STEATOHEPATITIS/METABOLIC LIVER DISEASE
Activation of Sphingosine Kinase 2 by Endoplasmic Reticulum Stress Ameliorates Hepatic Steatosis and Insulin Resistance in Mice Su-Yeon Lee,1* In-Kyung Hong,1* Bo-Rahm Kim,1 Soon-Mi Shim,2 Jae Sung Lee,3 Hui-Young Lee,3 Cheol Soo Choi,3 Bo-Kyung Kim,4 and Tae-Sik Park1 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)
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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.1-3 Elevated plasma free fatty acids (FFAs)
induce abnormal fatty acid (FA) uptake by liver, adipose tissue, and heart, which contributes to induction of hepatic steatosis, inflammation, and cardiac dysfunction, respectively.4-6 Increased tissue uptake of FFAs under conditions of obesity would trigger the
Abbreviations: ACC, acyl CoA carboxylase; Acer, alkaline ceramidase; ACOX1, acyl CoA oxidase 1; AdSphk2, adenovirus expressing human Sphk2; AKT, protein kinase B; ATF, activating transcription factor; CerS, ceramide synthase; CHOP, C/EBP homologous protein; CPT1, carnithine palmitoyltransferase 1; DGAT, diacylglycerol acyltransferase; DIO, diet-induced obese; ER, endoplasmic reticulum; FA, fatty acid; FAO, FA oxidation; FAS, fatty acid synthase; FFAs, free fatty acids; FOXO1, forkhead box protein O1; G6Pase, glucose 6-phophatase; HDL, high-density lipoprotein; H&E, hematoxilin and eosin; HFD, high-fat diet; HNMPA, hydroxy-2-naphthalenylmethylphosphonic acid; IP, intraperitoneally; IR, insulin receptor; IRE1a, inositol-requiring protein 1 alpha; IRS, insulin receptor substrate; LC-MS/MS, liquid chromatography/tandem mass spectrometry; LDL, low-density lipoprotein; LPS, lipopolysaccharide; mRNA, messenger RNA; NEFA, nonesterified fatty acid; OCR, oxygen consumption rate; ORO, Oil Red O; PCR, polymerase chain reaction; PEPCK, phosphoenolpyruvate carboxykinase; PERK, protein kinase RNA-like ER-associated kinase; PMHs, primary mouse hepatocytes; PPARa, peroxisome proliferation-activated receptor alpha; S1P, sphingosine 1 phosphate; SA, sphinganine; SEM, standard error of the mean; siRNA, small interfering RNA; SM, sphingomyelin; SO, sphingosine; Sphk, sphingosine kinase; SREBP, sterol responsive element-binding protein; sXBP1, spliced X-box-binding protein 1; TGs, triglycerides; UPR, unfolded protein response; uXBP1, unspliced XBP1; WT, wild type. From the 1Department of Life Science, Gachon University, Sungnam, Korea; 2Department of Food Science and Technology, Sejong University, Seoul, Korea; 3Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea; and 4Department of Physiology, Functional Genomics Institute, School of Medicine, Konkuk University, Seoul, Korea Received July 15, 2014; accepted March 20, 2015. Additional Supporting Information may be found at http://onlinelibrary.wiley.com/doi/10.1002/hep.27804/suppinfo 135
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synthesis of triglycerides (TGs), phospholipids, and sphingolipids. During this process, synthesis of specific lipid metabolites is known to provide the signaling molecules regulating the cascade of metabolic function.7 Lipid-mediated regulation of the cellular insulin-signaling cascade is associated with activation of serine/threonine kinases, leading to development of insulin resistance in insulin-sensitive tissues.8-10 Endoplasmic reticulum (ER) stress is induced by various cellular insults, including glucose deprivation, inflammatory state, and disruption of calcium homeostasis.11 Because ER is a principal organelle in cells, responsible for proper protein folding and maturation as well as protein trafficking to other cellular compartments, abnormal ER function by accumulation of unfolded protein elicits a signaling process termed the unfolded protein response (UPR) to alleviate ERassociated stress.11-13 UPR is initiated by release of glucose-regulated protein 78 from the ER and activation of three signaling cascades: the protein kinase RNA-like ER-associated kinase (PERK) pathway; the inositol-requiring protein 1 alpha (IRE1a) pathway; and the activating transcription factor (ATF)6 pathway.14 Chemical inducers of acute ER stress, such as tunicamycin and thapsigargin, induce these three UPR-signaling pathways, whereas the PERK pathway is activated in diet-induced obese (DIO) mice, indicating that each pathway has a distinctive role under different conditions of physiological stress.15-17 These transcription factors are implicated in expression of UPR genes, including chaperones and lipogenic genes. Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine and synthesize sphingosine 1 phosphate (S1P), a lysophospholipid.18 SphK was first identified from purification of 49-kDa protein from rat kidney, composed of Sphk1a and Sphk1b, which were cloned and characterized.19,20 A second isoform of Sphk2 is highly homologous to Sphk1, and both isoforms have five conserved domains found in lipid kinases.19 Whereas expression of Sphk1 is high in lung, spleen, kidney, and blood, Sphk2 is mainly expressed in
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liver, kidney, and heart.20,21 S1P levels are tightly regulated by synthesis catalyzed by SphKs, irreversible cleavage by S1P lyase, and dephosphorylation to produce sphingosine by S1P phosphatases.22 S1P is a ligand for a family of specific G-protein receptors (S1P1- S1P5). It exerts action by secretion and binding to the S1P G-protein receptors, a process called “inside-out” signaling.22 Despite their similarities and biochemical functions, Sphk2 has the opposite role as the antiapoptotic Sphk1, instead being proapoptotic.23 However, much less is known about the regulation of Sphk2 and its role in hepatic glucose and lipid metabolism. In this study, we investigated whether ER stress could trigger expression of Sphk2 and elevate hepatic S1P. Sphk2, a major SphK isotype in liver, was found to be up-regulated by acute ER stress. Hepatic overexpression of Sphk2 activated protein kinase B (AKT), independent of its proximal insulin signaling. As a result, glucose intolerance was improved and accumulation of lipid droplets was reduced by up-regulation of oxidative genes and increased FA oxidation (FAO). These results suggested that the UPR pathways regulate Sphk2 under distinctive physiological conditions, and that S1P production attenuates ER stress-mediated abnormalities through activation of FAO.
Materials and Methods Animal Experiments. All procedures were approved by the Gachon University (Sungnam, Korea) Institutional Animal Care and Use Committee. Detailed methods are provided in the Supporting Experimental Procedures. Preparation and Culture of Primary Hepatocytes. Eight week-old wild-type (WT) C57BL6 male mice were anesthetized and livers were isolated. Primary hepatocytes were prepared as described previously24 and are detailed in the Supporting Experimental Procedures. Preparation of Plasmids and Recombinant Adenovirus. The promoter sequence of Sphk2 was amplified by polymerase chain reaction (PCR) from mouse
This research was supported by the Basic Science Research Program and the Bio & Medical Technology Development Program through the National Research Foundation of Korea (NRF), funded by the Korean government (NRF-2011-0029583, NRF-2013R1A1A2006229, and NRF-2014M3A9B6069338; to T.S.P.). View this article online at wileyonlinelibrary.com. *These authors contributed equally to this study. Address reprint requests to: Tae-Sik Park, Ph.D., Department of Life Science, Gachon University, Sungnam, Gyeonggido 261-701, South Korea. E-mail: [email protected]; fax: 182-31-750-8573. C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27804 Potential conflict of interest: Nothing to report.
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genomic DNA and was inserted into the pGL3 basic vector (Promega, Madison, WI), generating a pSphk2pGL3 reporter construct. Sphk2 recombinant adenoviruses (AdSphk2) were constructed using an AdEasy Adenoviral Vector System (Stratagene, La Jolla, CA) and the pAdTrack CMV vector, as described previously.25 Transfection and Luciferase Reporter Assay. HepG2 cells were transiently cotransfected with pSphk2-pGL3 reporter construct and ER stress marker cloning vectors containing ATF4, ATF6, or sXBP1pcDNA3.0, respectively. An expression vector for Renilla luciferase, pTK-RL, was used to normalize transfection efficiency. RNA Preparation and Quantitative Real-Time PCR. Total RNA was isolated from mouse primary hepatocyte and liver tissues, and, subsequently, synthesized complementary DNA was analyzed to measure gene expression. Primer sequences used in this study are provided in Supporting Table 1. Western Blotting Analyses. Whole cell proteins were lysed using cell lysis buffer. Thirty micrograms of protein were used for immunoblotting, as described previously.26 The blots were developed with the enhanced chemiluminescent substrate (Millipore, Billerica, MA) and detected with a LAS4000 luminescent image analyzer (Fujifilm, Tokyo, Japan). Measurement of Metabolites. Blood glucose levels, plasma, and hepatic TGs, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and nonesterified fatty acid (NEFA) were measured as described in the Supporting Experimental Procedures. Sphingolipid levels in plasma and liver were measured by liquid chromatography/tandem mass spectrometry (LC-MS/MS), as described previously.26 Histology. For histological analyses, livers were isolated and frozen in optimal cutting temperature embedding medium. Paraffin embedded 5-lm sections of the liver were stained with hematoxilin and eosin (H&E; Sigma-Aldrich, St. Louis, MO) or Oil Red O (ORO). Statistical Analyses. Results are shown as mean 6 standard error of the mean (SEM). Comparison of different groups was carried out using the two-tailed unpaired Student t test. P values less than 0.05 (P < 0.05) were considered statistically significant.
Results ER Stress Up-Regulates Sphk2 Expression in Mouse Primary Hepatocytes and In Vivo. Recent reports suggested that ER stress activates hepatic lipid
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biosynthesis by up-regulating key lipogenic genes, such as DGAT2, sterol responsive element-binding protein (SREBP)1c, and LIPIN2.27 Given that sphingolipid biosynthesis is a nonoxidative FA pathway, we investigated whether ER stress could regulate biosynthesis of sphingolipids, which are known signaling lipids of metabolic dysregulation. To test this hypothesis in vivo, tunicamycin, an acute ER stress inducer, was intraperitoneally (IP) administered into WT mice and livers were isolated to measure expression of sphingolipid biosynthesizing genes. Among the sphingolipid biosynthetic genes, ceramide synthase (CerS)3, alkaline ceramidase (Acer)2, and Acer3 were up-regulated (Fig. 1A). Expression of UPR genes, including ATF4, ATF6, C/EBP homologous protein (CHOP), spliced X-box-binding protein 1 (sXBP1), and unspliced XBP1 (uXBP1), was up-regulated (Fig. 1B). Sphk2, the endpoint of this up-regulation in the sphingolipid biosynthetic pathway, was transcriptionally induced by tunicamycin in liver 6 hours after tunicamycin administration. When primary mouse hepatocytes (PMHs) were treated with tunicamycin, Sphk2, and messenger RNA (mRNA) and protein levels were induced in a time-dependent manner (Fig. 1C,D). In contrast, Sphk1, another isotype of SphK, was rarely detected in any of the conditions (Fig. 1B). These results suggested that Sphk2 is transcriptionally regulated by acute ER stress, aroused by tunicamycin. High-Fat Diet and Endotoxin Activate ER Stress, but Regulate Sphk2 Expression Differently. Because tunicamycin induces expression of all UPR genes, we aimed to investigate which ER stress-dependent signaling pathway is directly associated with Sphk2 upregulation. To test whether Sphk2 is regulated by ER stress in a physiological setting, mice were fed a highfat diet (HFD) for 4 weeks to generate hyperlipidemic ER stress conditions. Hepatic Sphk2 mRNA and protein were found to be down-regulated in livers of mice fed an HFD, compared to those of control mice (Fig. 2A,B). Whereas the longer period of an HFD feeding (8 and 12 weeks of HFD feeding; Supporting Fig. 1) did not alter UPR mediators, hepatic sXBP1 activation occurred in mice fed a 4-week HFD, but ATF4 was down-regulated with no change in ATF6 (Fig. 2A,B). HFD elevated ceramide and sphingomyelin (SM) levels in plasma and liver, but S1P was decreased with no statistical significance (Supporting Fig. 2). Another ER stress activator, lipopolysaccharide (LPS), induced UPR genes such as ATF4 and CHOP, but not sXBP1 (Fig. 2C,D). Despite the fact that most of the lipogenic genes were down-regulated, Sphk2 mRNA and protein levels were elevated (Fig. 2C,D). These results
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Fig. 1. ER stress induces expression of sphingolipid biosynthetic genes in liver. WT C57Bl/6 mice were injected IP with DMSO or tunicamycin (2.5 lg/g body weight; n 5 5 per group), and livers were isolated 6 hours after injection (A and B). Hepatic mRNA was extracted for quantitative PCR analyses. n 5 5, mean 6 SEM, *P < 0.05. Primary mouse hepatocytes were treated with tunicamycin (1.25 lg/mL) or DMSO for the indicated times before being harvested. Quantitative reverse-transcriptase PCR analysis was performed to determine Sphk2 expression (C). n 5 3, mean 6 SEM, *P < 0.05 versus controls. PMHs were treated with tunicamycin (1.25 lg/mL) or DMSO for 12 hours and harvested. Sphk2 protein amounts were analyzed by SDS-PAGE followed by immunoblotting (D). Abbreviations: AcCer, acid ceramidase; Acer1-3. alkaline ceramidase1-3; CerS2-5, ceramide synthase25; DMSO, dimethyl sulfoxide; NCer, neutral ceramidase; Smpd1, acid sphingomyelinase; Smpd2, neutral sphingomyelinase 1; Smpd3, neutral sphingomyelinase 2; TUN, tunicamycin.
suggested that LPS-mediated ER stress induces Sphk2, but regulation of Sphk2 is dependent on the pathophysiological conditions for ER stress induction. Activation of ATF4 Transcriptionally Up-Regulates Sphk2. To examine this differential regulation of Sphk2 by ATF4 and sXBP1, the reporter construct containing the Sphk2 promoter was cotransfected with ATF4, sXBP1, or CHOP, respectively, and measured Sphk2 expression. We found that ATF4 mediated transcriptional induction of Sphk2 by 6-fold, whereas
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sXBP1 suppressed Sphk2 expression (Fig. 3A). Because CHOP is a direct transcriptional target of ATF4, we overexpressed CHOP and examined its effects on Sphk2 expression. However, no change was found in Sphk2 promoter activity, suggesting that Sphk2 expression was only induced by ATF4 activation. In support of these results, adenoviral overexpression of ATF4 elevated Sphk2 protein levels in PMHs, whereas no alteration was found with sXBP1 overexpression (Fig. 3B). To confirm whether Sphk2 is a direct downstream target of ATF4, we transfected ATF4 small interfering RNA (siRNA). Down-regulation of ATF4 resulted in suppression of Sphk2 mRNA and protein levels (Fig. 3C,D). These results suggested that Sphk2 is transcriptionally regulated by ATF4. Sphk2-Mediated S1P Production Activates AKT Phosphorylation. The implication of ceramide and S1P in modulation of the insulin-signaling pathway has been reported on.28,29 S1P, a product of Sphk, is thought to improve glucose homeostasis in diabetic animal models.29 To test whether elevated Sphk2 expression regulates ER stress-dependent signaling pathways, adenovirus expressing human Sphk2 (AdSphk2) was constructed and used to infect PMHs. Phosphorylation of AKT was enhanced by AdSphk2 infection in a gene-dose–dependent manner, as reported previously (Fig. 4A).29 However, increased pAKT was not associated with the proximal insulinsignaling intermediate protein, (IRS)1 or IRS2, which is evidenced by the lack of alteration of pIRS1 or pIRS2 levels (Fig. 4A). Thus, the increased pAKT was independent of tyrosine phosphorylation of IRS. Whereas sphingolipid metabolites, such as sphinganine (SA), SO (sphingosine), ceramide, and SM, were not altered, only cellular S1P levels were elevated by Sphk2 expression (Fig. 4B-D). To confirm whether upstream insulin signaling is involved in phosphorylation of AKT in cells overexpressing Sphk2, we administered hydroxy-2-naphthalenylmethylphosphonic acid (HNMPA), an insulin receptor (IR) antagonist, and examined insulin response. We found that pAKT levels in response to insulin were not changed by IR inhibition in cells overexpressing Sphk2 (Fig. 4E). In contrast, phosphorylation of AKT by insulin was decreased in the control by HNMPA-mediated IR inhibition. These data support the notion that elevated hepatic S1P, induced by Sphk2, activates AKT independent of the insulin-signaling pathway. Hepatic Sphk2 Expression Alters Lipid Profiles in Plasma and Liver. To examine whether elevated hepatic expression of Sphk2 results in physiological alteration, WT mice fed an HFD for 4 weeks were
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Fig. 2. Hyperlipidemic and inflammatory ER stresses regulate Sphk2 differently. WT C57Bl/6 mice were fed a normal chow diet (NCD) or a 60 kcal% HFD for 4 weeks and livers were collected. Quantitative reverse-transcriptase PCR was performed with hepatic RNA (A). Immunoblotting analyses were performed with tissue lysates from mouse liver as described in Materials and Methods (B). WT C57Bl/6 mice were injected with either saline or LPS IP, and livers were collected 8 hours postinjection. Gene expression was measured by quantitative reverse-transcriptase PCR using hepatic mRNA as shown in (A) (C). Western blotting analysis was performed with tissue lysates from mouse livers (D). n 5 6, mean 6 SEM, *P < 0.05.
injected with AdSphk2 by the tail vein. Previous studies in rodents reported that delivery of recombinant adenoviruses resulted in preferential targeting of the transgene to the liver.30 Whereas plasma TG, HDL, and NEFA remained unchanged, total cholesterol and LDL levels were reduced. In addition, overexpression of Sphk2 did not change body weight or plasma glucose levels (Supporting Table 2). Interestingly, liver enzymes, such as alanine aminotransferase, were reduced, suggesting that hepatic function was improved. Levels of ceramide, SA, SO, SM, and glucosylceramide were all found to be reduced, whereas only S1P was elevated in plasma and liver of mice overexpressing Sphk2 (Supporting Fig. 3 and Fig. 5AE). In contrast to plasma levels, the observation that hepatic TG was reduced suggests that synthesis or oxidation was regulated by Sphk2 (Fig. 5F). All these findings of lipid profiling suggest that hepatic Sphk2 expression influences regulation of lipid metabolism in mice fed a HFD. Hepatic Sphk2 Up-Regulation Reduces Hepatic Lipid Accumulation. The result that hepatic TG levels were significantly diminished suggested that hepatic lipid metabolism may be regulated by Sphk2.
To verify the role of Sphk2 in hepatic lipid metabolism, expression of the genes involved in lipid biosynthesis and FAO was examined. Indeed, expression of genes related to FAO, such as PPARa (peroxisome proliferator-activated receptor alpha), CPT1 (carnitine palmitoyltransferase), and ACOX1 (acyl CoA oxidase), was up-regulated by Sphk2 overexpression, despite the lack of changes in lipogenic genes, including FA synthase (FAS), SREBP1c, acyl CoA carboxylase (ACC), and diacylglycerol acyltransferase (DGAT)2 (Fig. 6A). Immunoblotting analyses showed that levels of protein from these genes were elevated in Sphk2-overexpressed livers (Fig. 6B). In addition, ORO staining of liver tissues demonstrated that livers of AdSphk2-injected mice had a significant reduction in lipid droplets, compared to control, which is consistent with reduced hepatic TG levels (Figs. 5F and 6C). As a result of increased FAO, plasma b-hydroxybutyrate was elevated in AdSphk2-injected mice (Fig. 6D). To verify this further, we infected PMHs with AdSphk2 and measured the oxygen consumption rate (OCR). Addition of palmitic acid increases OCR because of increased substrate availability for FAO. We found that AdSphk2 infection increased OCR in the presence of palmitic
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Fig. 3. ATF4 up-regulates Sphk2. (A) HepG2 cells were cotransfected with Sphk2 reporter construct and 0.2 or 0.4 lg of pcDNA 3.0 containing ATF4, sXBP1, or CHOP. Promoter activity was measured by luciferase assay. Representative data from three independent experiments are shown (A). n 5 5, mean 6 SEM, *P < 0.05 versus control empty pcDNA3.0 control. #P < 0.05 versus 0.2 lg transfected group. PMHs were infected with AdGFP as a control, AdATF4, or AdsXBP1 for 24 hours. Immunoblotting analysis was performed with cell lysates (B). HepG2 cells were transfected with the control or ATF4 siRNA followed by tunicamycin treatment (1.25 lg/mL) for 6 hours. Then, cells were harvested, and mRNA expression was measured by quantitative reverse-transcriptase PCR. n 5 3, mean 6 SEM, *P < 0.05 versus control siRNA (C). Western blotting analysis was performed with cell lysates (D).
acid in a time-dependent manner, compared to either AdGFP-infected control or no palmitic acid control (Fig. 6E). These findings suggest that Sphk2 expression increases FAO and ameliorates the hepatic steatosis induced by HFD. Hepatic Sphk2 Expression Improves Glucose Intolerance. To explore the role of hepatic Sphk2 in insulin sensitivity and glucose metabolism, we analyzed glucose and insulin response in AdSphk2-injected mice. Despite the lack of change in basal blood glucose levels, mice overexpressing Sphk2 improved glucose intolerance when being fed an HFD (Fig. 7A). Plasma insulin levels were not significantly different between control and Sphk2-overexpressing mice during the glucose tolerance test, suggesting that the improved glucose intolerance was the result of the enhanced plasma glucose clearance without alteration of insulin secretion (Fig. 7B). Sphk2 overexpression also improved insulin tolerance (Fig. 7C). Improved glucose intolerance and insulin response suggest that ele-
vated Sphk2 expression is associated with regulation of insulin signaling in response to ER stress, which is induced by HFD. To verify the effects of Sphk2 upregulation on insulin signaling, immunoblotting analyses of the insulin-signaling proteins was performed. Although the basal conditions did not alter phosphorylation of AKT and forkhead box protein O1 (FOXO1), insulin caused increased phosphorylation of AKT and its downstream FOXO1 in mice overexpressing Sphk2 (Fig. 7D). Expression of gluconeogenic genes, including glucose 6-phophatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), were not changed (Fig. 7D and Supporting Fig. 4). To determine whether nonhepatic tissues are involved in improved glucose or insulin tolerance, we measured AKT phosphorylation in skeletal muscles and adipose tissues. However, we did not find any changes in pAKT levels (Fig. 7E,F). Sphk2 inhibition by K145, a selective Sphk2 inhibitor,31 did not alter glucose tolerance, hepatic cholesterol and TG levels, plasma S1P,
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Fig. 4. Sphk2-mediated S1P production increases Akt phosphorylation in hepatocytes. PMHs were infected with AdGFP or AdSphk2 for 24 hours in a gene-dose– dependent manner. Cells were harvested, and cell lysates were analyzed by immunoblotting (A). Representative data from three independent experiments are shown. PMHs were infected with AdGFP or AdSphk2 for 24 hours at 5 multiplicities of infection (MOI). Sphingoid bases (B), ceramide (C), and SM (D) were measured by LCMS/MS, as described in Materials and Methods. n 5 3, mean 6 SEM, *P < 0.05 versus AdGFP. HepG2 cells were infected with AdGFP or AdSphk2 for 24 hours at 5 MOI and treated with 50 nM of insulin for 10 minutes. Another set of cells were treated with HNMPA-(AM)3 for 6 hours and then insulin treatment for 10 minutes. Cells were harvested, and cell lysates were analyzed by immunoblotting (E). Abbreviation: Cer, ceramide.
and hepatic lipid droplets in AdSphk2-injected mice (Fig. 8A-D). These results suggest that Sphk2 expression did not reach the threshold levels that could regulate glucose metabolism in basal conditions, but the response to insulin became more sensitized by Sphk2mediated activation of the insulin-signaling pathway in conditions of insulin resistance.
Discussion ER is a principal cellular organelle responsible for protein maturation and trafficking to other cellular compartments. Accumulation of unfolded proteins stresses the ER, which is involved in development of metabolic dysfunction and inflammatory disease.32 Excessive intake of nutrients and infection-mediated
inflammatory response are physiological conditions that put stress on ER structure and function, then triggering the UPR to maintain ER homeostasis. When the condition of ER stress continues, lipid homeostasis would be perturbed and cellular messengers, such as diacylglycerol and FFA, disturb cellular signaling. In this study, to elucidate a link between ER stress and sphingolipid biosynthesis, we found the following: (1) Sphk2-mediated biosynthesis of S1P was activated by inflammatory ER stress in liver; (2) Sphk2 is upregulated by ATF4; and (3) hepatic up-regulation of Sphk2 activates FAO and improves hepatosteatosis as well as glucose metabolism. ER stress-induced UPR is mediated by three ER membrane-associated proteins: PERK, IRE1a, and ATF6. In these pathways, ER stress-activated transcription
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Fig. 5. Overexpession of Sphk2 elevates S1P, but decreases ceramide, SM, glucosylceramide, cholesterol, and TG in liver. WT C57Bl/6 mice fed an HFD (60 kcal% fat) for 4 weeks were infected with AdGFP or AdSphk2 (1 3 109 PFU) by tail vein injection. At 14 days postinjection, mice livers were isolated. Ceramide (A), dihydroceramide (B), sphingoid bases (C), SM (D), and glucosylceramide (E) in the liver were measured by LC-MS/MS, as described in Materials and Methods. After extraction of the lipids in liver tissue, total cholesterols and TG were measured by colorimetric methods (F). n 5 7-8, mean6 SEM, *P < 0.05 versus AdGFPinjected mouse. Abbreviation: PFU, plaque-forming units.
factors, such as ATF4, XBP1, and ATF6, induce various chaperones to correct the dysfunctions caused by accumulation of unfolded protein. Excessive UPR signaling leads to metabolic perturbation, including obesity and fatty liver. Each UPR-signaling pathway has a different function in metabolic regulation. For example, XBP1 deficiency results in suppression of de novo hepatic lipid biosynthesis, leading to decreased plasma TG, cholesterol, and FFAs.33 In contrast, ATF4 is activated in DIO mice, activating adipogenesis, and PERK-eIF2a(-ATF4) is involved in activation of inflammatory nuclear factor kappa B.34,35 Whereas there is crosstalk between these UPR branches, metabolic dysregulation and inflammation are the major fields for UPR pathways to be involved in stress-signaling pathways. A nonoxidative FA pathway is involved in biosynthesis of sphingolipids, and their metabolism leads to
biosynthesis of various bioactive lipid metabolites, including ceramide, SO, and S1P. These sphingolipid metabolites are important signaling messengers in metabolic regulation.36,37 Infusion of saturated FA results in elevation of ceramide levels in muscle and liver, contributing to peripheral insulin resistance.9 Pharmacological and genetic inhibition of de novo ceramide biosynthesis has been shown to improve glucose intolerance and insulin response in myriocin-treated DIO or in heterozygous Sptlc2-deficient mice.38,39 In contrast, adenoviral gene transfer of Sphk1 markedly reduced blood glucose levels, as well as TG and cholesterol.29 These findings implicate ceramide and S1P in hepatic glucose/lipid metabolism in response to the state of nutrition. Recent findings suggested that lipid metabolism is perturbed by ER stress-activated transcription factors,
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Fig. 6. Hepatic Sphk2 overexpression activates FA oxidation, decreases lipid droplets, and increases ketone body. WT C57bl6/J mice fed an HFD (60 kcal% fat) were injected with either AdGFP or AdSphk2 (1 3 109 PFU) by the tail vein. At 14 days postinjection, livers and plasma were isolated. Expression of genes involved in FA biosynthesis and oxidation were determined by quantitative reverse-transcriptase PCR (A). n 5 7, mean 6 SEM, *P < 0.05. At 14 days postinjection, immunoblotting analyses were performed using liver lysates (B). Livers were isolated and frozen sections were stained with ORO (upper) and alternatively with H&E (lower) (C). Representative pictures were taken at 403 magnification. b-hydroxybutyrate (ketone body) in plasma were measured as described in Materials and Methods (D). n 5 7, mean 6 SEM, *P < 0.05. PMHs were infected with AdGFP and AdSphk2 at 5 multiplicities of infection, then treated with bovine serum albumin/palmitate complexes and incubated for 2 hours, and oxygen consumption rate was measured at indicated time points (E). n 5 5, mean 6 SEM, *P < 0.05.
which implies that sphingolipid biosynthesis may also be regulated by ER stress in conjunction with other lipid biosynthesis. Feeding of an HFD to cause subchronic ER stress activation (4 weeks) resulted in different expression profiles of the UPR proteins from those observed in DIO mice (8 and 12 weeks; Supporting Fig. 1). Activation of sXBP1 and suppression
of ATF4 were observed in this subchronic condition (Fig. 2A,B). On the other hand, LPS-induced inflammatory ER stress only activated ATF4, leading to Sphk2 induction, suggesting that onset of ER stress is dependent on the various stimuli (Fig. 2C). This might be owing to the different activation of UPR pathways by hyperlipidemic and inflammatory ER
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Fig. 7. Sphk2 overexpression improves glucose and insulin intolerance in mice fed an HFD. WT C57Bl/6 mice fed an HFD (60 kcal% fat) for 4 weeks were injected with either AdGFP or AdSphk2 (1 3 109) by tail vein injection. Experiments were performed 7 days after adenoviral injection. Mice were fasted for 16 hours and glucose was injected IP at 1 g/kg body weight, then plasma glucose was measured at the indicated times. (A). Plasma insulin levels during the glucose tolerance test were measured (B). Insulin (1 U/kg body weight) was injected IP into mice after 4 hours of fasting, and plasma glucose was measured at the indicated times. n 5 7 for AdGFP and n 5 8 for AdSphk2, mean 6 SEM, *P < 0.05 vs. AdGFP (C). Mice were divided into two groups. One group was used as a control, and the other was injected with insulin (0.5 U/ kg body weight) IP for 10 minutes. Livers were isolated and western blotting analysis was performed on insulin-signaling proteins (D). Skeletal muscles (E) and adipocytes (F) were isolated and western blotting analysis was performed. Representative data from two independent experiments are shown (n 5 6 for each group).
stress, and each UPR pathway selectively modulates the cause-oriented target of stress signaling. In response to inflammatory ER stress, such as that caused by LPS, hepatocytes might request an urgent response through the combined effects of inflammatory response and ER stress by Sphk2 induction, mainly by ATF4. Although both XBP1 and ATF4 account for hepatic lipid metabolism, S1P biosynthesis is regulated differently, at least in the liver. We speculated that Sphk2 expression is differentially regulated by UPR transcription factors under different pathophysiological conditions. The stress-signaling pathways linked to
ATF4-mediated hepatic Sphk2 up-regulation deserve further studies to clarify the role of Sphk2 and its product, S1P, in metabolic/inflammatory regulation. Previous reports suggested that delivery of the Sphk1 gene improves insulin resistance and lipid abnormalities in KK/Ay diabetic mice by activation of insulin signaling, and these results may be through elevated hepatic S1P.29 However, Sphk1 is rarely expressed in liver and Sphk2 is the major Sphk isoform producing hepatic S1P. We found that adenoviral expression of Sphk2 activated the AKT pathway independent of IRS. This finding suggests that Sphk
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Fig. 8. Sphk2 inhibition by K145 did not alter glucose metabolism and lipid levels. Glucose tolerance tests were performed after HFD (45 kcal% fat) and injected with K145 (20 mg/ kg/2 days) IP for 2 weeks every other day. Mice were fasted for 16 hours and glucose was injected IP at 1 g/ kg body weight, then plasma glucose was measured at the indicated times (A). n 5 9 for control and n 5 7 for K145. TG and cholesterol were measured in mice liver by colorimetric assay (B). S1P level in plasma was measured by LC-MS/MS (C). Livers were isolated and frozen sections were stained with ORO (upper) and alternatively with H&E (lower) (D). Representative pictures were taken at 403 magnification.
contributes to regulation of hepatic glucose metabolism. Improvement of glucose intolerance in mice fed an HFD by hepatic Sphk2 expression supports the notion that elevated levels of S1P possibly act as an activator of insulin signaling in hyperglycemic conditions by increasing pAKT, as reported previously by Guan et al.40 However, adenoviral Sphk2 upregulation does not alter basal glucose levels in vivo, but only insulin response by activating the signaling pathway through increased pAKT. Independently, whereas plasma TG remained unchanged, hepatic TG accumulation was reduced by Sphk2 expression. We found that reduction of hepatic TG was the result of up-regulation of FA oxidative genes and increased FAO. The notion that AdSphk2-infected hepatocytes have increased oxygen consumption and elevated plasma ketone bodies suggests that Sphk2 expression is an important regulator of FAO. However, the results that pharmacological Sphk2 inhibition did not change glucose/FA metabolism suggest that there might be a compensatory mechanism by hepatic Sphk2. The mechanism of Sphk2-mediated activation of hepatic FAO and a role of S1P deserves further study.
In this study, we demonstrated that ER stressmediated Sphk2 up-regulation occurs through ATF4 activation under inflammatory conditions and improves hepatic glucose and lipid abnormalities. This raises the question of why ER stress-mediated Sphk2 activation improves glucose intolerance and FAO. One possibility is that UPR-signaling pathways are adaptive processes for alleviation of stressed states of ER by activating FAO. ATF4-mediated Sphk2 up-regulation in response to inflammatory ER stress modulates cellular signaling. As a result, Sphk2 up-regulation leads to reduction of lipid droplets by increased FAO. Additionally, Sphk2-mediated activation of AKT improved insulin signaling in pathophysiological conditions. Taken together, Sphk2 is regulated by the UPR sensors, depending on the causes of ER stress induction, and acts as a molecular messenger to relieve hepatic ER stress and maintain glucose and lipid homeostasis. Acknowledgment: The authors thank Dr. SeungHoi Koo (Korea University) for providing the sXBP1-, ATF4- and CHOP adenoviruses and overexpression vectors.
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References 1. Barr EL, Cameron AJ, Balkau B, Zimmet PZ, Welborn TA, Tonkin AM, Shaw JE. HOMA insulin sensitivity index and the risk of allcause mortality and cardiovascular disease events in the general population: the Australian Diabetes, Obesity and Lifestyle Study (AusDiab) study. Diabetologia 2010;53:79-88. 2. Hastie CE, Padmanabhan S, Slack R, Pell AC, Oldroyd KG, Flapan AD, et al. Obesity paradox in a cohort of 4880 consecutive patients undergoing percutaneous coronary intervention. Eur Heart J 2010;31: 222-226. 3. Roberts AW, Clark AL, Witte KK. Review article: left ventricular dysfunction and heart failure in metabolic syndrome and diabetes without overt coronary artery disease—do we need to screen our patients? Diab Vasc Dis Res 2009;6:153-163. 4. Bradbury MW. Lipid metabolism and liver inflammation. I. Hepatic fatty acid uptake: possible role in steatosis. Am J Physiol Gastrointest Liver Physiol 2006;290:G194-G198. 5. Zhou YT, Grayburn P, Karim A, Shimabukuro M, Higa M, Baetens D, et al. Lipotoxic heart disease in obese rats: implications for human obesity. Proc Natl Acad Sci U S A 2000;97:1784-1789. 6. Unger RH. Lipid overload and overflow: metabolic trauma and the metabolic syndrome. Trends Endocrinol Metab 2003;14:398-403. 7. Listenberger LL, Han X, Lewis SE, Cases S, Farese RV, Jr., Ory DS, Schaffer JE. Triglyceride accumulation protects against fatty acidinduced lipotoxicity. Proc Natl Acad Sci U S A 2003;100:3077-3082. 8. Chavez JA, Summers SA. Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes. Arch Biochem Biophys 2003;419:101-109. 9. Holland WL, Brozinick JT, Wang LP, Hawkins ED, Sargent KM, Liu Y, et al. Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturatedfat-, and obesity-induced insulin resistance. Cell Metab 2007;5:167-179. 10. Yu C, Chen Y, Cline GW, Zhang D, Zong H, Wang Y, et al. Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 2002;277:50230-50236. 11. Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin Invest 2002;110:1389-1398. 12. Hampton RY. ER stress response: getting the UPR hand on misfolded proteins. Curr Biol 2000;10:R518-R521. 13. Schroder M, Kaufman RJ. The mammalian unfolded protein response. Annu Rev Biochem 2005;74:739-789. 14. Malhi H, Kaufman RJ. Endoplasmic reticulum stress in liver disease. J Hepatol 2011;54:795-809. 15. Winnay JN, Boucher J, Mori MA, Ueki K, Kahn CR. A regulatory subunit of phosphoinositide 3-kinase increases the nuclear accumulation of X-box-binding protein-1 to modulate the unfolded protein response. Nat Med 2010;16:438-445. 16. Wang Y, Vera L, Fischer WH, Montminy M. The CREB coactivator CRTC2 links hepatic ER stress and fasting gluconeogenesis. Nature 2009;460:534-537. 17. Park SW, Zhou Y, Lee J, Lu A, Sun C, Chung J, et al. The regulatory subunits of PI3K, p85alpha and p85beta, interact with XBP-1 and increase its nuclear translocation. Nat Med 2010;16:429-437. 18. Maceyka M, Payne SG, Milstien S, Spiegel S. Sphingosine kinase, sphingosine-1-phosphate, and apoptosis. Biochim Biophys Acta 2002; 1585:193-201. 19. Taha TA, Hannun YA, Obeid LM. Sphingosine kinase: biochemical and cellular regulation and role in disease. J Biochem Mol Biol 2006; 39:113-131. 20. Billich A, Bornancin F, Devay P, Mechtcheriakova D, Urtz N, Baumruker T. Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases. J Biol Chem 2003;278:47408-47415. 21. Kihara A, Anada Y, Igarashi Y. Mouse sphingosine kinase isoforms SPHK1a and SPHK1b differ in enzymatic traits including stability, localization, modification, and oligomerization. J Biol Chem 2006;281:4532-4539.
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22. Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 2003;4:397-407. 23. Maceyka M, Sankala H, Hait NC, Le Stunff H, Liu H, Toman R, et al. SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism. J Biol Chem 2005;280:37118-37129. 24. Renton KW, Deloria LB, Mannering GJ. Effects of polyribonoinosinic acid polyribocytidylic acid and a mouse interferon preparation on cytochrome P-450-dependent monooxygenase systems in cultures of primary mouse hepatocytes. Mol Pharmacol 1978;14:672-681. 25. Luo J, Deng ZL, Luo X, Tang N, Song WX, Chen J, et al. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc 2007;2:1236-1247. 26. Lee SY, Kim JR, Hu Y, Khan R, Kim SJ, Bharadwaj KG, et al. Cardiomyocyte specific deficiency of serine palmitoyltransferase subunit 2 reduces ceramide but leads to cardiac dysfunction. J Biol Chem 2012;287:18429-18439. 27. Ryu D, Seo WY, Yoon YS, Kim YN, Kim SS, Kim HJ, et al. Endoplasmic reticulum stress promotes LIPIN2-dependent hepatic insulin resistance. Diabetes 2011;60:1072-1081. 28. Summers SA. Sphingolipids and insulin resistance: the five Ws. Curr Opin Lipidol 2010;21:128-135. 29. Ma MM, Chen JL, Wang GG, Wang H, Lu Y, Li JF, et al. Sphingosine kinase 1 participates in insulin signalling and regulates glucose metabolism and homeostasis in KK/Ay diabetic mice. Diabetologia 2007;50:891-900. 30. Nakagawa Y, Shimano H, Yoshikawa T, Ide T, Tamura M, Furusawa M, et al. TFE3 transcriptionally activates hepatic IRS-2, participates in insulin signaling and ameliorates diabetes. Nat Med 2006;12:107-113. 31. Liu K, Guo TL, Hait NC, Allegood J, Parikh HI, Xu W, et al. Biological characterization of 3-(2-amino-ethyl)25-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145) as a selective sphingosine kinase-2 inhibitor and anticancer agent. PLoS One 2013;8:e56471. 32. Hotamisligil GS. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 2010;140:900-917. 33. Lee AH, Scapa EF, Cohen DE, Glimcher LH. Regulation of hepatic lipogenesis by the transcription factor XBP1. Science 2008;320:1492-1496. 34. Seo J, Fortuno ES, 3rd, Suh JM, Stenesen D, Tang W, Parks EJ, et al. Atf4 regulates obesity, glucose homeostasis, and energy expenditure. Diabetes 2009;58:2565-2573. 35. Deng J, Lu PD, Zhang Y, Scheuner D, Kaufman RJ, Sonenberg N, et al. Translational repression mediates activation of nuclear factor kappa B by phosphorylated translation initiation factor 2. Mol Cell Biol 2004;24:10161-10168. 36. Hannun YA, Luberto C, Argraves KM. Enzymes of sphingolipid metabolism: from modular to integrative signaling. Biochemistry 2001; 40:4893-4903. 37. Park TS, Panek RL, Mueller SB, Hanselman JC, Rosebury WS, Robertson AW, et al. Inhibition of sphingomyelin synthesis reduces atherogenesis in apolipoprotein E-knockout mice. Circulation 2004;110:3465-3471. 38. Ussher JR, Koves TR, Cadete VJ, Zhang L, Jaswal JS, Swyrd SJ, et al. Inhibition of de novo ceramide synthesis reverses diet-induced insulin resistance and enhances whole-body oxygen consumption. Diabetes 2010;59:2453-2464. 39. Li Z, Zhang H, Liu J, Liang CP, Li Y, Teitelman G, et al. Reducing plasma membrane sphingomyelin increases insulin sensitivity. Mol Cell Biol 2011;31:4205-4218. 40. Guan H, Song L, Cai J, Huang Y, Wu J, Yuan J, et al. Sphingosine kinase 1 regulates the Akt/FOXO3a/Bim pathway and contributes to apoptosis resistance in glioma cells. PLoS One 2011;6:e19946.
Author names in bold designate shared co-first authorship.
Supporting Information Additional Supporting Information may be found at http://onlinelibrary.wiley.com/doi/10.1002/hep.27804/ suppinfo
Activation of Sphingosine Kinase 2 by Endoplasmic Reticulum Stress Ameliorates Hepatic Steatosis and Insulin Resistance in Mice Su-Yeon Lee,1* In-Kyung Hong,1* Bo-Rahm Kim,1 Soon-Mi Shim,2 Jae Sung Lee,3 Hui-Young Lee,3 Cheol Soo Choi,3 Bo-Kyung Kim,4 and Tae-Sik Park1 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)
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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.1-3 Elevated plasma free fatty acids (FFAs)
induce abnormal fatty acid (FA) uptake by liver, adipose tissue, and heart, which contributes to induction of hepatic steatosis, inflammation, and cardiac dysfunction, respectively.4-6 Increased tissue uptake of FFAs under conditions of obesity would trigger the
Abbreviations: ACC, acyl CoA carboxylase; Acer, alkaline ceramidase; ACOX1, acyl CoA oxidase 1; AdSphk2, adenovirus expressing human Sphk2; AKT, protein kinase B; ATF, activating transcription factor; CerS, ceramide synthase; CHOP, C/EBP homologous protein; CPT1, carnithine palmitoyltransferase 1; DGAT, diacylglycerol acyltransferase; DIO, diet-induced obese; ER, endoplasmic reticulum; FA, fatty acid; FAO, FA oxidation; FAS, fatty acid synthase; FFAs, free fatty acids; FOXO1, forkhead box protein O1; G6Pase, glucose 6-phophatase; HDL, high-density lipoprotein; H&E, hematoxilin and eosin; HFD, high-fat diet; HNMPA, hydroxy-2-naphthalenylmethylphosphonic acid; IP, intraperitoneally; IR, insulin receptor; IRE1a, inositol-requiring protein 1 alpha; IRS, insulin receptor substrate; LC-MS/MS, liquid chromatography/tandem mass spectrometry; LDL, low-density lipoprotein; LPS, lipopolysaccharide; mRNA, messenger RNA; NEFA, nonesterified fatty acid; OCR, oxygen consumption rate; ORO, Oil Red O; PCR, polymerase chain reaction; PEPCK, phosphoenolpyruvate carboxykinase; PERK, protein kinase RNA-like ER-associated kinase; PMHs, primary mouse hepatocytes; PPARa, peroxisome proliferation-activated receptor alpha; S1P, sphingosine 1 phosphate; SA, sphinganine; SEM, standard error of the mean; siRNA, small interfering RNA; SM, sphingomyelin; SO, sphingosine; Sphk, sphingosine kinase; SREBP, sterol responsive element-binding protein; sXBP1, spliced X-box-binding protein 1; TGs, triglycerides; UPR, unfolded protein response; uXBP1, unspliced XBP1; WT, wild type. From the 1Department of Life Science, Gachon University, Sungnam, Korea; 2Department of Food Science and Technology, Sejong University, Seoul, Korea; 3Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea; and 4Department of Physiology, Functional Genomics Institute, School of Medicine, Konkuk University, Seoul, Korea Received July 15, 2014; accepted March 20, 2015. Additional Supporting Information may be found at http://onlinelibrary.wiley.com/doi/10.1002/hep.27804/suppinfo 135
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synthesis of triglycerides (TGs), phospholipids, and sphingolipids. During this process, synthesis of specific lipid metabolites is known to provide the signaling molecules regulating the cascade of metabolic function.7 Lipid-mediated regulation of the cellular insulin-signaling cascade is associated with activation of serine/threonine kinases, leading to development of insulin resistance in insulin-sensitive tissues.8-10 Endoplasmic reticulum (ER) stress is induced by various cellular insults, including glucose deprivation, inflammatory state, and disruption of calcium homeostasis.11 Because ER is a principal organelle in cells, responsible for proper protein folding and maturation as well as protein trafficking to other cellular compartments, abnormal ER function by accumulation of unfolded protein elicits a signaling process termed the unfolded protein response (UPR) to alleviate ERassociated stress.11-13 UPR is initiated by release of glucose-regulated protein 78 from the ER and activation of three signaling cascades: the protein kinase RNA-like ER-associated kinase (PERK) pathway; the inositol-requiring protein 1 alpha (IRE1a) pathway; and the activating transcription factor (ATF)6 pathway.14 Chemical inducers of acute ER stress, such as tunicamycin and thapsigargin, induce these three UPR-signaling pathways, whereas the PERK pathway is activated in diet-induced obese (DIO) mice, indicating that each pathway has a distinctive role under different conditions of physiological stress.15-17 These transcription factors are implicated in expression of UPR genes, including chaperones and lipogenic genes. Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine and synthesize sphingosine 1 phosphate (S1P), a lysophospholipid.18 SphK was first identified from purification of 49-kDa protein from rat kidney, composed of Sphk1a and Sphk1b, which were cloned and characterized.19,20 A second isoform of Sphk2 is highly homologous to Sphk1, and both isoforms have five conserved domains found in lipid kinases.19 Whereas expression of Sphk1 is high in lung, spleen, kidney, and blood, Sphk2 is mainly expressed in
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liver, kidney, and heart.20,21 S1P levels are tightly regulated by synthesis catalyzed by SphKs, irreversible cleavage by S1P lyase, and dephosphorylation to produce sphingosine by S1P phosphatases.22 S1P is a ligand for a family of specific G-protein receptors (S1P1- S1P5). It exerts action by secretion and binding to the S1P G-protein receptors, a process called “inside-out” signaling.22 Despite their similarities and biochemical functions, Sphk2 has the opposite role as the antiapoptotic Sphk1, instead being proapoptotic.23 However, much less is known about the regulation of Sphk2 and its role in hepatic glucose and lipid metabolism. In this study, we investigated whether ER stress could trigger expression of Sphk2 and elevate hepatic S1P. Sphk2, a major SphK isotype in liver, was found to be up-regulated by acute ER stress. Hepatic overexpression of Sphk2 activated protein kinase B (AKT), independent of its proximal insulin signaling. As a result, glucose intolerance was improved and accumulation of lipid droplets was reduced by up-regulation of oxidative genes and increased FA oxidation (FAO). These results suggested that the UPR pathways regulate Sphk2 under distinctive physiological conditions, and that S1P production attenuates ER stress-mediated abnormalities through activation of FAO.
Materials and Methods Animal Experiments. All procedures were approved by the Gachon University (Sungnam, Korea) Institutional Animal Care and Use Committee. Detailed methods are provided in the Supporting Experimental Procedures. Preparation and Culture of Primary Hepatocytes. Eight week-old wild-type (WT) C57BL6 male mice were anesthetized and livers were isolated. Primary hepatocytes were prepared as described previously24 and are detailed in the Supporting Experimental Procedures. Preparation of Plasmids and Recombinant Adenovirus. The promoter sequence of Sphk2 was amplified by polymerase chain reaction (PCR) from mouse
This research was supported by the Basic Science Research Program and the Bio & Medical Technology Development Program through the National Research Foundation of Korea (NRF), funded by the Korean government (NRF-2011-0029583, NRF-2013R1A1A2006229, and NRF-2014M3A9B6069338; to T.S.P.). View this article online at wileyonlinelibrary.com. *These authors contributed equally to this study. Address reprint requests to: Tae-Sik Park, Ph.D., Department of Life Science, Gachon University, Sungnam, Gyeonggido 261-701, South Korea. E-mail: [email protected]; fax: 182-31-750-8573. C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27804 Potential conflict of interest: Nothing to report.
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genomic DNA and was inserted into the pGL3 basic vector (Promega, Madison, WI), generating a pSphk2pGL3 reporter construct. Sphk2 recombinant adenoviruses (AdSphk2) were constructed using an AdEasy Adenoviral Vector System (Stratagene, La Jolla, CA) and the pAdTrack CMV vector, as described previously.25 Transfection and Luciferase Reporter Assay. HepG2 cells were transiently cotransfected with pSphk2-pGL3 reporter construct and ER stress marker cloning vectors containing ATF4, ATF6, or sXBP1pcDNA3.0, respectively. An expression vector for Renilla luciferase, pTK-RL, was used to normalize transfection efficiency. RNA Preparation and Quantitative Real-Time PCR. Total RNA was isolated from mouse primary hepatocyte and liver tissues, and, subsequently, synthesized complementary DNA was analyzed to measure gene expression. Primer sequences used in this study are provided in Supporting Table 1. Western Blotting Analyses. Whole cell proteins were lysed using cell lysis buffer. Thirty micrograms of protein were used for immunoblotting, as described previously.26 The blots were developed with the enhanced chemiluminescent substrate (Millipore, Billerica, MA) and detected with a LAS4000 luminescent image analyzer (Fujifilm, Tokyo, Japan). Measurement of Metabolites. Blood glucose levels, plasma, and hepatic TGs, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and nonesterified fatty acid (NEFA) were measured as described in the Supporting Experimental Procedures. Sphingolipid levels in plasma and liver were measured by liquid chromatography/tandem mass spectrometry (LC-MS/MS), as described previously.26 Histology. For histological analyses, livers were isolated and frozen in optimal cutting temperature embedding medium. Paraffin embedded 5-lm sections of the liver were stained with hematoxilin and eosin (H&E; Sigma-Aldrich, St. Louis, MO) or Oil Red O (ORO). Statistical Analyses. Results are shown as mean 6 standard error of the mean (SEM). Comparison of different groups was carried out using the two-tailed unpaired Student t test. P values less than 0.05 (P < 0.05) were considered statistically significant.
Results ER Stress Up-Regulates Sphk2 Expression in Mouse Primary Hepatocytes and In Vivo. Recent reports suggested that ER stress activates hepatic lipid
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biosynthesis by up-regulating key lipogenic genes, such as DGAT2, sterol responsive element-binding protein (SREBP)1c, and LIPIN2.27 Given that sphingolipid biosynthesis is a nonoxidative FA pathway, we investigated whether ER stress could regulate biosynthesis of sphingolipids, which are known signaling lipids of metabolic dysregulation. To test this hypothesis in vivo, tunicamycin, an acute ER stress inducer, was intraperitoneally (IP) administered into WT mice and livers were isolated to measure expression of sphingolipid biosynthesizing genes. Among the sphingolipid biosynthetic genes, ceramide synthase (CerS)3, alkaline ceramidase (Acer)2, and Acer3 were up-regulated (Fig. 1A). Expression of UPR genes, including ATF4, ATF6, C/EBP homologous protein (CHOP), spliced X-box-binding protein 1 (sXBP1), and unspliced XBP1 (uXBP1), was up-regulated (Fig. 1B). Sphk2, the endpoint of this up-regulation in the sphingolipid biosynthetic pathway, was transcriptionally induced by tunicamycin in liver 6 hours after tunicamycin administration. When primary mouse hepatocytes (PMHs) were treated with tunicamycin, Sphk2, and messenger RNA (mRNA) and protein levels were induced in a time-dependent manner (Fig. 1C,D). In contrast, Sphk1, another isotype of SphK, was rarely detected in any of the conditions (Fig. 1B). These results suggested that Sphk2 is transcriptionally regulated by acute ER stress, aroused by tunicamycin. High-Fat Diet and Endotoxin Activate ER Stress, but Regulate Sphk2 Expression Differently. Because tunicamycin induces expression of all UPR genes, we aimed to investigate which ER stress-dependent signaling pathway is directly associated with Sphk2 upregulation. To test whether Sphk2 is regulated by ER stress in a physiological setting, mice were fed a highfat diet (HFD) for 4 weeks to generate hyperlipidemic ER stress conditions. Hepatic Sphk2 mRNA and protein were found to be down-regulated in livers of mice fed an HFD, compared to those of control mice (Fig. 2A,B). Whereas the longer period of an HFD feeding (8 and 12 weeks of HFD feeding; Supporting Fig. 1) did not alter UPR mediators, hepatic sXBP1 activation occurred in mice fed a 4-week HFD, but ATF4 was down-regulated with no change in ATF6 (Fig. 2A,B). HFD elevated ceramide and sphingomyelin (SM) levels in plasma and liver, but S1P was decreased with no statistical significance (Supporting Fig. 2). Another ER stress activator, lipopolysaccharide (LPS), induced UPR genes such as ATF4 and CHOP, but not sXBP1 (Fig. 2C,D). Despite the fact that most of the lipogenic genes were down-regulated, Sphk2 mRNA and protein levels were elevated (Fig. 2C,D). These results
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Fig. 1. ER stress induces expression of sphingolipid biosynthetic genes in liver. WT C57Bl/6 mice were injected IP with DMSO or tunicamycin (2.5 lg/g body weight; n 5 5 per group), and livers were isolated 6 hours after injection (A and B). Hepatic mRNA was extracted for quantitative PCR analyses. n 5 5, mean 6 SEM, *P < 0.05. Primary mouse hepatocytes were treated with tunicamycin (1.25 lg/mL) or DMSO for the indicated times before being harvested. Quantitative reverse-transcriptase PCR analysis was performed to determine Sphk2 expression (C). n 5 3, mean 6 SEM, *P < 0.05 versus controls. PMHs were treated with tunicamycin (1.25 lg/mL) or DMSO for 12 hours and harvested. Sphk2 protein amounts were analyzed by SDS-PAGE followed by immunoblotting (D). Abbreviations: AcCer, acid ceramidase; Acer1-3. alkaline ceramidase1-3; CerS2-5, ceramide synthase25; DMSO, dimethyl sulfoxide; NCer, neutral ceramidase; Smpd1, acid sphingomyelinase; Smpd2, neutral sphingomyelinase 1; Smpd3, neutral sphingomyelinase 2; TUN, tunicamycin.
suggested that LPS-mediated ER stress induces Sphk2, but regulation of Sphk2 is dependent on the pathophysiological conditions for ER stress induction. Activation of ATF4 Transcriptionally Up-Regulates Sphk2. To examine this differential regulation of Sphk2 by ATF4 and sXBP1, the reporter construct containing the Sphk2 promoter was cotransfected with ATF4, sXBP1, or CHOP, respectively, and measured Sphk2 expression. We found that ATF4 mediated transcriptional induction of Sphk2 by 6-fold, whereas
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sXBP1 suppressed Sphk2 expression (Fig. 3A). Because CHOP is a direct transcriptional target of ATF4, we overexpressed CHOP and examined its effects on Sphk2 expression. However, no change was found in Sphk2 promoter activity, suggesting that Sphk2 expression was only induced by ATF4 activation. In support of these results, adenoviral overexpression of ATF4 elevated Sphk2 protein levels in PMHs, whereas no alteration was found with sXBP1 overexpression (Fig. 3B). To confirm whether Sphk2 is a direct downstream target of ATF4, we transfected ATF4 small interfering RNA (siRNA). Down-regulation of ATF4 resulted in suppression of Sphk2 mRNA and protein levels (Fig. 3C,D). These results suggested that Sphk2 is transcriptionally regulated by ATF4. Sphk2-Mediated S1P Production Activates AKT Phosphorylation. The implication of ceramide and S1P in modulation of the insulin-signaling pathway has been reported on.28,29 S1P, a product of Sphk, is thought to improve glucose homeostasis in diabetic animal models.29 To test whether elevated Sphk2 expression regulates ER stress-dependent signaling pathways, adenovirus expressing human Sphk2 (AdSphk2) was constructed and used to infect PMHs. Phosphorylation of AKT was enhanced by AdSphk2 infection in a gene-dose–dependent manner, as reported previously (Fig. 4A).29 However, increased pAKT was not associated with the proximal insulinsignaling intermediate protein, (IRS)1 or IRS2, which is evidenced by the lack of alteration of pIRS1 or pIRS2 levels (Fig. 4A). Thus, the increased pAKT was independent of tyrosine phosphorylation of IRS. Whereas sphingolipid metabolites, such as sphinganine (SA), SO (sphingosine), ceramide, and SM, were not altered, only cellular S1P levels were elevated by Sphk2 expression (Fig. 4B-D). To confirm whether upstream insulin signaling is involved in phosphorylation of AKT in cells overexpressing Sphk2, we administered hydroxy-2-naphthalenylmethylphosphonic acid (HNMPA), an insulin receptor (IR) antagonist, and examined insulin response. We found that pAKT levels in response to insulin were not changed by IR inhibition in cells overexpressing Sphk2 (Fig. 4E). In contrast, phosphorylation of AKT by insulin was decreased in the control by HNMPA-mediated IR inhibition. These data support the notion that elevated hepatic S1P, induced by Sphk2, activates AKT independent of the insulin-signaling pathway. Hepatic Sphk2 Expression Alters Lipid Profiles in Plasma and Liver. To examine whether elevated hepatic expression of Sphk2 results in physiological alteration, WT mice fed an HFD for 4 weeks were
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Fig. 2. Hyperlipidemic and inflammatory ER stresses regulate Sphk2 differently. WT C57Bl/6 mice were fed a normal chow diet (NCD) or a 60 kcal% HFD for 4 weeks and livers were collected. Quantitative reverse-transcriptase PCR was performed with hepatic RNA (A). Immunoblotting analyses were performed with tissue lysates from mouse liver as described in Materials and Methods (B). WT C57Bl/6 mice were injected with either saline or LPS IP, and livers were collected 8 hours postinjection. Gene expression was measured by quantitative reverse-transcriptase PCR using hepatic mRNA as shown in (A) (C). Western blotting analysis was performed with tissue lysates from mouse livers (D). n 5 6, mean 6 SEM, *P < 0.05.
injected with AdSphk2 by the tail vein. Previous studies in rodents reported that delivery of recombinant adenoviruses resulted in preferential targeting of the transgene to the liver.30 Whereas plasma TG, HDL, and NEFA remained unchanged, total cholesterol and LDL levels were reduced. In addition, overexpression of Sphk2 did not change body weight or plasma glucose levels (Supporting Table 2). Interestingly, liver enzymes, such as alanine aminotransferase, were reduced, suggesting that hepatic function was improved. Levels of ceramide, SA, SO, SM, and glucosylceramide were all found to be reduced, whereas only S1P was elevated in plasma and liver of mice overexpressing Sphk2 (Supporting Fig. 3 and Fig. 5AE). In contrast to plasma levels, the observation that hepatic TG was reduced suggests that synthesis or oxidation was regulated by Sphk2 (Fig. 5F). All these findings of lipid profiling suggest that hepatic Sphk2 expression influences regulation of lipid metabolism in mice fed a HFD. Hepatic Sphk2 Up-Regulation Reduces Hepatic Lipid Accumulation. The result that hepatic TG levels were significantly diminished suggested that hepatic lipid metabolism may be regulated by Sphk2.
To verify the role of Sphk2 in hepatic lipid metabolism, expression of the genes involved in lipid biosynthesis and FAO was examined. Indeed, expression of genes related to FAO, such as PPARa (peroxisome proliferator-activated receptor alpha), CPT1 (carnitine palmitoyltransferase), and ACOX1 (acyl CoA oxidase), was up-regulated by Sphk2 overexpression, despite the lack of changes in lipogenic genes, including FA synthase (FAS), SREBP1c, acyl CoA carboxylase (ACC), and diacylglycerol acyltransferase (DGAT)2 (Fig. 6A). Immunoblotting analyses showed that levels of protein from these genes were elevated in Sphk2-overexpressed livers (Fig. 6B). In addition, ORO staining of liver tissues demonstrated that livers of AdSphk2-injected mice had a significant reduction in lipid droplets, compared to control, which is consistent with reduced hepatic TG levels (Figs. 5F and 6C). As a result of increased FAO, plasma b-hydroxybutyrate was elevated in AdSphk2-injected mice (Fig. 6D). To verify this further, we infected PMHs with AdSphk2 and measured the oxygen consumption rate (OCR). Addition of palmitic acid increases OCR because of increased substrate availability for FAO. We found that AdSphk2 infection increased OCR in the presence of palmitic
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Fig. 3. ATF4 up-regulates Sphk2. (A) HepG2 cells were cotransfected with Sphk2 reporter construct and 0.2 or 0.4 lg of pcDNA 3.0 containing ATF4, sXBP1, or CHOP. Promoter activity was measured by luciferase assay. Representative data from three independent experiments are shown (A). n 5 5, mean 6 SEM, *P < 0.05 versus control empty pcDNA3.0 control. #P < 0.05 versus 0.2 lg transfected group. PMHs were infected with AdGFP as a control, AdATF4, or AdsXBP1 for 24 hours. Immunoblotting analysis was performed with cell lysates (B). HepG2 cells were transfected with the control or ATF4 siRNA followed by tunicamycin treatment (1.25 lg/mL) for 6 hours. Then, cells were harvested, and mRNA expression was measured by quantitative reverse-transcriptase PCR. n 5 3, mean 6 SEM, *P < 0.05 versus control siRNA (C). Western blotting analysis was performed with cell lysates (D).
acid in a time-dependent manner, compared to either AdGFP-infected control or no palmitic acid control (Fig. 6E). These findings suggest that Sphk2 expression increases FAO and ameliorates the hepatic steatosis induced by HFD. Hepatic Sphk2 Expression Improves Glucose Intolerance. To explore the role of hepatic Sphk2 in insulin sensitivity and glucose metabolism, we analyzed glucose and insulin response in AdSphk2-injected mice. Despite the lack of change in basal blood glucose levels, mice overexpressing Sphk2 improved glucose intolerance when being fed an HFD (Fig. 7A). Plasma insulin levels were not significantly different between control and Sphk2-overexpressing mice during the glucose tolerance test, suggesting that the improved glucose intolerance was the result of the enhanced plasma glucose clearance without alteration of insulin secretion (Fig. 7B). Sphk2 overexpression also improved insulin tolerance (Fig. 7C). Improved glucose intolerance and insulin response suggest that ele-
vated Sphk2 expression is associated with regulation of insulin signaling in response to ER stress, which is induced by HFD. To verify the effects of Sphk2 upregulation on insulin signaling, immunoblotting analyses of the insulin-signaling proteins was performed. Although the basal conditions did not alter phosphorylation of AKT and forkhead box protein O1 (FOXO1), insulin caused increased phosphorylation of AKT and its downstream FOXO1 in mice overexpressing Sphk2 (Fig. 7D). Expression of gluconeogenic genes, including glucose 6-phophatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), were not changed (Fig. 7D and Supporting Fig. 4). To determine whether nonhepatic tissues are involved in improved glucose or insulin tolerance, we measured AKT phosphorylation in skeletal muscles and adipose tissues. However, we did not find any changes in pAKT levels (Fig. 7E,F). Sphk2 inhibition by K145, a selective Sphk2 inhibitor,31 did not alter glucose tolerance, hepatic cholesterol and TG levels, plasma S1P,
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Fig. 4. Sphk2-mediated S1P production increases Akt phosphorylation in hepatocytes. PMHs were infected with AdGFP or AdSphk2 for 24 hours in a gene-dose– dependent manner. Cells were harvested, and cell lysates were analyzed by immunoblotting (A). Representative data from three independent experiments are shown. PMHs were infected with AdGFP or AdSphk2 for 24 hours at 5 multiplicities of infection (MOI). Sphingoid bases (B), ceramide (C), and SM (D) were measured by LCMS/MS, as described in Materials and Methods. n 5 3, mean 6 SEM, *P < 0.05 versus AdGFP. HepG2 cells were infected with AdGFP or AdSphk2 for 24 hours at 5 MOI and treated with 50 nM of insulin for 10 minutes. Another set of cells were treated with HNMPA-(AM)3 for 6 hours and then insulin treatment for 10 minutes. Cells were harvested, and cell lysates were analyzed by immunoblotting (E). Abbreviation: Cer, ceramide.
and hepatic lipid droplets in AdSphk2-injected mice (Fig. 8A-D). These results suggest that Sphk2 expression did not reach the threshold levels that could regulate glucose metabolism in basal conditions, but the response to insulin became more sensitized by Sphk2mediated activation of the insulin-signaling pathway in conditions of insulin resistance.
Discussion ER is a principal cellular organelle responsible for protein maturation and trafficking to other cellular compartments. Accumulation of unfolded proteins stresses the ER, which is involved in development of metabolic dysfunction and inflammatory disease.32 Excessive intake of nutrients and infection-mediated
inflammatory response are physiological conditions that put stress on ER structure and function, then triggering the UPR to maintain ER homeostasis. When the condition of ER stress continues, lipid homeostasis would be perturbed and cellular messengers, such as diacylglycerol and FFA, disturb cellular signaling. In this study, to elucidate a link between ER stress and sphingolipid biosynthesis, we found the following: (1) Sphk2-mediated biosynthesis of S1P was activated by inflammatory ER stress in liver; (2) Sphk2 is upregulated by ATF4; and (3) hepatic up-regulation of Sphk2 activates FAO and improves hepatosteatosis as well as glucose metabolism. ER stress-induced UPR is mediated by three ER membrane-associated proteins: PERK, IRE1a, and ATF6. In these pathways, ER stress-activated transcription
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Fig. 5. Overexpession of Sphk2 elevates S1P, but decreases ceramide, SM, glucosylceramide, cholesterol, and TG in liver. WT C57Bl/6 mice fed an HFD (60 kcal% fat) for 4 weeks were infected with AdGFP or AdSphk2 (1 3 109 PFU) by tail vein injection. At 14 days postinjection, mice livers were isolated. Ceramide (A), dihydroceramide (B), sphingoid bases (C), SM (D), and glucosylceramide (E) in the liver were measured by LC-MS/MS, as described in Materials and Methods. After extraction of the lipids in liver tissue, total cholesterols and TG were measured by colorimetric methods (F). n 5 7-8, mean6 SEM, *P < 0.05 versus AdGFPinjected mouse. Abbreviation: PFU, plaque-forming units.
factors, such as ATF4, XBP1, and ATF6, induce various chaperones to correct the dysfunctions caused by accumulation of unfolded protein. Excessive UPR signaling leads to metabolic perturbation, including obesity and fatty liver. Each UPR-signaling pathway has a different function in metabolic regulation. For example, XBP1 deficiency results in suppression of de novo hepatic lipid biosynthesis, leading to decreased plasma TG, cholesterol, and FFAs.33 In contrast, ATF4 is activated in DIO mice, activating adipogenesis, and PERK-eIF2a(-ATF4) is involved in activation of inflammatory nuclear factor kappa B.34,35 Whereas there is crosstalk between these UPR branches, metabolic dysregulation and inflammation are the major fields for UPR pathways to be involved in stress-signaling pathways. A nonoxidative FA pathway is involved in biosynthesis of sphingolipids, and their metabolism leads to
biosynthesis of various bioactive lipid metabolites, including ceramide, SO, and S1P. These sphingolipid metabolites are important signaling messengers in metabolic regulation.36,37 Infusion of saturated FA results in elevation of ceramide levels in muscle and liver, contributing to peripheral insulin resistance.9 Pharmacological and genetic inhibition of de novo ceramide biosynthesis has been shown to improve glucose intolerance and insulin response in myriocin-treated DIO or in heterozygous Sptlc2-deficient mice.38,39 In contrast, adenoviral gene transfer of Sphk1 markedly reduced blood glucose levels, as well as TG and cholesterol.29 These findings implicate ceramide and S1P in hepatic glucose/lipid metabolism in response to the state of nutrition. Recent findings suggested that lipid metabolism is perturbed by ER stress-activated transcription factors,
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Fig. 6. Hepatic Sphk2 overexpression activates FA oxidation, decreases lipid droplets, and increases ketone body. WT C57bl6/J mice fed an HFD (60 kcal% fat) were injected with either AdGFP or AdSphk2 (1 3 109 PFU) by the tail vein. At 14 days postinjection, livers and plasma were isolated. Expression of genes involved in FA biosynthesis and oxidation were determined by quantitative reverse-transcriptase PCR (A). n 5 7, mean 6 SEM, *P < 0.05. At 14 days postinjection, immunoblotting analyses were performed using liver lysates (B). Livers were isolated and frozen sections were stained with ORO (upper) and alternatively with H&E (lower) (C). Representative pictures were taken at 403 magnification. b-hydroxybutyrate (ketone body) in plasma were measured as described in Materials and Methods (D). n 5 7, mean 6 SEM, *P < 0.05. PMHs were infected with AdGFP and AdSphk2 at 5 multiplicities of infection, then treated with bovine serum albumin/palmitate complexes and incubated for 2 hours, and oxygen consumption rate was measured at indicated time points (E). n 5 5, mean 6 SEM, *P < 0.05.
which implies that sphingolipid biosynthesis may also be regulated by ER stress in conjunction with other lipid biosynthesis. Feeding of an HFD to cause subchronic ER stress activation (4 weeks) resulted in different expression profiles of the UPR proteins from those observed in DIO mice (8 and 12 weeks; Supporting Fig. 1). Activation of sXBP1 and suppression
of ATF4 were observed in this subchronic condition (Fig. 2A,B). On the other hand, LPS-induced inflammatory ER stress only activated ATF4, leading to Sphk2 induction, suggesting that onset of ER stress is dependent on the various stimuli (Fig. 2C). This might be owing to the different activation of UPR pathways by hyperlipidemic and inflammatory ER
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Fig. 7. Sphk2 overexpression improves glucose and insulin intolerance in mice fed an HFD. WT C57Bl/6 mice fed an HFD (60 kcal% fat) for 4 weeks were injected with either AdGFP or AdSphk2 (1 3 109) by tail vein injection. Experiments were performed 7 days after adenoviral injection. Mice were fasted for 16 hours and glucose was injected IP at 1 g/kg body weight, then plasma glucose was measured at the indicated times. (A). Plasma insulin levels during the glucose tolerance test were measured (B). Insulin (1 U/kg body weight) was injected IP into mice after 4 hours of fasting, and plasma glucose was measured at the indicated times. n 5 7 for AdGFP and n 5 8 for AdSphk2, mean 6 SEM, *P < 0.05 vs. AdGFP (C). Mice were divided into two groups. One group was used as a control, and the other was injected with insulin (0.5 U/ kg body weight) IP for 10 minutes. Livers were isolated and western blotting analysis was performed on insulin-signaling proteins (D). Skeletal muscles (E) and adipocytes (F) were isolated and western blotting analysis was performed. Representative data from two independent experiments are shown (n 5 6 for each group).
stress, and each UPR pathway selectively modulates the cause-oriented target of stress signaling. In response to inflammatory ER stress, such as that caused by LPS, hepatocytes might request an urgent response through the combined effects of inflammatory response and ER stress by Sphk2 induction, mainly by ATF4. Although both XBP1 and ATF4 account for hepatic lipid metabolism, S1P biosynthesis is regulated differently, at least in the liver. We speculated that Sphk2 expression is differentially regulated by UPR transcription factors under different pathophysiological conditions. The stress-signaling pathways linked to
ATF4-mediated hepatic Sphk2 up-regulation deserve further studies to clarify the role of Sphk2 and its product, S1P, in metabolic/inflammatory regulation. Previous reports suggested that delivery of the Sphk1 gene improves insulin resistance and lipid abnormalities in KK/Ay diabetic mice by activation of insulin signaling, and these results may be through elevated hepatic S1P.29 However, Sphk1 is rarely expressed in liver and Sphk2 is the major Sphk isoform producing hepatic S1P. We found that adenoviral expression of Sphk2 activated the AKT pathway independent of IRS. This finding suggests that Sphk
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Fig. 8. Sphk2 inhibition by K145 did not alter glucose metabolism and lipid levels. Glucose tolerance tests were performed after HFD (45 kcal% fat) and injected with K145 (20 mg/ kg/2 days) IP for 2 weeks every other day. Mice were fasted for 16 hours and glucose was injected IP at 1 g/ kg body weight, then plasma glucose was measured at the indicated times (A). n 5 9 for control and n 5 7 for K145. TG and cholesterol were measured in mice liver by colorimetric assay (B). S1P level in plasma was measured by LC-MS/MS (C). Livers were isolated and frozen sections were stained with ORO (upper) and alternatively with H&E (lower) (D). Representative pictures were taken at 403 magnification.
contributes to regulation of hepatic glucose metabolism. Improvement of glucose intolerance in mice fed an HFD by hepatic Sphk2 expression supports the notion that elevated levels of S1P possibly act as an activator of insulin signaling in hyperglycemic conditions by increasing pAKT, as reported previously by Guan et al.40 However, adenoviral Sphk2 upregulation does not alter basal glucose levels in vivo, but only insulin response by activating the signaling pathway through increased pAKT. Independently, whereas plasma TG remained unchanged, hepatic TG accumulation was reduced by Sphk2 expression. We found that reduction of hepatic TG was the result of up-regulation of FA oxidative genes and increased FAO. The notion that AdSphk2-infected hepatocytes have increased oxygen consumption and elevated plasma ketone bodies suggests that Sphk2 expression is an important regulator of FAO. However, the results that pharmacological Sphk2 inhibition did not change glucose/FA metabolism suggest that there might be a compensatory mechanism by hepatic Sphk2. The mechanism of Sphk2-mediated activation of hepatic FAO and a role of S1P deserves further study.
In this study, we demonstrated that ER stressmediated Sphk2 up-regulation occurs through ATF4 activation under inflammatory conditions and improves hepatic glucose and lipid abnormalities. This raises the question of why ER stress-mediated Sphk2 activation improves glucose intolerance and FAO. One possibility is that UPR-signaling pathways are adaptive processes for alleviation of stressed states of ER by activating FAO. ATF4-mediated Sphk2 up-regulation in response to inflammatory ER stress modulates cellular signaling. As a result, Sphk2 up-regulation leads to reduction of lipid droplets by increased FAO. Additionally, Sphk2-mediated activation of AKT improved insulin signaling in pathophysiological conditions. Taken together, Sphk2 is regulated by the UPR sensors, depending on the causes of ER stress induction, and acts as a molecular messenger to relieve hepatic ER stress and maintain glucose and lipid homeostasis. Acknowledgment: The authors thank Dr. SeungHoi Koo (Korea University) for providing the sXBP1-, ATF4- and CHOP adenoviruses and overexpression vectors.
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Author names in bold designate shared co-first authorship.
Supporting Information Additional Supporting Information may be found at http://onlinelibrary.wiley.com/doi/10.1002/hep.27804/ suppinfo
