Three forms of PPARs are expressed in the heart. In animal models, PPARγ agonist treatment improves lipotoxic cardiomyopathy; however, PPARγ agonist treatment of humans is associated with peripheral edema and increased heart failure. To directly assess effects of increased PPARγ on heart function, we created transgenic mice expressing PPARγ1 in the heart via the cardiac α-myosin heavy chain (α-MHC) promoter. PPARγ1-transgenic mice had increased cardiac expression of fatty acid oxidation genes and increased lipoprotein triglyceride (TG) uptake. Unlike in cardiac PPARα-transgenic mice, heart glucose transporter 4 (GLUT4) mRNA expression and glucose uptake were not decreased. PPARγ1-transgenic mice developed a dilated cardiomyopathy associated with increased lipid and glycogen stores, distorted architecture of the mitochondrial inner matrix, and disrupted cristae. Thus, while PPARγ agonists appear to have multiple beneficial effects, their direct actions on the myocardium have the potential to lead to deterioration in heart function.

Background-In clinical studies, sphingomyelin (SM) plasma levels correlated with the occurrence of coronary heart disease independently of plasma cholesterol levels. We hypothesized that inhibition of SM synthesis would have antiatherogenic effects. To test this hypothesis, apolipoprotein E (apoE)-knockout (KO) mice were treated with myriocin, a potent inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in SM biosynthesis. Methods and Results-Diet-admix treatment of apoE-KO mice with myriocin in Western diet for 12 weeks lowered SM and sphinganine plasma levels. Decreases in sphinganine and SM concentrations were also observed in the liver and aorta of myriocin-treated animals compared with controls. Inhibition of de novo sphingolipid biosynthesis reduced total cholesterol and triglyceride plasma levels. Cholesterol distribution in lipoproteins demonstrated a decrease in ␤-VLDL and LDL cholesterol and an increase in HDL cholesterol. Oil red O staining of total aortas demonstrated reduction of atherosclerotic lesion coverage in the myriocin-treated group. Atherosclerotic plaque area was also reduced in the aortic root and brachiocephalic artery. Conclusions-Inhibition of de novo SM biosynthesis in apoE-KO mice lowers plasma cholesterol and triglyceride levels, raises HDL cholesterol, and prevents development of atherosclerotic lesions.

Sphingolipid metabolism is implicated to play an important role in apoptosis. Here we show that dihydrosphingosine (DHS) and phytosphingosine (PHS), two major sphingoid bases of fungi, have potent fungicidal activity with remarkably high structural and stereochemical specificity against Aspergillus nidulans. In fact, only naturally occurring DHS and PHS are active. Further analysis revealed that DHS and PHS induce rapid DNA condensation independent of mitosis, large-scale DNA fragmentation, and exposure of phosphatidylserine, all common morphological features characteristic of apoptosis, suggesting that DHS and PHS induce apoptosis in A. nidulans. The finding that DNA fragmentation requires protein synthesis, which implies that an active process is involved, further supports this proposition. The induction of apoptosis by DHS and PHS is associated with the rapid accumulation of reactive oxygen species (ROS). However, ROS are not required for apoptosis induced by DHS and PHS, as scavenging of ROS by a free radical spin trap has no effect. We further demonstrate that apoptosis induced by DHS and PHS is independent of metacaspase function but requires mitochondrial function. Together, the results suggest that DHS and PHS induce a type of apoptosis in A. nidulans most similar to the caspase-independent apoptosis observed in mammalian systems. As A. nidulans is genetically tractable, this organism should be an ideal model system for dissecting sphingolipid signaling in apoptosis and, importantly, for further elucidating the molecular basis of caspase-independent apoptosis.In recent years, mounting evidence has indicated that sphingolipid metabolism is an important cell signaling system. Rapid and transient changes in sphingolipid metabolism are closely associated with a wide range of cellular activities, including the stress response, apoptosis, inflammation, cell cycle regulation, and cancer development (19,20,33). For instance, stress signals rapidly and transiently elevate the level of cellular ceramide; conversely, growth factors stimulate the rapid, transient generation of sphingosine-1-phosphate (S-1-P). Importantly, the treatment of cells with short-chain ceramide analogs or the expression of sphingomyelinases reproduces most of the effects of endogenous ceramide, particularly in the stress response and apoptosis (19,20,33). Furthermore, S-1-P, as a highaffinity ligand of the edg family of G-protein-coupled receptors, promotes cell survival and proliferation by antagonizing ceramide-mediated apoptosis (52, 61). Thus, ceramide, as an important regulatory component in the stress response and apoptosis, and S-1-P, involved in cell survival and proliferation, have attracted enormous scientific interest in recent years. In particular, a large body of evidence has now accumulated to implicate an important role of ceramide in the stress response and apoptosis. It is proposed that the relative levels of cellular ceramide and S-1-P determine whether cells undergo apoptosis or continue to proliferate (61).The sphingoid ...

It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency-or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity.Metabolic syndrome is a collection of abnormalities in metabolism, including obesity, nonalcoholic fatty liver disease, macrophage inflammation, impaired fasting glucose clearance, dyslipidemia, and hypertension. Insulin resistance appears to be a key feature in metabolic syndrome (47). The de novo sphingolipid synthesis pathway is considered a promising target for pharmacological intervention in insulin resistance. It has been shown that inhibition of serine palmitoyltransferase (SPT; the first enzyme for sphingolipid biosynthesis) increases insulin sensitivity (17). However, the mechanism is incompletely understood, since such an inhibition decreases many bioactive sphingolipids, including sphingomyelin (44), ceramide, and glycosphingolipids. Ceramide levels appear to be important in mediating inflammation, obesity, and insulin sensitivity (4, 17, 18). Sphingomyelin (SM) levels also appear to be important in mediating inflammation and atherosclerosis (11,27,34). However, few in vivo studies have been conducted to investigate the functions of these two metabolism-related sphingolipids separately, since animal models are lacking.The biochemical synthesis of SM occurs through the actions of SPT, 3-ketosphinganine reductase, ceramide synthase, dihydroceramide desaturase, and sphingomyelin synthase (SMS) (36). Mammalian SPT contains two subunits, Sptlc1 and Sptlc2, encoding 53-and 63-kDa proteins, respectively (13, 64). These subunits are homologous, sharing roughly 20% sequence identity (13, 64), and form a heterodimer. A third subunit, Sptlc3, has also been reported (19), but its function remains to be elucidated. Recently, the discovery of two proteins, ssSPTa and ssSPTb, was reported. Each substantially enhances the activity of mammalian SPT, expressed in either yeast or mammalian cells, and...

Sphingolipids are major components of the plasma membrane of eukaryotic cells and were once thought of merely as structural components of the membrane. We have investigated effects of inhibiting sphingolipid biosynthesis, both in germinating spores and growing hyphae of Aspergillus nidulans. In germinating spores, genetic or pharmacological inactivation of inositol phosphorylceramide (IPC) synthase arrests the cell cycle in G 1 and also prevents polarized growth during spore germination. However, inactivation of IPC synthase not only eliminates sphingolipid biosynthesis but also leads to a marked accumulation of ceramide, its upstream intermediate. We therefore inactivated serine palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway, to determine effects of inhibiting sphingolipid biosynthesis without an accumulation of ceramide. This inactivation also prevented polarized growth but did not affect nuclear division of germinating spores. To see if sphingolipid biosynthesis is required to maintain polarized growth, and not just to establish polarity, we inhibited sphingolipid biosynthesis in cells in which polarity was already established. This inhibition rapidly abolished normal cell polarity and promoted cell tip branching, which normally never occurs. Cell tip branching was closely associated with dramatic changes in the normally highly polarized actin cytoskeleton and found to be dependent on actin function. The results indicate that sphingolipids are essential for the establishment and maintenance of cell polarity via control of the actin cytoskeleton and that accumulation of ceramide is likely responsible for arresting the cell cycle in G 1 .

The endoplasmic reticulum (ER) is the principal organelle in the cell for protein folding and trafficking, lipid synthesis, and cellular calcium homeostasis. Perturbation of ER function results in activation of the unfolded protein response (UPR) and is implicated in abnormal lipid biosynthesis and development of insulin resistance. In this study, we investigated whether transcription of sphingosine kinase (Sphk)2 is regulated by ER stress-mediated UPR pathways. Sphk2, a major isotype of sphingosine kinase in the liver, was transcriptionally up-regulated by tunicamycin and lipopolysaccharides. Transcriptional regulation of Sphk2 was mediated by activation of activating transcription factor (ATF)4 as demonstrated by promoter assays, immunoblotting, and small interfering RNA analyses. In primary hepatocytes, adenoviral Sphk2 expression elevated cellular sphingosine 1 phosphate (S1P) and activated protein kinase B phosphorylation, with no alteration of insulin receptor substrate phosphorylation. Hepatic overexpression of Sphk2 in mice fed a high-fat diet (HFD) led to elevated S1P and reduced ceramide, sphingomyelin, and glucosylceramide in plasma and liver. Hepatic accumulation of lipid droplets by HFD feeding was reduced by Sphk2-mediated up-regulation of fatty acid (FA) oxidizing genes and increased FA oxidation in liver. In addition, glucose intolerance and insulin resistance were ameliorated by improved hepatic insulin signaling through Sphk2 up-regulation. Conclusion: Sphk2 is transcriptionally up-regulated by acute ER stress through activation of ATF4 and improves perturbed hepatic glucose and FA metabolism. (HEPATOLOGY 2015;62:135-146) O besity developed by consumption of excess nutrients is a major contributor to the development of metabolic dysfunctions, such as insulin resistance, hypertension, and cardiovascular events.

The nucleotide-dependent tetramerization of purified native URA7-encoded CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) from the yeast Saccharomyces cerevisiae was characterized. CTP synthetase existed as a dimer in the absence of ATP and UTP. In the presence of saturating concentrations of ATP and UTP, the CTP synthetase protein existed as a tetramer. Increasing concentrations of ATP and UTP caused a dosedependent conversion of the dimeric species to a tetramer. The kinetics of enzyme tetramerization correlates with the kinetics of enzyme activity. The tetramerization of CTP synthetase was dependent on UTP and Mg 2؉ ions. ATP facilitated the UTP-dependent tetramerization of CTP synthetase by a mechanism that involved the ATP-dependent phosphorylation of UTP catalyzed by the enzyme. The glutaminase reaction that is catalyzed by the enzyme was not required for enzyme tetramerization. CTP, a potent inhibitor of CTP synthetase activity, did not inhibit the ATP/UTP-dependent tetramerization of the enzyme. Phosphorylation of the purified native CTP synthetase with protein kinase A and protein kinase C facilitated the nucleotide-dependent tetramerization. Dephosphorylation of native CTP synthetase with alkaline phosphatase prevented the nucleotide-dependent tetramerization of the enzyme. This correlated with the inactivation of CTP synthetase activity. Rephosphorylation of the dephosphorylated enzyme with protein kinase A and protein kinase C resulted in a partial restoration of the nucleotidedependent tetramerization of the enzyme. This tetramerization correlated with the partial restoration of CTP synthetase activity. Taken together, these results indicated that enzyme tetramerization was required for CTP synthetase activity and that enzyme phosphorylation played an important role in the tetramerization and regulation of the enzyme.The nucleotide CTP is required for the synthesis of RNA, DNA, phospholipids, and sialoglycoproteins (1). CTP is synthesized from UTP via the reaction catalyzed by the cytosolicassociated enzyme CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) (2, 3) (Fig. 1). In eukaryotic cells, the regulation of CTP synthetase activity plays an important role in the balance of nucleotide pools (4 -9) and in the synthesis of membrane phospholipids (10, 11). Moreover, unregulated CTP synthetase activity is a common property of leukemic cells (12) and rapidly growing tumors found in liver (13), colon (14), and lung (15). Overall, these observations emphasize the importance of studies to understand the regulation of CTP synthetase activity.Our laboratory utilizes the yeast Saccharomyces cerevisiae as a model eukaryote to study the regulation of CTP synthetase activity. The yeast enzyme is encoded by the URA7 (7) and URA8 (8) genes. The URA7-(9) and URA8-encoded (16) CTP synthetases have been purified to apparent homogeneity and characterized with respect to their enzymological and kinetic properties. These CTP synthetases exhibit positive cooperative kinetics with respect to UT...

Objective Sphingomyelin synthase (SMS) catalyzes the conversion of ceramide to sphingomyelin (SM), and sits at the crossroads of sphingolipid biosynthesis. SMS has two isoforms: SMS1 and SMS2. Although they have the same SMS activity, they are different enzymes with distinguishable subcellular localizations and cell expression patterns. It is conceivable that these differences could yield different consequences, in terms of sphingolipid metabolism and its related atherogenesis. Methods and Results we created Sms1 gene knockout (KO) mice and found that Sms1 deficiency significantly decreased plasma, liver, and macrophage SM (59%, 45%, and 54%, respectively), but had only a marginal effect on ceramide levels. Surprisingly, we found that Sms1 deficiency dramatically increased glucosylceramide and GM3 levels in plasma, liver, and macrophages (4 to 12 fold), while Sms2 deficiency had no such effect. We evaluated total SMS activity in tissues and found that Sms1 deficiency causes 77% reduction of SMS activity, indicating SMS1 is the major SMS in macrophages. Moreover, Sms1 deficient-macrophages have significantly higher glucosylceramide synthase activity. We also found that Sms1 deficiency significantly attenuated toll-like 4 receptor-mediated NF-κB and MAP kinase activation after LPS treatment. To evaluate atherogenicity, we transplanted Sms1 KO mouse bone marrow into LDL receptor KO mice (Sms1−/−→Ldlr−/−). After 3 months on a Western diet, these animals showed a significant decrease of atherosclerotic lesions in the root and the entire aorta (35% and 44%, P<0.01, respectively), and macrophage content in lesions (51%, P<0.05), compared with WT→Ldlr−/−) mice. Conclusions Sms1 deficiency decreases SM, but dramatically increases the levels of glycosphingolipids. Atherosclerosis in Sms1−/−→Ldlr−/− mice is significantly decreased.