Judy K. Shigenaga

The Acute Phase Response Is Associated with Retinoid X Receptor Repression in Rodent Liver

The acute phase response (APR) is associated with decreased hepatic expression of many proteins involved in lipid metabolism. The nuclear hormone receptors peroxisome proliferator-activated receptor α (PPARα) and liver X receptor (LXR) play key roles in regulation of hepatic lipid metabolism. Because heterodimerization with RXR is crucial for their action, we hypothesized that a decrease in RXR may be one mechanism to coordinately down-regulate gene expression during APR. We demonstrate that lipopolysaccharide (LPS) induces a rapid, dose-dependent decrease in RXRα, RXRβ, and RXRγ proteins in hamster liver. Maximum inhibition was observed at 4 h for RXRα (62%) and RXRβ (50%) and at 2 h for RXRγ (61%). These decreases were associated with a marked reduction in RXRα, RXRβ, and RXRγ mRNA levels. Increased RNA degradation is likely responsible for the repression of RXR, because LPS did not decreaseRXRβ and RXRγ transcription and only marginally inhibited (38%) RXRα transcription. RXR repression was associated with decreased LXRα and PPARα mRNA levels and reduced RXR·RXR, RXR·PPAR and RXR·LXR binding activities in nuclear extracts. Furthermore, LPS markedly decreased both basal and Wy-14,643-induced expression of acyl-CoA synthetase, a well characterized PPARα target. The reduction in hepatic RXR levels alone or in association with other nuclear hormone receptors could be a mechanism for coordinately inhibiting the expression of multiple genes during the APR.

Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor)in mouse liver during the acute phase response

Expression of P-450 (Cyp) enzymes is reduced in liver during the acute phase response, contributing to the decrease in bile acid levels and drug metabolism during infection. Nuclear hormone receptors CAR and PXR are key transactivators of Cyp2b and Cyp3a genes, respectively. Injection of bacterial lipopolysaccharide (LPS) induced the expected reduction in Cyp2b10 and Cyp3a mRNA levels in mouse liver. These decreases were associated with a marked reduction in CAR and PXR mRNA levels within 4 h following treatment. LPS-induced CAR and PXR repression were dose-dependent and sustained for at least 16 h. LPS treatment also reversed the up-regulation of Cyp3a in mice pre-treated with PXR ligand RU486. In addition, we observed a concomitant decrease in RXR (retinoid X receptor) mRNA levels, the obligatory partner of both CAR and PXR for high affinity binding to DNA. These findings represent one possible molecular mechanism underlying sepsis-induced repression of Cyp enzymes.

Endotoxin and Cytokines Increase Hepatic Sphingolipid Biosynthesis and Produce Lipoproteins Enriched in Ceramides and Sphingomyelin

Alterations in triglyceride and cholesterol metabolism often accompany inflammatory diseases and infections. We studied the effects of endotoxin (lipopolysaccharide [LPS]) and cytokines on hepatic sphingolipid synthesis, activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid synthesis, and lipoprotein sphingolipid content in Syrian hamsters. Administration of LPS induced a 2-fold increase in hepatic SPT activity. The increase in activity first occurred at 16 hours, peaked at 24 hours, and was sustained for at least 48 hours. Low doses of LPS produced maximal increases in SPT activity, with half-maximal effect seen at approximately 0.3 microg LPS/100 g body weight. LPS increased hepatic SPT mRNA levels 2-fold, suggesting that the increase in SPT activity was due to an increase in SPT mRNA. LPS treatment also produced 75% and 2.5-fold increases in hepatic sphingomyelin and ceramide synthesis, respectively. Many of the metabolic effects of LPS are mediated by cytokines. Interleukin 1 (IL-1), but not tumor necrosis factor, increased both SPT activity and mRNA levels in the liver of intact animals, whereas both IL-1 and tumor necrosis factor increased SPT mRNA levels in HepG2 cells. IL- produced a 3-fold increase in SPT mRNA in HepG2 cells, and the half-maximal dose was 2 ng/mL. IL-1 also increased the secretion of sphingolipids into the medium. Analysis of serum lipoprotein fractions demonstrated that very low density lipoprotein, intermediate density lipoprotein, and low density lipoprotein isolated from animals treated with LPS contained significantly higher amounts of ceramide, glucosylceramide, and sphingomyelin. Taken together, these results indicate that LPS and cytokines stimulate hepatic sphingolipid synthesis, which results in an altered structure of circulating lipoproteins and may promote atherogenesis.

Endotoxin down-regulates ABCG5 and ABCG8 in mouse liver and ABCA1 and ABCG1 in J774 murine macrophages differential role of LXR

Several of the ATP binding cassette (ABC) transporters have recently been shown to play important roles in reverse cholesterol transport (RCT) and prevention of atherosclerosis. In the liver, ABCG5 and ABCG8 have been proposed to efflux sterols into the bile for excretion. ABCG5 and ABCG8 also limit absorption of dietary cholesterol and plant sterols in the intestine. In macrophages, ABCA1 and ABCG1 mediate cholesterol removal from these cells to HDL. Many of these ABC transporters are regulated by the liver X receptor (LXR). We have previously shown that endotoxin (lipopolysaccharide) down-regulates LXR in rodent liver. In the present study, we examined the in vivo and in vitro regulation of these ABC transporters by endotoxin. We found that endotoxin significantly decreased mRNA levels of ABCG5 and ABCG8 in the liver, but not in the small intestine. When endotoxin or cytokines (tumor necrosis factor and interleukin-1) were incubated with J774 murine macrophages, the mRNA levels of ABCA1 were decreased. This effect was rapid and sustained, and was associated with a reduction in ABCA1 protein levels. Endotoxin and cytokines also decreased ABCG1 mRNA levels in J774 cells. Although LXR is a positive regulator of ABCA1 and ABCG1, we did not observe a reduction in protein levels of LXR or in binding of nuclear proteins to an LXR response element in J774 cells. The decrease in ABCG5 and ABCG8 levels in the liver as well as a reduction in ABCA1 and ABCG1 in macrophages during the host response to infection and inflammation coupled with other previously described changes in the RCT pathway may aggravate atherosclerosis.

Adipocyte Fatty Acid–Binding Protein Expression and Lipid Accumulation Are Increased During Activation of Murine Macrophages by Toll-Like Receptor Agonists

Objective—Toll-like receptors (TLRs) recognize pathogens and mediate signaling pathways important for host defense. Recent studies implicate TLR polymorphisms in atherosclerosis risk in humans. Adipocyte fatty acid–binding protein (aP2) is present in macrophages and has an important role in atherosclerotic plaque development. We investigated aP2 expression in RAW 264.7 cells treated with lipopolysaccharide (LPS) and other TLR agonists and assessed lipid accumulation in these activated murine macrophages. Methods and Results—Stimulation with LPS, a TLR4 ligand, resulted in a 56-fold increase in aP2 mRNA expression, and zymosan, a TLR2 ligand, induced an ≈1500-fold increase. Polyinosine: polycytidylic acid (poly I:C), a TLR3 ligand, led to a 9-fold increase. Levels of aP2 protein were significantly increased in LPS or zymosan-treated macrophages compared with control or poly I:C–treated cells. In addition, the cholesteryl ester content of LPS or zymosan-treated macrophages was ≈5-fold greater in the presence of low-density lipoprotein, and triglyceride content was ≈2-fold greater in the absence of exogenous lipid than control or poly I:C–treated cells. Conclusions—Expression of macrophage aP2 is induced on TLR activation and parallels increases in cholesteryl ester and triglyceride levels. These results provide a molecular link between the known roles of TLR and aP2 in foam cell formation.

Inflammation stimulates the expression of PCSK9

Inflammation induces marked changes in lipid and lipoprotein metabolism. Proprotein convertase subtilisin kexin 9 (PCSK9) plays an important role in regulating LDL receptor degradation. Here, we demonstrate that LPS decreases hepatic LDL receptor protein but at the same time hepatic LDL receptor mRNA levels are not decreased. We therefore explored the effect of LPS on PCSK9 expression. LPS results in a marked increase in hepatic PCSK9 mRNA levels (4h 2.5-fold increase; 38h 12.5-fold increase). The increase in PCSK9 is a sensitive response with 1microg LPS inducing a (1/2) maximal response. LPS also increased PCSK9 expression in the kidney. Finally, zymosan and turpentine, other treatments that induce inflammation, also stimulated hepatic expression of PCSK9. Thus, inflammation stimulates PCSK9 expression leading to increased LDL receptor degradation and decreasing LDL receptors thereby increasing serum LDL, which could have beneficial effects on host defense.

The acute phase response stimulates the expression of angiopoietin like protein 4.

The acute phase response is characterized by elevations in serum triglyceride levels due to both an increase in hepatic VLDL production and a delay in the clearance of triglyceride rich lipoproteins secondary to a decrease in lipoprotein lipase (LPL) activity. Recently there has been a marked increase in our understanding of factors that regulate LPL activity. GPIHBP1 facilitates the interaction of LPL and lipoproteins thereby allowing lipolysis to occur. Angiopoietin like proteins (ANGPTL) 3 and 4 inhibit LPL activity. In the present study, treatment of mice with LPS, an activator of TLR4 and a model of Gram-negative infections, did not alter the expression of GPIHBP1 in heart or adipose tissue. However, LPS decreased the expression of ANGPTL3 in liver and increased the expression of ANGPTL4 in heart, muscle, and adipose tissue. Serum ANGPTL4 protein levels were markedly increased at 8 and 16h following LPS treatment. Administration of zymosan, an activator of TLR2 and a model of fungal infections, also increased serum ANGPTL4 protein and mRNA levels in liver, heart, muscle, and adipose tissue. Finally, treatment of 3T3-L1 adipocytes with LPS or cytokines (TNF alpha, IL-1 beta, and interferon gamma) stimulated ANGPTL4 expression. These studies demonstrate that ANGPTL4 is a positive acute phase protein and the increase in ANGPTL4 could contribute to the hypertriglyceridemia that characteristically occurs during the acute phase response by inhibiting LPL activity.

LPS decreases fatty acid oxidation and nuclear hormone receptors in the kidney.

Inflammation produces marked changes in lipid metabolism, including increased serum fatty acids (FAs) and triglycerides (TGs), increased hepatic TG production and VLDL secretion, increased adipose tissue lipolysis, and decreased FA oxidation in liver and heart. Lipopolysaccharide (LPS) also increases TG and cholesteryl ester levels in kidneys. Here we confirm these findings and define potential mechanisms. LPS decreases renal FA oxidation by 40% and the expression of key proteins required for oxidation of FAs, including FA transport protein-2, fatty acyl-CoA synthase, carnitine palmitoyltransferase-1, medium-chain acyl-CoA dehydrogenase, and acyl-CoA oxidase. Similar decreases were observed in peroxisome proliferator-activated receptor α (PPARα)-deficient mice. LPS also caused a reduction in renal mRNA levels of PPARα (75% decrease), thyroid hormone receptor α (TRα) (92% decrease), and TRβ (84% decrease), whereas PPARβ/δ and γ were not altered. Expression of PGC1 α and β, coactivators required for PPARs and TR, was also decreased in kidneys of LPS-treated mice, as were mitochondrial genes regulated by PGC1 (Atp5g1, COX5a, Idh3a, and Ndufs8). Decreased renal FA oxidation could be a by-product of the systemic coordinated host response to increase FAs and TGs available for host defense and/or tissue repair. However, the kidney requires energy to support its transport functions, and the inability to generate energy via FA oxidation might contribute to the renal failure seen in severe sepsis.

Effect of interleukin-1 on lipid metabolism in the rat. Similarities to and differences from tumor necrosis factor.

Infection and inflammation are associated with hypertriglyceridemia, which is thought to be mediated by cytokines. Previous studies at our laboratory and others have shown that tumor necrosis factor acutely increases serum triglyceride levels primarily by stimulating hepatic lipid synthesis and secretion. The role of interleukin-1 (IL-1), a cytokine that is also secreted by stimulated macrophages and that has many actions that overlap those of tumor necrosis factor, has not been studied in depth. The present study demonstrates that IL-1, at doses similar to those that cause fever and anorexia and that stimulate adrenocorticotropic hormone secretion, rapidly increases serum triglyceride levels; this elevation persists for at least 17 hours. Serum cholesterol levels are not altered by IL-1. Neither is the clearance of triglyceride-rich lipoproteins affected by IL-1. However, hepatic triglyceride secretion, measured by the Triton WR-1339 technique, is increased in IL-1-treated animals. Accompanying this stimulation in hepatic lipid secretion is an increase in de novo fatty acid synthesis in the liver. IL-1 does not increase serum free fatty acid and glycerol levels, suggesting that IL-1 does not stimulate lipolysis in vivo. Additionally, inhibition of lipolysis does not prevent the increase in serum triglyceride levels, providing further evidence that lipolysis does not play a crucial role in the increased hepatic lipid synthesis and secretion induced by IL-1. In contrast, tumor necrosis factor increases lipolysis, which contributes to the increase in serum triglycerides. That multiple cytokines rapidly elevate plasma triglyceride levels suggest that these changes in lipid metabolism may play an important role in the organisms response to infection and inflammation.

Endotoxin increases parathyroid hormone-related protein mRNA levels in mouse spleen. Mediation by tumor necrosis factor.

Parathyroid hormone-related protein (PTHrP) causes hypercalcemia in malignancy. However, the role and regulation of PTHrP in normal physiology is just beginning to be explored. PTHrP is found in the spleen and has several other features common to cytokines. Since endotoxin (LPS) causes many of its effects indirectly by inducing cytokines, studies were undertaken to determine whether LPS might also induce splenic PTHrP expression. LPS (100 ng/mouse) increased splenic PTHrP mRNA levels 3.6-fold in C3H/OuJ mice. This effect was maximal at 2 h and returned to baseline by 4 h. PTHrP peptide levels also increased 3.3-fold in splenic extracts in response to LPS (1 microgram/mouse). Murine TNF-alpha and human IL-1 beta, cytokines that mediate many of the effects of LPS, also increased splenic PTHrP mRNA levels. LPS-resistant C3H/HeJ mice, which produce minimal amounts of TNF and IL-1 in response to LPS, were resistant to LPS induction of splenic PTHrP mRNA, while TNF-alpha and IL-1 beta readily increased PTHrP mRNA levels in C3H/HeJ mice. Anti-TNF antibody blocked LPS induction of splenic PTHrP mRNA in C3H/OuJ mice by 68%, indicating that TNF is a mediator of the LPS induction of PTHrP levels. In contrast, an IL-1 receptor antagonist (IL-1ra) was ineffective. The increase in PTHrP in the spleen during the immune response suggests that PTHrP may play an important role in immune modulation, perhaps by mediating changes in lymphocyte proliferation and/or function.