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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell Metab. Author manuscript; available in PMC Feb 1, 2009.
Published in final edited form as:
PMCID: PMC2276699
NIHMSID: NIHMS39855

Metabolomics Reveals that Hepatic Stearoyl-CoA Desaturase 1 Downregulation Exacerbates Inflammation and Acute Colitis

SUMMARY

To investigate the pathogenic mechanism of ulcerative colitis, a dextran sulfate sodium (DSS)-induced acute colitis model was examined by serum metabolomic analysis. Higher levels of stearoyl lysophosphatidylcholine and lower levels of oleoyl lysophosphatidylcholine in DSS-treated mice compared to controls led to the identification of DSS-elicited inhibition of stearoyl-CoA desaturase 1 (SCD1) expression in liver. This decrease occurred prior to the symptoms of acute colitis and was well correlated with elevated expression of proinflammatory cytokines. Furthermore, Citrobacter rodentium-induced colitis and lipopolysaccharide treatment also suppressed SCD1 expression in liver. Scd1 null mice were more susceptible to DSS treatment than wild-type mice, while oleic acid feeding and in vivo SCD1 rescue with SCD1 adenovirus alleviated the DSS-induced phenotype. This study reveals that inhibition of SCD1-mediated oleic acid biogenesis exacerbates proinflammatory responses to exogenous challenges, suggesting that SCD1 and its related lipid species may serve as potential targets for intervention or treatment of inflammatory diseases.

INTRODUCTION

The incidence of inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is rising globally. In the United States, IBD affects about 1.4 million people (Loftus, 2004). Genetic predisposition and environmental exposures have been accepted as two major contributing factors in the initiation of IBD (Loftus, 2004; Schreiber and Hampe, 2000), while autoimmune events, such as elevated production of proinflammatory cytokines and antibodies as well as increased activation of immune cells, are the driving forces behind the progression of IBD, leading to chronic inflammation, the destruction of intestinal mucosa, and the manifestation of clinical symptoms (Levine and Fiocchi, 2000). In spite of scientific advances in understanding the pathogenesis of IBD, especially in the fields of pathology and immunology (Xavier and Podolsky, 2007), many molecular events related to the pathophysiology of IBD remain unknown.

Metabolomics, a rapidly evolving tool in systems biology, aims to define small-molecule metabolomes in biofluids, cells, tissues, and organisms and to monitor the metabolic flux following genetic modification, pathophysiological changes, or exogenous challenges (Nicholson et al., 1999; Griffin, 2006). Since multivariate data analysis (MDA), the data processing platform of metabolomics, possesses the capacity to detect subtle changes in a large data set, the potential application of metabolomics in the diagnosis of disease (Brindle et al., 2002) and the characterization of disease-related animal models (Dumas et al., 2006; Jones et al., 2005) has been explored. However, the power of metabolomics in establishing convincing biomarkers and identifying novel molecular mechanisms has not been fully demonstrated.

To understand the pathogenesis of IBD and to explore new therapeutic strategies, several IBD animal models based on chemical treatment or genetic modification have been developed (Byrne and Viney, 2006). Dextran sulfate sodium (DSS)-induced acute colitis is one of the most widely used models because of its robustness and reproducibility (Okayasu et al., 1990). The influences of DSS treatment on the digestive tract and immune system have been extensively examined (Cooper et al., 1993; Egger et al., 2000; Ni et al., 1996), while its impact on blood chemistry and the liver has not been fully explored. In the present study, a liquid chromatography-mass spectrometry (LC-MS)-based metabolomic approach was adopted to investigate this model. A dramatic inhibitory effect of DSS treatment on stearoyl-CoA desaturase 1 (SCD1) in the liver was identified through multivariate model construction and structural elucidation of serum biomarkers and further characterized as an early event prior to the clinical symptoms. DSS-induced colitis was accelerated in Scd1 null mice but attenuated by a high-oleic acid diet and SCD1 overexpression, suggesting that SCD1-mediated oleic acid biogenesis possesses a protective function against proinflammatory signaling.

RESULTS

Identification of SCD1 Inhibition by Metabolomic Analysis of DSS-Elicited Changes in Mouse Serum

The occurrence of acute colitis after 7 day DSS treatment was confirmed before conducting serum metabolomic analysis. Both 2.5% and 5% DSS treatments significantly decreased body weight and colon length (see Figure S1A available online) and elicited severe diarrhea and rectal bleeding (Figure S1B). Histological analysis revealed inflammation in mucosa, submucosa, and muscularis propria with loss of crypts and surface epithelia, as indicated by disease activity index (DAI) scores (Figure S1B). Overall, the pathological phenotype of the 5% DSS group was more severe than in the 2.5% DSS group.

To examine the DSS-induced biochemical changes, an LC-MS-based metabolomic analysis of deproteinized serum from control and DSS-treated mice was performed. A two-component model was constructed by a supervised partial least square discriminant analysis (PLS-DA) using the values of mass, retention time, and relative abundance of detected serum compounds and further validated by a permutation method (Figure S1C). Dose-dependent separation of the three mouse groups was observed in the scores scatter plot and was mainly defined by the first principal component (PC) of the model (Figure 1A). Contribution of individual ions to the PCs was further examined in the loadings scatter plot (Figure 1B). Among all detected serum ions, one ion high in the DSS-treated samples (m/z = 524.372+ at 5.66 min) and two ions high in the controls (m/z = 522.355+ at 5.07 min and m/z = 520.339+ at 4.68 min) made the most important contributions to the first PC. Tandem mass spectrometry (MS/MS) fragmentation and accurate mass measurements showed that all three compounds resembled each other as revealed by the presence of 184.073+ (C5H15NO4P+) and 104.107+ (C5H14NO+) daughter ions (Figures S1D–S1F), characteristic fragments formed by the cleavage of the phosphodiester bond in the phosphocholine moiety (Pulfer and Murphy, 2003). The appearance of ion 258.110+ (C8H21NO6P+) further indicated the existence of a glycerophosphocholine backbone and a potential monoacyl moiety in all three compounds. Based on the aforementioned structural information as well as the complementary nature of fragments 341.306+ (Figure S1D), 339.293+ (Figure S1E), and 337.271+ (Figure S1F) to the 184.073+ phosphocholine moiety, the structures of three lysophosphatidylcholines (LPCs) were determined as 1-stearoyl-sn-glycero-3-phosphorylcholine (stearoyl-LPC; 18:0-LPC; accurate mass of [M+H]+ = 524.3711), 1-oleoyl-sn-glycero-3-phosphorylcholine (oleoyl-LPC; 18:1-LPC; [M+H]+ = 522.3554), and 1-linoleoyl-sn-glycero-3-phosphorylcholine (linoleoyl-LPC; 18:2-LPC; [M+H]+ = 520.3398), respectively, and then confirmed by authentic standards. Because of their profound influence on the multivariate model, the relative abundances of these three C18 acyl-LPC species in the three mouse groups were compared. In a dose-dependent manner, the relative abundance of saturated stearoyl-LPC increased significantly, whereas both unsaturated oleoyl-LPC and linoleoyl-LPC decreased significantly after DSS treatment (Figure 1C; Figure S1G).

Figure 1
Metabolomic Analysis of DSS-Induced Acute Colitis and Identification of SCD1 Inhibition

The DSS-elicited imbalance between saturated and unsaturated lipids, especially the increased ratio between stearoyl-LPC and oleoyl-LPC, bears a striking resemblance to the phenotype of Scd1 null mice, which are deficient in stearoyl-CoA desaturase 1 (SCD1), an endoplasmic reticulum (ER) enzyme responsible for the biosynthesis of oleic acid (18:1) and palmitoleic acid (16:1) from stearic acid (18:0) and palmitic acid (16:0), respectively (Miyazaki et al., 2000). Western blot analysis of mouse liver microsomes confirmed that SCD1 protein expression in liver was abolished by DSS treatment (Figure 1D). Moreover, cytochrome P450 3A (CYP3A), another ER protein, was also significantly reduced, while calnexin and GRP78, two constitutive proteins in the ER, were not affected by the treatment, suggesting that DSS selectively affects ER proteins (Figure 1D). Results from mRNA measurement further revealed that SCD1 suppression occurred at the transcriptional level (Figure 1E). To correlate the diminished SCD1 expression with the decreased oleoyl-LPC and increased stearoyl-LPC levels, serum metabolomic profiles of wild-type and Scd1 null mice were compared. Scd1 null mice were clearly separated from wild-type mice in the first PC of an unsupervised principal components analysis (PCA) model (Figure 1F), and the three top-ranking ions contributing to the separation were still stearoyl-LPC (18:0-LPC), which is high in the Scd1 null mice, and oleoyl-LPC (18:1-LPC) and linoleoyl-LPC (18:2-LPC), which are high in the wild-type mice (Figure 1G). In addition, the ratio of palmitoleoyl-LPC (16:1-LPC) to palmitoyl-LPC (16:0-LPC) in both DSS-treated mice and Scd1 null mice was also dramatically reduced compared to wild-type mice (data not shown). Thus, all of our observations supported the view that loss of hepatic SCD1 is the main cause of the altered balance between stearic acid-derived lipid species and oleic acid-derived lipid species after DSS treatment.

Correlation of SCD1 Suppression with Proinflammatory Events

Dose-response experiments identified the inhibition of SCD1 expression after 7 day DSS treatment (Figures 1D and 1E). However, whether this dramatic suppression of gene expression was a secondary effect or a direct response to DSS treatment was still unknown. Thus, a time-course experiment was conducted to examine the correlation between SCD1 expression and disease progression. The localized effects of 5% DSS on the colon, including the reduction of colon length and histological damage, appeared from day 1, while the clinical symptoms of acute colitis, including body weight loss, diarrhea, and rectal bleeding, did not become prominent until day 5 (Figures 2A and 2B). In contrast to the mild symptoms in the first 3 days, SCD1 mRNA and protein levels in liver were dramatically reduced from day 1 and almost completely suppressed on day 5 (Figures 2C and 2D). SCD1 inhibition in liver was further reflected by the progressive decrease of oleoyl-LPC and the increase of stearoyl-LPC in serum (Figure 2E). This rapid decrease of SCD1 expression and function in liver was achieved without apparent hepatotoxicity as indicated by serum aminotransferase activities (Figure 2F).

Figure 2
Time-Course Responses to 5% DSS Treatment

Sterol regulatory element-binding protein 1 (SREBP1), a transcription factor controlling lipogenesis, is recognized as both a regulator and an effector of SCD1 (Miyazaki et al., 2004; Tabor et al., 1999). To determine the role of SREBP1 in DSS-induced SCD1 suppression, SREBP1 expression in liver was examined. Although DSS also reduced SREBP1 mRNA and protein in liver, the decrease of SREBP1 proceeded in a much less dramatic pattern than the decrease of SCD1 (Figures S2A and S2B). Similarly, the expression levels of acetyl-CoA carboxylase (Acc) and fatty acid synthase (Fas), two SREBP1-targeting genes, were also gradually reduced by DSS (Figures S2C and S2D). Since it has been reported that Scd1 null mice have much lower SREBP1 expression than wild-type mice (Miyazaki et al., 2004), it is possible that downregulation of SREBP1 might be a downstream event of SCD1 inhibition instead of an upstream event. Moreover, because the regulation of SCD1 by insulin has been demonstrated previously (Waters and Ntambi, 1994), serum insulin levels were examined. The results indicated that DSS treatment has no significant influence on insulin levels (Figure S2E).

Because production of proinflammatory cytokines is a critical event in the initiation and progression of IBD, the relationship between SCD1 suppression and cytokine expression was explored. Hepatic levels of interleukin 1β (IL-1β) and tumor necrosis factor α(TNFα) were not significantly induced until day 3, although their expression levels in colon rose rapidly from day 1 (Figures 2G and 2H). Overall, the inhibition of hepatic SCD1 expression was an early event in DSS-induced colitis, occurring before the induction of proinflammatory cytokines in liver and the appearance of systemic IBD symptoms.

Suppression of SCD1 Expression by Bacterially Induced IBD and by Endotoxin

The size of the DSS molecule (35–44 kDa) used for inducing IBD excludes its direct adsorption in the intestine, except for some local uptake by macrophage-mediated phagocytosis in the colonic mucosa (Kitajima et al., 1999). Therefore, the downregulation of SCD1 expression in liver was probably achieved by signals derived from the gut instead of a direct effect of DSS on the liver. Since DSS treatment can significantly affect the bacterial population in the colon and also inflict damage to the epithelial layer (Okayasu et al., 1990), it is possible that signals from disrupted intestinal microflora may play a role in SCD1 inhibition in liver. To examine this hypothesis, a bacterially induced IBD model was adopted. After infection with Citrobacter rodentium, a rodent-specific pathogenic bacterium (Higgins et al., 1999), colonic hyperplasia represented by dramatic mucosal thickening developed, and other IBD symptoms including diarrhea, slight rectal bleeding, and body weight loss were observed (data not shown). The serum oleoyl-LPC level was dramatically reduced following C. rodentium infection (Figure 3A), corresponding to the diminished protein and gene expression of hepatic SCD1 (Figures 3B and 3C). Furthermore, lipopolysaccharide (LPS) treatment also abolished SCD1 expression in liver (Figure 3D), suggesting a potential contribution by bacterial endotoxins to the DSS- and C. rodentium-elicited SCD1 inhibition.

Figure 3
Suppression of SCD1 Expression by Bacterially Induced Inflammatory Bowel Disease and Endotoxin Treatment

Acceleration of DSS-Induced Acute Colitis in Scd1 Null Mice

To examine the role of SCD1 in DSS-induced colitis, Scd1 null mice were challenged with DSS. Compared to wild-type mice, Scd1 null mice were much more susceptible to DSS, as indicated by increased body weight loss (Figure 4A), shorter colon length (Figure 4B), and more severe diarrhea (Figure 4C) and rectal bleeding (Figure 4D). These results suggest that SCD1 inhibition might be not only a passive event following DSS treatment but a contributing factor to the progression of colitis.

Figure 4
Acceleration of DSS-Induced Colitis in Scd1 Null Mice

Attenuation of DSS-Induced Acute Colitis and Proinflammatory Signaling by a High-Oleic Acid Diet

A direct consequence of SCD1 inhibition is the imbalance between saturated LPCs and monounsaturated LPCs in serum, as shown in DSS treatment and Scd1 null mice (Figure 1; Figure 2; Figure 3). Previous studies on palmitoyl-LPC (16:0-LPC) and stearoyl-LPC (18:0-LPC) have largely defined saturated LPCs as proinflammatory (Asaoka et al., 1992; Huang et al., 2005) and atherogenic phospholipids (Wu et al., 1998), consistent with our observation that the progression of DSS-induced colitis was accompanied by a simultaneous increase in the serum stearoyl-LPC level. However, the significance of reduced serum oleoyl-LPC levels in the pathogenesis of acute colitis was still unclear. One interesting observation, reported in two independent studies on LPCs, is that IL-1β production from monocytes (Liu-Wu et al., 1998) and microglia (Stock et al., 2006) is significantly induced by both palmitoyl-LPC and stearoyl-LPC, but not by monounsaturated oleoyl-LPC, suggesting a potential functional difference between saturated LPCs and unsaturated LPCs. To examine this hypothesis, the response of mice on a high-oleic acid diet to 2.5% DSS treatment was compared with that of mice on a control diet. Diarrhea and rectal bleeding appeared on day 2 in the control group but did not become prominent in the high-oleic acid group until day 4 and day 5 of DSS treatment, respectively (Figure 5A). Microscopic examination of colon tissues indicated that high-oleic acid feeding alone did not alter colon histology in comparison to the control group (Figure 5B). However, DSS-induced colon damage in mice on the high-oleic acid diet (histology DAI 2.15 ± 0.55) was much less severe than that in mice on the control diet (histology DAI 3.85 ± 0.66) after a 7 day treatment, and total loss of colonic crypts and surface epithelia in the control diet group was partially reversed by the high-oleic acid feeding (Figure 5B).

Figure 5
Attenuation of DSS-Induced Colitis by a High-Oleic Acid Diet

Serum metabolomics showed a clear separation of the four mouse groups in a PCA model, in which the first PC determined the responses to DSS treatment while the second PC separated high-oleic acid feeding from control feeding (Figure 5C). As expected, oleoyl-LPC and stearoyl-LPC were the two ions with the greatest contribution to the first PC in the loadings scatter plot, while the higher abundances of linoleoyl-LPC and palmitoyl-LPC in mice on the control diet helped define the second PC (Figure 5D). High-oleic acid feeding raised the serum oleoyl-LPC level to about 2.5-fold of the level in the untreated control group (Figure 5E). In fact, after 7 days of DSS treatment, the serum oleoyl-LPC level in the high-oleic acid group was still comparable to its level in the untreated control group. However, it should be noted that the high-oleic acid feeding did not alter the basal expression level of hepatic SCD1 or the inhibitory effects of DSS treatment on SCD1, as shown by the gene expression level of Scd1 in liver (Figure 5F) and the fact that serum oleoyl-LPC levels in the high-oleic acid group were still significantly decreased following DSS treatment (Figure 5G). This result further confirmed that the regulation of serum oleoyl-LPC levels following DSS treatment is a downstream event of SCD1 activity. Moreover, DSS-induced Il1b gene expression in colon was significantly reduced by high-oleic acid feeding (Figure 5G). Similarly, DSS-elicited increase of serum IL-1β levels was also partially reversed by high-oleic acid feeding (Figure 5H). Overall, higher levels of serum oleoyl-LPC corresponded to reduced production of proinflammatory cytokines and attenuated progression of DSS-induced acute colitis.

Attenuation of DSS-Induced Acute Colitis by SCD1 Overexpression

Both the susceptibility of Scd1 null mice to DSS (Figure 4) and the resistance of mice fed a high-oleic acid diet (Figure 5) suggested that SCD1 might possess a protective function against proinflammatory signals. To validate this hypothesis, SCD1 was overexpressed by adenovirus-mediated in vivo transfection. Compared to mice transfected with control adenovirus, the initiation and progression of clinical symptoms of DSS-induced colitis were much more delayed in mice transfected with SCD1 adenovirus (Ad-SCD1) during a 7 day 5% DSS treatment, as indicated by body weight, colon length, and the DAI scores of rectal bleeding and diarrhea (Figures 6A–6D). Examination of SCD1 protein levels in liver showed that Ad-SCD1 mice not only had higher basal SCD1 expression than control mice but also sustained higher SCD1 levels even after 5 days of DSS treatment (Figure 6E). The protective effect of SCD1 transfection was further reflected in the fact that at day 5 of DSS treatment, both untreated and treated Ad-SCD1 mice had significantly higher serum oleoyl-LPC levels than corresponding controls (Figure 6F).

Figure 6
Attenuation of DSS-Induced Colitis by SCD1 Overexpression

DISCUSSION

Although Crohn’s disease and ulcerative colitis, two subtypes of IBD, are mainly localized ailments, pathological phenotypes of IBD are not limited to the digestive tract. Clinical data have suggested that IBD can inflict severe damage to the hepatobiliary system (Desmet and Geboes, 1987). For example, two prominent side effects of IBD are the high incidence of both primary sclerosing cholangitis (Broome and Bergquist, 2006) and abnormal liver function (Mendes et al., 2007). However, the underlying mechanism is still largely unknown, even after extensive pathological and immunological investigations. To obtain further insights into the etiology and clinical symptoms of IBD, a metabolomic approach was adopted in this study to examine the IBD-related biochemical changes. Serum, as the major destination for small molecules originating from the liver, was used as the sample. DSS-induced acute colitis was chosen as the IBD animal model because of its robustness and resemblance to human IBD regarding the disruption of the colonic epithelial layer and the progressive immune and inflammatory responses (Arseneau et al., 2000; Podolsky, 2000). Serum metabolomic analysis revealed that DSS treatment shifted the balance between saturated LPCs and monounsaturated LPCs in serum. Since SCD1 is the sole enzyme responsible for the biogenesis of both oleic acid and palmitoleic acid (two major monounsaturated fatty acids in vivo) and since the liver is a major site of biogenesis for serum LPCs (Baisted et al., 1988; Sekas et al., 1985), this change in the serum lipid profile was subsequently attributed to the rapid and complete inhibition of Scd1 gene expression in liver. In fact, the results from this study indicated that the ratio between serum stearoyl-LPC and serum oleoyl-LPC is a sufficient in vivo biomarker of SCD1 activity. Overall, the identification of SCD1 inhibition in this study demonstrates the power of LC-MS-based metabolomics as a tool for unraveling disease-associated biochemical events and their underlying mechanisms. The combination of high-resolution chromatography and accurate mass measurement can function as an efficient technical platform for defining the metabolomic profile of complex biomatrices.

Despite the insights obtained from this metabolomics-based study, it remains to be determined how DSS treatment, which is thought to be a localized event in the digestive tract, can so dramatically affect SCD1 enzyme levels in the liver. It is known that DSS treatment has a significant influence on the microbial population in the intestine (Okayasu et al., 1990), which in turn can affect the initiation and progression of inflammatory responses (Lupp et al., 2007). To examine the potential role of bacteria in the regulation of hepatic SCD1 expression, a C. rodentium-induced IBD model was further adopted in this study. Similar to the DSS model, C. rodentium infection led to the downregulation of SCD1 in liver and the dramatic decrease of oleoyl-LPC species in serum, even though the phenotype and symptoms of this model were different from those of the DSS model in many aspects. Furthermore, intraperitoneal injection of endotoxin also abolished hepatic SCD1 expression. Therefore, it is highly likely that disruption of intestinal microflora and the release of endotoxins following DSS treatment contribute to the inflammatory responses, and the downregulation of SCD1 expression should therefore be considered a general event in IBD instead of a DSS-specific event (Figure 7). Interestingly, SCD1 is not the sole enzyme in the ER that is affected by DSS, C. rodentium, and LPS treatments. Members of the cytochrome P450 enzyme family, including CYP3A in this study, are also significantly downregulated by DSS (Masubuchi and Horie, 2004), bacteria (Richardson et al., 2006), and endotoxins (Morgan, 1989). As direct downstream targets of endotoxins and bacterial infection, proinflammatory cytokines, such as TNFα and interleukins, can also suppress the expression of ER enzymes (Monshouwer et al., 1996), including SCD1 (Weiner et al., 1991). Since IL-1β expression and TNFα expression in colon were dramatically induced from day 1 of DSS treatment (Figures 2G and 2H), it is likely that portal vein delivery of proinflammatory cytokines from the colon, as revealed by the elevated IL-1β levels in serum, may contribute to suppression of SCD1 expression in liver. Overall, SCD1 inhibition following DSS, C. rodentium, and LPS treatments should be considered the consequence of multiple proinflammatory events. The exact roles of bacteria, endotoxins, and cytokines in these events remain to be investigated.

Figure 7
Role of SCD1 and Its Related Lipid Species in DSS-Induced Proinflammatory Signaling

LPCs, which mainly originate from the phospholipase A2 (PLA2)-mediated hydrolysis of phosphatidylcholine and lecithin-cholesterol acyltransferase-catalyzed transesterification, are major lipid species in serum (Croset et al., 2000; Glomset, 1968). Under conditions of normal homeostasis, the bioactivities of serum LPCs are suppressed by forming complexes with albumin and lipoproteins (Croset et al., 2000). However, under pathological conditions, the serum concentrations of free LPCs can be significantly elevated due to the accumulation of LPCs (Mehta et al., 1990; Okita et al., 1997; Sasagawa et al., 1999; Sobel et al., 1978) and the depletion of albumin in serum (Liao et al., 1986), leading to higher LPC-related bioactivities. It has been reported that ovarian cancer patients have higher levels of serum palmitoyl- and stearoyl-LPC and lower levels of serum oleoyl-and linoleoyl-LPC compared to healthy subjects (Okita et al., 1997), which resembles the LPC profile produced by DSS and C. rodentium infection. Therefore, future studies on the serum metabolome of human IBD and cancer patients should provide more information on the clinical relevance of SCD1 inhibition.

As for the role of individual LPCs in the pathogenesis of colitis, the correlation between increased concentrations of palmitoyl-and stearoyl-LPC and elevated levels of IL-1β and TNFα in this study is consistent with previous in vitro observations (Liu-Wu et al., 1998; Stock et al., 2006; Takabe et al., 2004) and further suggests that saturated LPCs can promote the release of proinflammatory cytokines in vivo and exacerbate the pathological signs of inflammation. On the other hand, monounsaturated LPCs, especially oleoyl-LPC, are potentially protective since the low serum oleoyl-LPC levels in Scd1 null and DSS-treated mice corresponded with susceptibility to acute colitis and the progression of inflammation whereas the high serum oleoyl-LPC levels achieved by the high-oleic acid diet and SCD1 overexpression were associated with resistance to DSS. This conclusion is further supported by previous observations that oleic acid-derived lipids are significantly decreased in the trinitrobenzenesulfonic acid (TNBS)-induced acute colitis model (Nieto et al., 2002) and that supplemental olive oil in the diet attenuates DSS-induced toxicity (Camuesco et al., 2005). It is possible that oleoyl-LPC, which does not induce the release of proinflammatory cytokines from immune cells (Liu-Wu et al., 1998; Stock et al., 2006), might counter the cytokine-inducing effect of saturated LPCs by competitively blocking their binding to potential LPC receptors in endothelial cells (Lum et al., 2003). Therefore, instead of concluding that all LPCs are proinflammatory, as in previous studies using saturated LPCs for chemical treatments (Huang et al., 2005; Lum et al., 2003; Ousman and David, 2000; Takabe et al., 2004), the results from our study suggest that there are functional differences among LPC species with regard to the activation of the immune system. In addition to LPCs, free fatty acids and their other derivatives can also mediate the immune responses (Zurier, 1993). For example, nitrated oleic acid has been identified as an endogenous ligand of peroxisome proliferator-activated receptor γ (PPARγ), an important transcription factor in anti-inflammatory signaling (Baker et al., 2005). However, the exact role of oleic acid, nitrated oleic acid, and oleoyl-LPC in maintaining the homeostasis of the immune system and the underlying mechanisms of inflammation still requires further study. Overall, the results of this study support a model in which SCD1 plays a suppressive role in proinflammatory signaling by converting saturated fatty acids to monounsaturated fatty acids (Figure 7). This conclusion provides a possible mechanistic explanation for the widely reported observation that consumption of unsaturated fat, such as the olive oil-rich Mediterranean diet, has anti-inflammatory benefits for diseases associated with inflammation, such as cardiovascular disease, rheumatoid arthritis, and IBD (Alarcon de la Lastra et al., 2001) while the consumption of saturated fat is largely proinflammatory (Nicholls et al., 2006).

In summary, LC-MS-based metabolomic analysis identified a dramatic inhibition of SCD1 expression, potentially mediated by endotoxins and cytokine signaling, as an early event in DSS-induced acute colitis. The accumulation of saturated LPCs through the inhibition or deficiency of SCD1 exacerbated the immune response, whereas the increase of monounsaturated lipids through dietary supplementation and SCD1 overexpression attenuated DSS-induced inflammation, suggesting that SCD1 functions as a suppressive regulator of the immune system under normal homeostasis (Figure 7). Uncovering pathogenic modifications of the transcriptome and proteome through a metabolomic approach offers great promise for the use of metabolomics as a key component of systems pathobiology.

EXPERIMENTAL PROCEDURES

Chemicals and Diets

DSS (35–44 kDa) was purchased from MP Biomedicals. Lysophosphatidylcholines were obtained from Avanti Polar Lipids, and other reagents were obtained from Sigma-Aldrich. For the oleic acid supplementation experiment, an AIN93M purified diet containing 40 g/kg soybean oil was used as the control diet, and a modified AIN93M diet containing 30 g/kg oleic acid and 10 g/kg soybean oil was used as the high-oleic acid diet (Dyets, Inc.). Mice were acclimated to the AIN93M control diet or high-oleic acid diet for 1 week prior to DSS treatment.

DSS-Induced Colitis and Tissue Evaluation

Male C57BL/6 mice, 5 to 8 weeks old, were purchased from Charles River Laboratories. Scd1 null mice were described previously (Miyazaki et al., 2001). Maintenance and handling were in accordance with animal study protocols approved by the NCI Animal Care and Use Committee. Mice were administered 2%–5% (w/v) DSS in drinking water. Daily changes in body weight and pathological signs of colitis, including rectal bleeding, diarrhea, and bloody stool, were assessed by an experienced pathologist and reported as the disease activity index (DAI) on a scale from 0 to 4. Serum samples were collected by retro-orbital bleeding at the end of DSS treatment. Liver and colon samples were harvested immediately following CO2 euthanization. For microscopic and macroscopic examination of colon damage, colons were opened longitudinally, flushed with PBS, and fixed in 10% buffered formalin. After colon length measurement, colons were Swiss rolled and processed in paraffin. Colon sections stained with hematoxylin and eosin were scored according to the histological criteria of colitis (Cooper et al., 1993). Liver function was evaluated by measuring serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities (Catachem).

Bacterially Induced IBD Model and Endotoxin Treatment

Establishment of the bacterially induced IBD model was described previously (Richardson et al., 2006). Briefly, C3H/HeOuJ mice were infected with 2.0 × 108 colony-forming units of C. rodentium (ATCC) in saline by oral gavage. The control group was administered saline by gavage. Mice were sacrificed after apparent clinical symptoms of IBD were observed. Serum, liver, and colon samples were collected for further analysis. For endotoxin treatment, two groups of C57BL/6 mice were administered saline or 4 mg/kg LPS in saline by intraperitoneal injection and sacrificed 24 hr later for liver collection.

LC-MS Analysis of Mouse Serum

Serum samples were mixed with 20 volumes of 66% aqueous acetonitrile and centrifuged at 18,000 × g for 10 min to remove protein and particulates. For LC-MS analysis, a 5 μl aliquot was injected into a Waters UPLC-QTOFMS system. An Acquity UPLC BEH C18 column (Waters) was used to separate serum metabolites at 35°C. The mobile phase flow rate was 0.5 ml/min, with a gradient ranging from 5% to 99% aqueous acetonitrile containing 0.1% formic acid over a 10 min run. The QTOF Premier mass spectrometer was operated in the positive electrospray ionization (ESI) mode. Capillary voltage and cone voltage were maintained at 3 kV and 20 V, respectively. Source temperature and desolvation temperature were set at 120°C and 350°C, respectively. Nitrogen was used as both cone gas (50 l/hr) and desolvation gas (600 l/hr), and argon was used as collision gas. For accurate mass measurement, the mass spectrometer was calibrated with sodium formate solution (range m/z 100–1000) and then monitored by intermittent injection of the lock mass sulfadimethoxine ([M+H]+ = 311.0814 m/z) in real time. Mass chromatograms and mass spectral data were acquired by MassLynx software (Waters) in centroided format. The structures of serum biomarkers were elucidated by MS/MS fragmentation with collision energies ranging from 15 to 35 eV. Serum concentrations of stearoyl-LPC and oleoyl-LPC were quantified using QuanLynx (Waters). Standard curves of both LPCs were linear in the range of 0.5–20 μM (r2 > 0.95).

Data Processing and Multivariate Data Analysis

Chromatographic and spectral data of serum samples were processed using MarkerLynx (Waters) to generate a multivariate data matrix by centroiding, integration, normalization, and deconvolution. The data matrix was further exported into SIMCA-P+ software (Umetrics) and transformed by an appropriate scaling technique for model construction. Major latent variables in the data matrix, represented as principal components, were generated by principal components analysis (PCA) and partial least square discriminant analysis (PLS-DA) and described in a scores scatter plot. Serum biomarkers were identified by analyzing ions in the loadings scatter plot.

Western Blot Analysis

Microsomes, whole-cell lysates, and nuclear extracts of mouse liver were prepared following standard procedures. Targeted proteins were detected using corresponding antibodies, including CYP3A (mAb 2-13-1) (Park et al., 1986), SCD1, calnexin, GRP78, SREBP1, and GAPDH (Santa Cruz Biotechnology).

RNA Analysis

Quantitative real-time PCR (qPCR) was performed using SYBR green PCR master mix (Superarray) in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Primer sequences are listed in Table S1. Expression levels of targeted genes were normalized to β-actin.

Insulin and IL-1β Analysis

Serum insulin levels were measured by radioimmunoassay (RIA) (Linco Research). Serum IL-1β levels were determined by enzyme-linked immunosorbent assay (ELISA) (R&D Systems).

In Vivo Overexpression of SCD1 by Adenovirus-Mediated Transfection

Adenovirus expressing rat SCD1 (Ad-SCD1) was kindly provided by V.I. Zannis (Boston University), and control adenovirus was kindly provided by S. Kimura (National Cancer Institute). Construction of Ad-SCD1 using pAdTrackCMV vector was described previously (Drosatos et al., 2007). Ad-SCD1 was amplified in QBI293 cells and purified by two cesium chloride ultracentrifugation steps followed by dialysis and titration. For in vivo transfection, male C57BL/6 mice were injected with 2 × 109 plaque-forming units of control adenovirus or Ad-SCD1 per mouse. Second and third injections were conducted at the third and sixth day of DSS treatment.

Statistics

Experimental values are expressed as mean ± SD. Statistical analysis was performed by two-tailed Student’s t test for unpaired data, with p < 0.05 considered statistically significant.

Supplementary Material

Supplemental References and Figures

Supplemental Table

Acknowledgments

We thank V.I. Zannis for providing adenovirus expressing SCD1 and S. Kimura for control adenovirus. We also appreciate the technical assistance of J. Buckley, U. Jaowattana, and T. Tanabe. J.R.I. is grateful to US Smokeless Tobacco Company for a grant for collaborative research. This study was supported by the NCI Intramural Research Program of the NIH.

Footnotes

Supplemental Data include one table, Supplemental References, and two figures and can be found with this article online at http://www.cellmetabolism.org/cgi/content/full/7/2/135/DC1/.

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