphingosine 1-phosphate (S1P) synthesis was upregulated and S1P was elevated. Collectively, the fermented GSC extract activates FAO through elevation of S1P synthesis and has potential as a therapeutic for hepatosteatosis.

Inhibition of lactate dehydrogenase A suppresses inflammatory response in RAW 264.7 macrophages.

Lactate is an important metabolite in cellular metabolism and fluctuates in certain disease conditions including cancer and immune diseases. It was hypothesized that a decrease in lactate would modulate the inflammatory response elicited by lipopolysaccharides (LPS) in macrophages. When RAW 264.7 macrophages were treated with FX11, a specific lactate dehydrogenase (LDHA) inhibitor, the expression of the cytokines, inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX­2) was downregulated due to reduced cellular lactate levels. Genetic suppression of LDHA by small interfering RNA (siRNA) downregulated the LPS­activated expression of interleukin (IL)­6, iNOS, and COX­2, and reduced the production of IL­6 and nitrites. Pharmacological and genetic suppression of LDHA inhibited the phosphorylation of p38 mitogen­activated protein kinase. Microarray gene expression profile demonstrated that the genes involved in cell proliferation and inflammation were mainly altered by siRNA­mediated LDHA suppression. Collectively, the present observations suggest that lactate may be an important metabolite and implicated in regulation of inflammatory response.

Adipocyte-Specific Deficiency of De Novo Sphingolipid Biosynthesis Leads to Lipodystrophy and Insulin Resistance.

Sphingolipids have been implicated in the etiology of chronic metabolic diseases. Here, we investigated whether sphingolipid biosynthesis is associated with the development of adipose tissues and metabolic diseases. SPTLC2, a subunit of serine palmitoyltransferase, was transcriptionally upregulated in the adipose tissues of obese mice and in differentiating adipocytes. Adipocyte-specific SPTLC2-deficient (aSPTLC2 KO) mice had markedly reduced adipose tissue mass. Fatty acids that were destined for the adipose tissue were instead shunted to liver and caused hepatosteatosis. This impaired fat distribution caused systemic insulin resistance and hyperglycemia, indicating severe lipodystrophy. Mechanistically, sphingosine 1-phosphate (S1P) was reduced in the adipose tissues of aSPTLC2 KO mice, and this inhibited adipocyte proliferation and differentiation via the downregulation of S1P receptor 1 and decreased activity of the peroxisome proliferator-activator receptor γ. In addition, downregulation of SREBP (sterol regulatory element-binding protein)-1c prevented adipogenesis of aSPTLC2 KO adipocytes. Collectively, our observations suggest that the tight regulation of de novo sphingolipid biosynthesis and S1P signaling plays an important role in adipogenesis and hepatosteatosis.

Celecoxib-mediated activation of endoplasmic reticulum stress induces de novo ceramide biosynthesis and apoptosis in hepatoma HepG2 cells mobilization.

Ceramides are the major sphingolipid metabolites involved in cell survival and apoptosis. When HepG2 hepatoma cells were treated with celecoxib, the expression of the genes in de novo sphingolipid biosynthesis and sphingomyelinase pathway was upregulated and cellular ceramide was elevated. In addition, celecoxib induced endoplasmic reticulum (ER) stress in a time-dependent manner. SPTLC2, a subunit of serine palmitoyltransferase, was overexpressed by adenovirus. Adenoviral overexpression of SPTLC2 (AdSPTLC2) decreased cell viability of HEK293 and HepG2 cells. In addition, AdSPTLC2 induced apoptosis via the caspase-dependent apoptotic pathway and elevated cellular ceramide, sphingoid bases, and dihydroceramide. However, overexpression of SPTLC2 did not induce ER stress. Collectively, celecoxib activates de novo sphingolipid biosynthesis and the combined effects of elevated ceramide and transcriptional activation of ER stress induce apoptosis. However, activation of de novo sphingolipid biosynthesis does not activate ER stress in hepatoma cells and is distinct from the celecoxib-mediated activation of ER stress. [BMB Reports 2017; 50(3): 144-149].

Immunomodulatory Efficacy of Standardized Annona muricata (Graviola) Leaf Extract via Activation of Mitogen-Activated Protein Kinase Pathways in RAW 264.7 Macrophages.

Annona muricata, commonly known as Graviola, has been utilized as a traditional medicine to treat various human diseases. The aim of this study was to examine the immune-enhancing activity of Graviola leaf extracts in RAW 264.7 macrophage cells. Active ingredients in Graviola leaf extracts (GE) were identified as kaempferol-3-O-rutinoside and quercetin-3-O-rutinoside by LC-MS/MS. When treated with steam or 50% ethanol GE, cell morphology was altered due to initiation of cell differentiation. While the cell viability was not altered by the steam GE, it was reduced by the ethanol GE. Both steam and ethanol GE induced the transcriptional expression of cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1ß, but only the steam extract upregulated inducible nitric oxide synthase (iNOS). In consistence with mRNA expression, the production of TNF-α and nitrite was elevated by both steam and ethanol extracts of Graviola leaves. This is mainly due to activation of mitogen-activated protein (MAP) kinase signaling pathways. These results suggest that Graviola leaves enhance immunity by activation of the MAP kinase pathways. These bioactive properties of Graviola indicate its potential as a health-promoting ingredient to boost the immune system.

Bioefficacy of Graviola leaf extracts in scavenging free radicals and upregulating antioxidant genes.

The aims of this study were to determine bioactive components of Graviola leaf extracts and to examine the radical scavenging capacity, gene expression and transcription factors of antioxidant enzymes. Rutin, kaempferol-rutinoside, and vitamin U were identified from the steaming and 50% EtOH extracts of Graviola leaves. Graviola leaf extracts effectively scavenged peroxy and nitrogen radicals. 50% EtOH of Graviola leaves provided a 1-2.9 times higher trolox equivalent than the steaming extract. It also had a higher VCEAC. Graviola leaf extracts reduced the generation of reactive oxygen species (ROS) induced by H2O2 in a dose-dependent manner. The 50% EtOH extract of Graviola leaves upregulated SOD1 and Nrf2, but catalase and HMOX1 were not altered by the 50% EtOH extract of Graviola leaves.