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Physiol. Genomics 31: 216-227, 2007. First published June 12, 2007; doi:10.1152/physiolgenomics.00264.2006
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Received 4 December 2006; accepted in final form 5 June 2007.
Physiological Genomics 31:216-227 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

TNF-{alpha} interferes with lipid homeostasis and activates acute and proatherogenic processes

Klementina Fon Tacer1, Drago Kuzman2, Matej Seliskar1, Denis Pompon3 and Damjana Rozman1

1 Center for Functional Genomics and Biochips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia
2 Lek Pharmaceuticals, Ljubljana, Slovenia
3 Laboratoire d'Ingénierie des Protéines Membranaires, Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The interaction between disrupted lipid homeostasis and immune response is implicated in the pathogenesis of several diseases, but the molecular bridges between the major players are still a matter of controversy. Our systemic study of the inflammatory cytokine tumor necrosis factor-alpha (TNF-{alpha}) in the livers of mice exposed to 20-h cytokine/fasting for the first time shows that TNF-{alpha} interferes with adaptation to fasting and activates harmful proatherogenic pathways, partially through interaction with the insulin-Insig-sterol regulatory element binding protein (Srebp) signaling pathway. In addition to the increased expression of acute-phase inflammatory genes, the most prominent alterations represent modified lipid homeostasis observed on the gene expression and metabolite levels. These include reduction of HDL-cholesterol, increase of LDL-cholesterol, and elevated expression of cholesterogenic genes, accompanied by increase of potentially harmful precholesterol metabolites and suppression of cholesterol elimination through bile acids, likely by farnesoid X receptor-independent mechanisms. On the transcriptional level, a shift from fatty oxidation toward fatty acid synthesis is observed. The concept of the influence of TNF-{alpha} on the Srebp regulatory network, followed by downstream effects on sterol metabolism, is novel. Observed acute alterations in lipid metabolism are in agreement with chronic disturbances found in patients.

transcriptome; inflammation; acute response; cholesterol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE INTERPLAY BETWEEN INFLAMMATION and lipid metabolism has recently been the focus of research aimed at understanding the development of metabolic syndrome and mechanisms of atherogenesis (33, 74). One of the difficulties in studying the interaction between inflammation and lipid metabolism in vivo is anorexia triggered by proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-{alpha}), interleukins (IL), and the endotoxin lipopolysaccharide (LPS) (37). Studies that discuss the effects of inflammatory agents have so far mostly neglected the metabolic adaptation to fasting, which contributed to controversial conclusions (26, 29, 38, 39, 48).

Cholesterol synthesis and uptake are regulated by feedback inhibition (72) that depends on the transcriptional activators sterol regulatory element-binding proteins (Srebp-1a, -1c, and -2) (12). Under conditions of sterol depletion, the activation of membrane-bound Srebps and their transport from endoplasmic reticulum (ER) to Golgi is mediated by the Srebp cleavage activating protein (Scap) (24). In contrast, when cholesterol content rises, cholesterol binds to the sterol-sensing domain of Scap (65). This enables Scap to bind the anchored Insig proteins, which prevents intracellular transport and disables activation of Srebps (11, 78, 80). Insig-1 and Insig-2 are closely related membrane proteins of the ER. They regulate cholesterol homeostasis by binding in a sterol-dependent manner to two ER proteins, the rate-limiting enzyme of cholesterol biosynthesis, HMG-CoA reductase, and Scap. In this manner they prevent activation of Srebp on one side and direct degradation of HMG-CoA reductase on the other side (34).

Cholesterol is eliminated from the body through conversion to bile acids in the liver. Liver X receptors (Lxrs) and farnesoid X receptor (Fxr) are nuclear receptors that function as intracellular sensors for sterols and bile acids, respectively. In response to their ligands, these receptors induce transcriptional responses that maintain a balanced, finely tuned regulation of cholesterol and bile acid metabolism (16, 45). Lxrs are sterol sensors and bind oxysterols to regulate genes critical to cholesterol efflux (Abca1, Abcg1, and ApoE), bile acid synthesis (Cyp7A1), and cholesterol secretion into bile for excretion (Abcg5/g8) (67). Fxr is activated by bile acids that negatively regulate their synthesis to prevent bile acid toxicity and maintain bile acid homeostasis (45).

Data suggest that inflammation might be one of the processes that permit the bypass of the cholesterol negative feedback loop. An increased rate of de novo hepatic cholesterol synthesis has been observed in different animal models of infection (26, 28, 30, 60), which would suggest a coordinate upregulation of cholesterogenic genes/enzymes and repression of those responsible for cholesterol excretion. However, other studies reported that LPS, TNF-{alpha}, and IL-1 provoke a discordant regulation of cholesterogenic genes (26, 28, 61). In contrast to the above, a recent transcriptome study of the acute-phase response to LPS in vivo reported a coordinate decrease of cholesterogenic gene expression (81).

We used a systemic approach to evaluate the effect of TNF-{alpha} treatment in the mouse in vivo by separating the acute-phase inflammatory effects from the effects of metabolic adaptation in liver to fasting. Our findings provide novel evidence that TNF-{alpha} disturbs adaptation processes to caloric restriction and mediates acute lipid-related and other proatherogenic changes including modulation of the Srebp regulatory pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials.
Recombinant human TNF-{alpha} with a specific activity of 3.3 x 107 U/mg was generously provided by V. Menart (Lek Pharmaceuticals). TNF-{alpha} was freshly diluted in sterile 0.9% saline to 0.15 mg/ml. Rabbit polyclonal antibodies anti-human Cyp51 (59), anti-human HMG-CoA reductase protein (Hmgcr) (Upstate), and anti-actin (Sigma-Aldrich) were applied together with goat-anti-rabbit-horseradish peroxidase (HRP) (Amersham), and mouse monoclonal anti-Srebp-2 antibody (BD Pharmingen) with goat-anti-mouse-HRP (Amersham). Cholesterol, desmosterol, 7-dehydrocholesterol, lanosterol, and lathosterol were from Steraloids. Follicular fluid meiosis-activating sterol (FF-MAS) and testis meiosis-activating sterol (T-MAS) are laboratory standards from A. G. Byskov (Laboratory of Reproductive Biology, University Hospital of Copenhagen).

Animals and treatment.
All in vivo procedures were in accordance with the Amsterdam Protocol on Animal Protection and Welfare and were approved by the Veterinary Administration of the Republic of Slovenia. Experiments were performed on C57BL/6 male mice (Harlan) between 10 and 12 wk of age. Mice were injected intravenously with human recombinant TNF-{alpha} (30 µg/animal) or with a corresponding volume (200 µl) of saline and euthanized 20 h later. In fasted and fasted/TNF-{alpha} groups food was withdrawn after TNF-{alpha} application (37). Details are provided in the supplementary data for this article.1

Plasma parameter analyses.
Plasma total cholesterol, HDL-cholesterol, triglycerides, free fatty acids, and glucose were measured by enzymatic assays using commercially available kits (CHOD/PAP, Roche; HDL-cholesterol direct, Randox; Infinity Triglycerides, Thermo Scientific; free fatty acids colorimetric assay kit, Roche; Autokit Glucose 2, Waco). Insulin concentrations were measured with an ultrasensitive rat insulin ELISA kit (Crystal Chem). Very low-density lipoprotein, intermediate-density lipoprotein (IDL)/LDL, and HDL were isolated by fast performance liquid chromatography (FPLC). Pooled plasma (120 µl) from five mice was applied to a Superose 6 HR 10/30 (Pharmacia) column. Cholesterol and triglyceride content in fractions were measured enzymatically.

RNA isolation and quantitative real-time PCR.
Total RNA was isolated with TRI reagent (Sigma), subjected to RNeasy Cleanup column (Qiagen), and processed for real-time PCR on an ABI Prism 7900 HT system (Applied Biosystems) (57). Details are provided in the supplementary material for this article.

cDNA microarray hybridization and data analysis.
Ten micrograms of total RNA was labeled with a Agilent direct labeling kit with Cy5 (fasted and TNF-{alpha} treated) and Cy3 (control) fluorescent dyes (Amersham) and hybridized on Agilent cDNA arrays (G4104A) according to manufacturer's instructions. Individual samples from fasted and TNF-{alpha}-treated animals were hybridized versus the pooled control sample. Statistical significance of differential gene expression change was evaluated according to the local pooled error test (42) and the z-test (52). Data were deposited in the GEO database with accession number GSE6317. Details are provided in the supplementary materials for this article.

Immunoblot analyses of liver microsomal proteins and nuclear extracts.
Protein extracts were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with primary polyclonal antibodies against Cyp51 (1:300), Hmgcr (1:1,000), actin (1:1,000), and anti-Srebp-2 antibody (1 µg/ml), followed by 1-h incubation with a peroxidase-conjugated anti-rabbit IgG. Peroxidase activity was measured with goat anti-rabbit IgG (1:1,000) or anti-mouse IgG (1:1,000) (Amersham), using the CL-HRP substrate system (Pierce Biotechnology). Quantification was performed with UVI Soft-UVI Band software (UVI Tec). Details are provided in the supplementary materials for this article.

Liver sterol extraction and liquid chromatography-mass spectrometry analysis.
Total liver sterols were extracted as previously described (3, 31) and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis. Details are provided in the supplementary materials for this article.

Statistics.
One-way ANOVA followed by Tukey's multiple comparison test was used to compare the three groups. Statistical analysis was performed with Graph Pad Prism 4.02 (Graph Pad Software, San Diego, CA), and a value of P < 0.05 was considered to be significant for all parameters measured.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TNF-{alpha} decreases HDL-cholesterol level in plasma.
Serum parameters were analyzed as part of a systematic analysis to explore the effect of TNF-{alpha} on lipid metabolism. After a single dose of TNF-{alpha} administration, food was withdrawn in TNF-{alpha}/fasted and fasted groups, whereas the control group received food ad libitum. Twenty hours after TNF-{alpha} administration, animals were euthanized. As expected, glucose and insulin levels were lower in the fasted group compared with the normal fed group (Table 1). TNF-{alpha} did not result in significant changes of serum glucose and insulin levels. Serum triglycerides and free fatty acids were higher in the TNF-{alpha}-treated group, but the changes were not statistically significant.


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  		Table 1. Serum parameters in control, fasted, and TNF-{alpha}-treated mice

 
For total plasma cholesterol level, no statistically significant increase was observed after TNF-{alpha} treatment compared with the fasting group (Table 1). However, the effect of TNF-{alpha} on lipoprotein distribution was significant. FPLC lipoprotein analysis showed that TNF-{alpha} induced a shift from HDL to LDL + IDL (Fig. 1), resulting in significantly lower HDL-cholesterol in TNF-{alpha}-treated animals compared with fasting counterparts.


Figure 1
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  		Fig. 1. TNF-{alpha} decreases serum HDL level and increases serum levels of LDL and intermediate-density lipoprotein (IDL). Pooled plasma (120 µl) from 5 mice from control (x), fasted ({blacksquare}), and TNF-{alpha}-treated ({blacktriangleup}) groups was applied to a Superose 6 HR 10/30 (Pharmacia) column for lipoprotein separation. Cholesterol and triglyceride content in fractions were measured enzymatically. Arrows indicate TNF-{alpha}-induced change in lipoprotein composition from decrease in HDL to increase in LDL + IDL.

 
TNF-{alpha} interferes with metabolic adaptation to fasting on transcriptional level.
To understand the molecular responses to TNF-{alpha} in fasting conditions, microarray analysis was applied using mouse Agilent cDNA chips and mRNA from liver of fasted, TNF-{alpha}-treated, and control animals. The effect of fasting on the liver transcriptome was evaluated by direct comparison (fasted animals/normal fed controls), while the effect of TNF-{alpha} in fasted conditions was evaluated by indirect comparison using the normal fed control group as a common reference. Approximately 10% of the 9,595 genes present on the microarray were expressed above the level of detection. Fasting modulated 12% of genes expressed above the detection level, 46 being upregulated and 64 downregulated, whereas TNF-{alpha} treatment resulted in modulation of 22% of genes, 50 being upregulated and 107 repressed compared with fasted counterparts (Fig. 1 in supplementary materials). The expression of selected genes was confirmed by quantitative RT-PCR (qRT-PCR) (Figs. 2 and 3).


Figure 2
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  		Fig. 2. TNF-{alpha} activates expression of genes involved in cholesterol synthesis and inhibits expression of bile acid synthesis genes. Equal amounts of liver RNA from 3 animals/group were pooled and converted to cDNA. Expression of genes involved in cholesterol synthesis (A), cholesterol negative feedback loop (B), and bile acid synthesis (C) was measured by quantitative RT-PCR (qRT-PCR) in livers of normal-fed (open bars), fasted (gray bars), and TNF-{alpha}-treated/fasted (filled bars) animals. mRNA values were normalized to 18S rRNA and plotted relative to normal-fed controls as means ± SD of triplicate measurements. UniGene symbols are used as abbreviations.

 

Figure 3
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  		Fig. 3. TNF-{alpha} modulates expression of genes involved in bile acid and fatty acid homeostasis, lipoprotein metabolism, and acute-phase response. Expression of genes involved in bile acid (BA) homeostasis (A), fatty acid synthesis (B) and oxidation (C), and lipoprotein metabolism and acute-phase response (D) was measured by qRT-PCR in livers of normal-fed (open bars), fasted (gray bars), and TNF-{alpha}-treated/fasted (filled bars) animals. mRNA values were normalized to 18S rRNA and plotted relative to normal-fed controls as means ± SD of triplicate measurements. UniGene symbols are used as abbreviations. FXR, farnesoid X receptor; LXR, liver X receptor; APR, acute phase response.

 
TNF-{alpha} activates expression of cholesterol biosynthetic genes in fasting.
As expected, during fasting the expression of cholesterogenic genes is coordinately downregulated because disconnection of anabolic pathways (cholesterol metabolism in Table 2, cholesterol biosynthesis in Fig. 2A). We also observed lower expression of LDL receptor mRNA (Fig. 2A) and altered mRNA levels of regulatory proteins Srebp (Fig. 2B) and Insig (Fig. 2B, Table 2). The mRNA expression of the three Srebps, as well as Insig-1 and Insig-2b, is repressed, whereas Insig-2a mRNA is expectedly upregulated (Ref. 78; Table 2, Fig. 2B).


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  		Table 2. Differentially expressed genes in mouse liver in fasted condition and after TNF-{alpha} treatment as detected by microarray analysis

 
In contrast, despite fasting, TNF-{alpha} treatment results in the upregulation of genes involved in cholesterol synthesis. This cytokine coordinately mobilizes the expression of genes involved in the early (Hmgcs, Hmgcr, Fdps, Fdft1) and late (Cyp51, Sc4mol, Dhcr7, Sc5d, Dhcr7, Dhcr24) parts of cholesterol synthesis (Table 2, Fig. 2A). The expression of LDL receptor mRNA was not changed (Fig. 2A). In addition to cholesterogenic genes, TNF-{alpha} induces alterations in the expression of genes encoding regulatory proteins, Srebps and Insigs, respectively. Compared with the fasting response, Srebp-1a, Srebp-2, and Insig-1 mRNAs are increased, while Insig-2a mRNA level is reduced by TNF-{alpha} (Fig. 2B). The only exception to the rule is Srebp-1c, the transcription of which is not activated by TNF-{alpha} (Fig. 2B).

TNF-{alpha} represses genes involved in reverse cholesterol transport and bile acid synthesis.
Bile acid synthesis occurs via either the neutral (classic) or the acidic (alternate) pathway (reviewed in Ref. 68). TNF-{alpha} strongly represses three enzymes critical to bile acid synthesis, Cyp7a and Cyp8b from the neutral pathway and Cyp27 from the acidic pathway (Fig. 2C). We observed a slightly decreased Cyp7a1 mRNA level in fasting conditions, but after TNF-{alpha} treatment its expression was almost completely blocked (Fig. 2C). Another gene from the neutral branch of bile acid synthesis, Cyp8b1, increases by exposing mice to starvation (Ref. 21; Fig. 2C). Similar to Cyp7a1, TNF-{alpha} also strongly repressed mRNA level of Cyp8b1 as well as Cyp27, the rate-limiting enzyme of the acidic branch of bile acid synthesis. However, the expression of the other enzyme from this branch, Cyp7b1, was increased by TNF-{alpha}, which is consistent with previous reports (20).

The bile acid synthesizing enzymes Cyp7a1, Cyp8b1, and Cyp27 are subject to negative feedback regulation by bile acids that serve as ligands for nuclear receptors Fxr and Pxr. Fxr mediates negative feedback regulation of bile acid synthesis involving activation of another nuclear receptor, Shp (2, 35). The expression level of Fxr is downregulated by inflammation (49), which was observed also in our study (Fig. 3A). The Fxr target genes Shp and Pxr are not changed by fasting and TNF-{alpha} (Fig. 3A). Hepatic transporters are responsible for uptake and efflux of bile acids. Two other Fxr target genes, bile salt export pump (Bsep), which is induced by Fxr, and Na+-taurocholate cotransporting polypeptide (Ntcp, Slc10a1), which is repressed by Fxr, are both strongly downregulated by TNF-{alpha} (Fig. 3A). Thus our data support the hypothesis that TNF-{alpha} strongly inhibits bile acid synthesis and transport by the mechanism that is most probably independent of Fxr.

Storage of cholesterol and conversion of excess cholesterol to bile acids is regulated by nuclear receptors Lxrs (67). Although no decrease in Lxr and its heterodimer partner Rxr was observed, downregulation of Lxr target genes involved in reverse cholesterol transport (Abcg5, ApoE; Fig. 3B) has been detected. This is consistent with previously observed reduced transcriptional activity of Lxr-Rxr dimers during acute-phase response (7).

Despite fasting, TNF-{alpha} activates fatty acid synthesis and decreases fatty acid oxidation.
Since Lxrs are also key regulators of hepatic fatty acid synthesis through transcriptional activation of Srebp-1c (66), we investigated some of the Lxr target genes involved in this pathway. In fasting, we observed downregulation of two key enzymes of fatty acid synthesis, fatty acid synthase (Fas) and acetyl-CoA carboxylase (Acc-{alpha}) (Fig. 3B). Inhibition is most probably through inhibition of Srebp-1c mRNA level (Fig. 2B). In contrast, TNF-{alpha} activates the expression of two lipogenic genes, Fas and Acc-{alpha} (Fig. 3B), despite fasting and independently of Lxr and SREBP-1c (Fig. 2B). These data suggest that TNF-{alpha} activates fatty acid synthesis through activation of another isoform, likely Srebp-1a (Fig. 2B).

Nuclear receptor Ppar-{alpha}, which is activated by free fatty acids, plays a crucial role in coordinating metabolic changes caused by fasting and starvation (46). When activated, it turns on fatty acid oxidation and ketone body synthesis. We observed that several genes encoding enzymes involved in fatty acid ß-oxidation (Cpt1-{alpha}, Cpt2, Peci, Ehhadh, Aco, Hadh2) and {omega}-oxidation (Cyp4a10, Cyp4a14) as well as fatty acid transport and uptake by liver (Fabp 1 and 2, Acsl1, Slc27a2) are highly upregulated in fasting (Table 2, Fig. 3C). The {omega}-hydroxylases of fatty acids from the cytochrome P-450 superfamily, Cyp4a10 and Cyp4a14, are among the most upregulated genes in fasting (Table 2, Fig. 3C) (6). Despite nutrient deprivation, the same genes are repressed by TNF-{alpha} (Table 2, Fig. 3C), most probably because of the repression of Ppar-{alpha} expression itself (Fig. 3C) (50).

Lipoprotein metabolism, acute-phase response, and inflammation.
Increased expression of genes encoding HDL-apolipoproteins A-V and C-II (Table 2) has been observed during fasting, while the expression of ApoF, an inhibitor of cholesteryl ester transfer protein (Cetp) (77), is diminished. Fasting provokes decline in acute-phase and other inflammatory proteins. The expression of Saa3 mRNA, a well-known acute-phase response protein marker, is clearly reduced (Table 2, Fig. 3D). In addition, TNF-{alpha} interferes with lipoprotein metabolism. We have observed downregulation of ApoA1, ApoE, and hepatic lipase (Lipc) mRNA expression and elevated levels of Acat mRNA (Table 2, Fig. 3D). HDL-associated apolipoproteins (ApoA-I, A-II, C-IV) as well as paraoxonase 1 (Pon1) represent one of the most strongly downregulated groups of genes after TNF-{alpha} application (Table 2, Fig. 3D). In addition, five members of the serine protease inhibitor (Serpin) protein superfamily, important in hemostasis, clotting, complement system, and inflammation (reviewed in Ref. 32), were also significantly downregulated by fasting. TNF-{alpha} markedly induces the mRNA expression of many acute-phase proteins, such as Saa3, serum amyloid P-component, orosomucoid 2, orosomucoid 1, hemopexin, and complement component 3. Saa3 is the most upregulated gene in our study.

Amount of Cyp51 and mature SREBP-2 protein is increased by TNF-{alpha}.
The quantity of the postlanosterol enzyme Cyp51 is significantly diminished in fasted animals and increased after TNF-{alpha}-treatment (Fig. 4, A and C). This is in accordance with the Cyp51 mRNA expression data (Fig. 2A), suggesting that the TNF-{alpha}-mediated upregulation of Cyp51 mRNA leads to the elevated amount of the Cyp51 protein. The protein seems to be enzymatically active since an increase of sterol FF-MAS, which is the product of the Cyp51-catalyzed reaction, is also observed (Fig. 5B). This indicates that Cyp51 is regulated primarily on the transcriptional level.


Figure 4
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  		Fig. 4. TNF-{alpha} augments Cyp51 protein level and nuclear content of SREBP-2. Representative immunoblots of liver microsomal (A) and nuclear (B) proteins from control (lane 1, open bars), fasted (lane 2, gray bars) and fasted/TNF-{alpha}-treated (lane 3, filled bars) mice are shown. A: 40 µg of microsomal proteins was subjected to SDS-PAGE and immunoblot analysis against HMG-CoA-reductase (Hmgcr) and lanosterol 14{alpha}-demethylase (Cyp51). Data represent pools from 3 animals. B: 60 µg of pooled nuclear extracts from 2 animals were subjected to SDS-PAGE, and immunoblot analysis against Srebp-2 was performed. C and D: intensity of bands from at least 2 independent replicates were quantified and normalized to actin, and fold induction was calculated relative to control, which was set to 1. AU, arbitrary unit.

 

Figure 5
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  		Fig. 5. Outline of the late part of cholesterol biosynthesis (A) and the liver sterol metabolome (B) in fasting (gray bars) and after TNF-{alpha} application (filled bars). A: enzymatic steps are labeled by numbers. Sterols measured in our study are represented by formulas. Two enzymes, Cyp51 (lanosterol 14{alpha}-demethylase, 1) and Dhcr24 (24-dehydrocholesterol reductase, 7), compete for lanosterol, the 1st cyclic intermediate of the late part of cholesterol synthesis. This leads to 2 possible branches with sterol intermediates containing either saturated (right) or unsaturated (left) {Delta}24 bond. 2, Dhcr14 (14-dehydrocholesterol reductase); 3, C-4 demethylation by Sc4mol (C-4 demethylation by sterol C4-methyl oxidase), Nsdhl (3-ß-hydroxy-{Delta}5-steroid dehydrogenase), and Hsd3b3 (3ß-keto-steroid reductase); 4, Ebp-{Delta}8, {Delta}7-sterol isomerase; 5, Sc5d-sterol-C5 desaturase; 6, Dhcr7 (7-dehydrocholesterol reductase). Abbreviations of enzyme names correspond to UniGene symbols. FF-MAS, follicular fluid meiosis-activating sterol (4,4-dimethyl-5{alpha}-cholesta-8,14,24-triene-3ß-ol); T-MAS, testis meiosis-activating sterol (4,4-dimethyl-5{alpha}-cholesta-8,24-diene-3ß-ol). B: liver sterol metabolome represented as log 2 ratio of fasted/normally fed animals (gray bars) and TNF-{alpha}/fasted vs. fasted animals (filled bars). Data represent mean ± SD log 2 ratio of 6 animals of each sex. *P < 0.05, 1-way ANOVA followed by Tukey's multiple comparison test. #Sterols were below level of detection in fasted animals. Fold change was calculated according to the detection level threshold for each sterol. Lathosterol was below level of detection in our samples.

 
Surprisingly (see Ref. 28), the quantity of HMG-CoA reductase protein (Hmgcr) was not significantly altered in fasting or 20 h after the TNF-{alpha} stimulus (Fig. 4, A and C). Since Hmgcr is the rate-limiting enzyme of cholesterol synthesis, the kinetics of Hmgcr might precede the changes of other enzymes. Additionally, it is established that Hmgcr is regulated primarily by posttranscriptional and posttranslational sterol-dependent events (64). When sterols accumulate in the cell, Hmcgr protein undergoes a sterol-accelerated degradation, a process mediated by Insig proteins. And, remarkably, the cholesterol precursor lanosterol, which accumulates after TNF-{alpha} treatment (Fig. 5B), is a more potent inducer of Hmgcr degradation than cholesterol itself (reviewed in Ref. 34).

Repeated Western blots with 100,000 g membrane factions of the mouse liver with the anti-Srebp-2 antibody did not show a detectable signal at 125 kDa, where membrane-bound Srebp-2 should reside (12). However, the amount of 68-kDa nuclear Srebp-2 was reduced by fasting (40) and elevated by TNF-{alpha} (Fig. 4, B and D). This is in accordance with the observed changes in expression of cholesterogenic genes that are regulated by Srebp-2 where diminished expression in starvation and upregulation after TNF-{alpha} treatment has been observed. Our data suggest that TNF-{alpha} stimulates cholesterol biosynthesis at least in part by stimulating transcription and activation of the regulatory proteins from the cholesterol negative feedback loop.

TNF-{alpha} alters liver sterol metabolic profile.
To our knowledge, there is no comprehensive study of the effects of inflammation or inflammatory cytokine TNF-{alpha} on the liver sterol profile. Figure 5A shows a schematic outline of the late part of cholesterol synthesis. Two enzymes (Cyp51 and Dhcr24) compete for the first cyclic intermediate, lanosterol. Consequently, the late part of the cholesterol pathway has two branches with {Delta}24 desaturated or saturated intermediates. In addition to cholesterol, we measured several cholesterol synthesis intermediates: FF-MAS, T-MAS, lathosterol, zymosterol, desmosterol, and 7-dehydrocholesterol. As expected (see Ref. 75), in livers of normally fed animals the level of sterol intermediates is very low, cholesterol representing 99.5% of the total sterol pool (data not shown).

Interestingly, fasting did not provoke a significant change in the liver sterol profile (Fig. 5B). The quantity of cholesterol and lanosterol was not changed significantly, whereas FF-MAS, T-MAS, zymosterol, and lathosterol were even below the level of detection (0.05 ng/mg for FF-MAS and T-MAS; 0.01 ng/mg for zymosterol and lathosterol). TNF-{alpha} treatment resulted in an increase of all measured liver sterols except 7-dehydrocholesterol and lathosterol (Fig. 5B), indicating activation of cholesterol biosynthesis primarily through the branch with unsaturated {Delta}24 intermediates. The increased sterol intermediate level was statistically significant for FF-MAS, T-MAS, zymosterol, and desmosterol. It is important to note that changes in the metabolic sterol profile agree with changes of respective postlanosterol genes, as observed by real-time PCR and microarray analysis (Fig. 2A, Table 2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
TNF-{alpha} is a multifunctional cytokine with different roles ranging from proliferation to inflammatory effects and mediation of the immune responses (reviewed in Ref. 58) and in general induces anorexia. Our systemic approach enabled separation of the acute TNF-{alpha} response from the metabolic adjustment of liver to fasting. This is an important issue since experiments in vivo include food removal after induction of inflammation.

Mammals have evolved a complex mechanism to survive food deprivation. Generally, the hallmark of fasting is upregulation of processes that are linked to energy production for gluconeogenesis, together with disconnection of all anabolic pathways. In accordance with this, genes involved in the ß- and {omega}-oxidation of fatty acids as well as their transport are highly upregulated, while expression of cholesterol and bile acid synthesis genes is reduced (Table 2, Figs. 2 and 6). Fasting increases the expression of the protective HDL-apolipoproteins (A-V, C-II) and inhibits the expression of the inflammatory apolipoprotein gene Saa3, which displaces ApoA-I from HDL and converts HDL from protective to proatherogenic (14). Expression of the acute-phase proteins is also reduced, which supports the view that caloric restriction induces protective processes (5). Adaptation to fasting includes downregulation of Srebp proteins that coordinately regulate the expression of genes involved in cholesterol and fatty acid biosynthesis. Downregulation of Srebp-2-dependent cholesterogenic genes is expected since fasting also diminishes the amount of the nuclear Srebp-2 protein (Fig. 4, B and D).


Figure 6
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  		Fig. 6. Schematic overview of cholesterol homeostasis and the effects of TNF-{alpha} in the liver of fasted mice. A: cholesterol biosynthesis is regulated by the negative cholesterol feedback loop and transcription factors Srebps. Activated Srebps trigger transcription of cholesterol biosynthetic genes leading to higher cholesterol production. In contrast, cholesterol loading prevents Srebp activation mediated by Insigs. Excess cholesterol is eliminated from peripheral organs through reverse cholesterol transport (RCT) by HDL particles. Cholesterol is converted to bile acids for excretion. Fasting (gray arrows) inhibits cholesterol and fatty acid synthesis by inhibiting expression of regulatory proteins involved in the cholesterol negative feedback loop (Srebps, Insig2a). This consequently leads to reduced expression of cholesterogenic genes and Cyp51 protein. To provide energy, fatty acid oxidation is activated in the nutrient-depleted condition. In contrast, TNF-{alpha} (black arrows) induces the expression of genes encoding regulatory proteins Srebp-1a and -2 that leads to upregulation of cholesterogenic genes as well as genes involved in fatty acid synthesis. The expression of 2 major liver Insig proteins is reciprocal. In fasting, Insig-2a is upregulated and Insig-1 is reduced, while TNF-{alpha} reverses the expression of both. TNF-{alpha} inhibits expression of genes even further in bile acid synthesis and transport. At the same time, TNF-{alpha} blocks elimination of cholesterol through bile acid synthesis and inhibits fatty acid oxidation. VLDL, very low-density lipoprotein; FFA, free fatty acids. B: in contrast to fasting, which provokes protective, antiatherogenic, protective changes, TNF-{alpha} induces proatherogenic alterations in the liver.

 
In contrast, TNF-{alpha} exerts multiple lipid-related proatherogenic and prooxidative alterations that can be mediated through receptors TNF receptor (TNFR)I and TNFRII (4). Human recombinant TNF-{alpha} was injected in our study; the effects are likely due to the activation of TNFRI, since mouse TNFRII shows strong specificity for murine TNF-{alpha} (53). First of all, a coordinate upregulation of the early and late genes of cholesterol synthesis was observed (Fig. 2A), similarly as in rats (71) and after chronic LPS treatment in the mouse (81). At least three genes of this pathway (Fdps, Fdft, Cyp51) are upregulated also by lead nitrate, which induces hypercholesterolemia through TNF-{alpha} and IL-1 (51). Importantly, the observed transcriptional changes of the postlanosterol genes are in accordance with the observed sterol metabolic profile (Fig. 5B). This indicates that changes on the transcriptional level (increased expression of cholesterogenic genes mediated by elevated levels of nuclear Srebp-2) are followed by modulation of the respective enzyme activities, suggesting further that postlanosterol cholesterol synthesis is regulated primarily on the level of transcription. TNF-{alpha} results in increase of {Delta}24 unsaturated postlanosterol cholesterol intermediates that are not detected in the livers of regularly fed (75) and fasted (Fig. 5B) animals. It is worth mentioning that these sterols are potentially harmful since they are not incorporated into the membranes because of redundant methyl groups (8). The exceptions are 7-dehydrocholesterol, found in membranes of patients with Smith-Lemli-Opitz syndrome (69), and desmosterol, residing in spermatozoa and astrocyte membranes (56, 63). Other sterols, such as FF-MAS and T-MAS, have not yet fully understood signaling properties. They reinitiate oocyte meiosis in vitro (13), contribute to maturation of the oocyte in humans in vivo (9), and are synthesized in sperm in situ (17). They are also nonspecific ligands of the Lxr (44) and are suggested to work through membrane G protein-coupled receptors (36). In addition to a higher rate of cholesterol production, our studies (Fig. 2C) and those of others (47) show that cholesterol elimination through bile acid synthesis and export is strongly inhibited. Consequently, cholesterol and its precursors (10) can be subjected to harmful oxidation that can intensify atherogenesis (55). Our data indicate that the oxidation potential is increased after TNF-{alpha} stimulus, since many genes involved in defense mechanisms against oxidative stress are downregulated (Table 1 in supplementary data).

Furthermore, response to TNF-{alpha} includes the shift from fatty acid oxidation (Table 2, Figs. 3C and 6) to fatty acid synthesis (Table 2, Figs. 3B and 6). Notably, the decrease of genes from fatty acid oxidation is a result of repressed Ppar-{alpha} expression (Fig. 3C; Ref. 50) that plays a central role in fatty acid metabolism in fasting by directly stimulating the transcription of genes involved in fatty acid oxidation (46). Fatty acid synthesis is normally activated in the high-fed state through insulin-mediated activation of Lxr-Srebp-1c transcription factors (66). However, the TNF-{alpha}-induced activation of fatty acid synthesis in a low insulin-to-glucagon ratio seems to be Lxr- and Srebp-1c independent and might rely on activation of Srebp-1a (Fig. 2A).

On the systemic level, the transcriptome data seem to be in accordance with the plasma lipoprotein profile. TNF-{alpha} administration results in increased expression of genes involved in cholesterol synthesis and reduced expression of genes responsible for cholesterol elimination through bile acids, which together contribute to increase in LDL-cholesterol and reduction of HDL-cholesterol. Importantly, TNF-{alpha} inhibits the expression of paraoxonase (Pon1; Table 2, Fig. 3D), the major apolipoprotein that protects LDL from oxidative stress (27) and is a valuable marker of atherogenic changes also in humans (54). A possible mechanism to explain the observed lipid imbalance is interaction of TNF-{alpha} with the insulin-Insig-Srebp pathway that represents a bridge between cholesterol and energy homeostasis (23). The expression profile of Srebps and Insigs in the liver of fasted and TNF-{alpha}-treated animals resembles that during a fasting-refeeding protocol (79). During fasting the expression of the three Srebps is repressed, but insulin stimulus during carbohydrate diet refeeding (22, 34, 79), or TNF-{alpha} despite low insulin-to-glucagon ratio (our study), activates their expression (Fig. 2B), with the exception of Srebp-1c. The expression of Srebp-1c is induced by insulin (19, 73) but not by TNF-{alpha} (Fig. 2B). In addition to Srebps, the expression of Insig mRNA after TNF-{alpha} stimulus also follows the pattern of Insig mRNA expression after refeeding. Insig-2a expression is inhibited by insulin and is repressed by refeeding as well as by TNF-{alpha} (Fig. 2B). Insig-1 can replace Insig-2a (22, 34, 79) (Fig. 2B). Expression of the two Insigs is regulated in a different manner. Insig-1 is an obligatory Srebp target gene (43). Elevated levels of Insig-1 indicate a higher Srebp transcriptional activity after TNF-{alpha} application, which coincides with a higher Srebp-2 protein level (Fig. 4, B and D).

Even if we have not measured all proteins and enzyme activities, the correlation between mRNA and sterol metabolite levels suggests the regulation of postlanosterol cholesterol synthesis primarily at the transcriptional level. However, this seems not to be true for the early part of the pathway. While HMG-CoA reductase transcription is regulated by Srebp-2 (downregulation of Srebp-2 and HMG-CoA reductase mRNAs in starvation) the protein is further subjected to sterol-dependent regulation. In sterol-depleted conditions (fasting) the stability of HMG-CoA reductase protein is likely increased (Fig. 4A), and TNF-{alpha} in fasting does not seem to have an influence on HMG-CoA reductase protein.

The observed TNF-{alpha}-mediated perturbations are in agreement with metabolic changes in obese subjects (62) and patients with metabolic syndrome (15). It is important to emphasize that the TNF-{alpha} action on liver can be either direct or indirect through stimulated lipolysis in adipose tissue (70), since this cytokine is implicated in the insulin resistance of adipose tissue in obese animals and humans (41, 76). Additionally, the pathophysiology of other diseases (cancer, sepsis, HIV, etc.) includes TNF-{alpha}-induced anorexia and cachexia (1, 25).

In conclusion, our systemic study indicates that TNF-{alpha} severely disrupts lipid homeostasis under fasting conditions, which modulates metabolism of cholesterol, fatty acids, bile acids, and lipoproteins. Despite caloric restriction, TNF-{alpha} coordinately induces expression of genes involved in synthesis of cholesterol and at the same time abolishes its elimination. The TNF-{alpha} activation of cholesterogenic genes results at least in part by interacting with the insulin-Insig-Srebp signaling pathway. In contrast to fasting, which is accompanied with protective alterations, the acute TNF-{alpha} inflammatory response activates harmful metabolic pathways that are involved also in chronic proatherogenic and related pathological processes.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work was supported by the Slovenian Research Agency (Grants J1-6713, P1-0527, Z1-7562-0381) and funds from Lek Pharmaceuticals, d.d. K. Fon Tacer was supported by a fellowship from the Slovenian Research Agency. Work was generated in the context of the STEROLTALK project, funded by the European Community as contract no. LSHG-CT-2005-512096 under the 6th Framework Programme for Research and Technological Development. This work reflects only the authors' views, and the European Community is not liable for any use that may be made of the information contained therein.


   ACKNOWLEDGMENTS
 
We thank Mogens Baltsen and Dr. A. G. Byskov (Laboratory of Reproductive Biology, University Hospital of Copenhagen) for FF-MAS and T-MAS standards, Martina Perse (Institute of Pathology, Medical Experimental Centre, Faculty of Medicine, University of Ljubljana), Dr. Manica Cerne (Lek Pharmaceuticals, d.d.) and Dr. Srdjan Novakovic (Institute of Oncology, Ljubljana) for help with animals, Tadeja Rezen and Helena Klavzar (Center for Functional Genomics and Biochips, Faculty of Medicine, University of Ljubljana) for technical help, and Viktor Menart (Lek Pharmaceuticals, d.d.) for the human recombinant TNF-{alpha}. We also thank Vladka Serbec-Curin (Blood Transfusion Centre of Slovenia) for access to the qRT-PCR apparatus. We give special thanks to the Mangelsdorf-Kliewer laboratory, University of Texas Southwestern Medical Center (Dallas, TX), which allowed K. Fon Tacer to perform experiments for the revised manuscript.


   FOOTNOTES
 
Address for reprint requests and other correspondence: D. Rozman, Center for Functional Genomics and Biochips, Inst. of Biochemistry, Faculty of Medicine, Univ. of Ljubljana, Zaloska 4, 1000 Ljubljana, Slovenia (e-mail: damjana.rozman{at}mf.uni-lj.si).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

1 The online version of this article contains supplemental material. Back


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 DISCUSSION
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