Role for monokines in the metabolic effects of endotoxin. Interferon-gamma restores responsiveness of C3H/HeJ mice in vivo.

To examine the role of cytokines in mediating the lipogenic effects of endotoxin (LPS), we studied the effects of LPS and cytokines on hepatic fatty acid synthesis in LPS-sensitive C3H/OuJ mice and in LPS-resistant C3H/HeJ mice, whose macrophages are defective in the ability to produce tumor necrosis factor (TNF) and IL-1 in response to LPS. HeJ mice were 16-fold less sensitive than OuJ mice to the lipogenic effect of LPS. In OuJ mice, 10 micrograms of LPS caused a maximal increase in hepatic lipogenesis (3.86 +/- 0.41-fold), whereas in HeJ mice the maximal increase was only 1.79 +/- 0.32-fold after 100 micrograms of LPS. This lipogenic response paralleled the decreased ability of LPS to increase hepatic and splenic levels of mRNAs for TNF and IL-1 and serum levels of TNF in HeJ mice. In contrast, the maximal effect of TNF on lipogenesis was greater and the sensitivity to TNF was increased 2.4-fold in HeJ mice compared to OuJ mice. Administration of IFN-gamma before LPS in HeJ mice had no effect on IL-1 mRNA, but partially restored the LPS-induced increase in hepatic and splenic mRNA for TNF and serum TNF levels, which may account for the partial restoration of sensitivity to the lipogenic effect of LPS after IFN-gamma treatment. These results indicate that cytokines produced by mononuclear leukocytes mediate the lipogenic effects of LPS.

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.

Autoantibodies against the insulin receptor. Dissociation of the acute effects of the antibodies from the desensitization seen with prolonged exposure.

The effect of antibodies against the insulin receptor (anti-R) found in a patient with the type B syndrome of insulin resistance and acanthosis nigricans was characterized using 3T3-L1 cultured fat cells. Anti-R acutely mimicked the action of insulin by stimulating deoxyglucose uptake. With more prolonged exposure, this insulinomimetic effect decayed, glucose metabolism returned to basal levels, and the cells became severely resistant to the actions of insulin. As seen with anti-R from a previous patient, desensitization consisted of both a dramatic decrease in the maximal responsiveness of the cells to insulin and a shift in the dose-response curve for insulin-stimulated glucose oxidation. The acute and chronic effects of anti-R were then compared. The concentration of anti-R required to half-maximally inhibit insulin binding averaged more than twice that required for half-maximal stimulation of deoxyglucose uptake, consistent with the amount of spare receptors in 3T3-L1 cells. After prolonged exposure, the insulinomimetic activity was completely lost at all concentrations of anti-R, even at those that did not completely induce insulin resistance. Thus, loss of the insulinomimetic activity of anti-R is necessary, but not sufficient, to cause desensitization. Less anti-R was required to desensitize cells to insulin than would have been predicted on the basis of the acute inhibition of binding and the number of spare receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin induces parathyroid hormone-related protein gene expression in splenic stromal and smooth muscle cells, not in splenic lymphocytes.

PTH-related protein (PTHrP), the peptide that is responsible for most cases of hypercalcemia of malignancy, is also produced under normal circumstances by a variety of tissues. Its role and regulation at these sites are not well understood. Recently, we have shown that PTHrP is induced in the spleen during the host response to endotoxin (LPS) and that tumor necrosis factor (TNF) is a major mediator of this effect. Given the large body of in vitro evidence suggesting that PTHrP can be produced by lymphocytes and act in an autocrine loop to alter their function, studies were undertaken to determine whether lymphocytes were the cells responsible for PTHrP production in the spleen. Both constitutive and LPS-induced PTHrP messenger RNA (mRNA) levels were the same in mice lacking mature T cells (nude mice) and in mice lacking natural killer (NK) cells (due to pretreatment with antibody against NK 1.1) compared to levels in normal mice, suggesting that neither mature T cells nor NK cells were the splenic source of PTHrP. Even scid mice that lack functioning T and B cells responded to TNF with the induction of splenic PTHrP mRNA levels comparable to those in control mice. Localization of PTHrP mRNA in subfractions of rat spleens after in vivo treatment with LPS confirmed the results of the murine studies; PTHrP mRNA was barely detectable in the lymphocyte-rich single cell fraction of the spleen. In contrast, the stromal fraction of the spleen was enriched with PTHrP mRNA both in the basal state and in response to LPS. A similar pattern of distribution was seen for interleukin-6; LPS only increased mRNA levels of this TNF-inducible cytokine in the splenic stroma. In addition, mRNA for the PTH/PTHrP receptor, which decreased in response to LPS, colocalized with PTHrP mRNA in the stromal fraction of the spleen. Immunohistochemical studies identified PTHrP in two populations of splenic cells: 1) smooth muscle cells located in the splenic capsule and trabeculae and 2) a subpopulation of stromal cells located in the red pulp of the spleen, primarily in a subcapsular distribution. Consistent with the localization of PTHrP mRNA, lymphocytes in the white pulp of the spleen did not stain for PTHrP.

Identification of the intact insulin receptor using a sequence-specific antibody directed against the C-terminus of the beta-subunit.

An antibody was raised against a synthetic peptide corresponding to the carboxyl-terminal amino acids of the human insulin receptor (Anti-R beta C). Immunoprecipitation of the human insulin receptor and immunoblotting to the beta-subunit by Anti-R beta C could be inhibited by competition with the corresponding peptide. However, even at saturating concentrations, anti-R beta C could not completely immunoprecipitate or immunodeplete insulin receptors compared to a human autoantibody (anti-R B2). Using receptor labeled directly by 125I, evidence of multiple forms of the beta-subunit was found. When the receptor could be immunoprecipitated by anti-R beta C, the beta-subunit migrated with an apparent mol wt (MW) of 96,000 (at or above the phosphorylase b MW marker). However, in preparations where anti-R beta C was not able to immunoprecipitate the insulin receptor, the beta-subunit migrated at a significantly lower MW of 91,000 (below phosphorylase b), as detected by immunoprecipitation with Anti-R B2. Intermediate forms could also be detected. Phosphorylation of partially purified insulin receptor did not affect is ability to be immunoprecipitated by anti-R beta C, although insulin-stimulated phosphorylation increased the apparent MW of the beta-subunit. However, insulin receptor that was phosphorylated in solubilized extracts of whole cells had a beta-subunit that migrated at lower MW and was not immunoprecipitated by anti-R beta C. One possible explanation for this is that the beta-subunit may be degraded during preparation. When the MW of insulin receptor that has been purified to homogeneity from human placenta is compared to our data, it is clear that many of these insulin receptor preparations contain lower MW beta-subunits. These results must be taken into account when the sites of phosphorylation and kinase activity of purified insulin receptor preparations are studied.

Cytokine regulation of parathyroid hormone-related protein messenger ribonucleic acid levels in mouse spleen: paradoxical effects of interferon-gamma and interleukin-4.

Under normal physiological conditions, PTH-related protein (PTHrP) is produced in a wide variety of tissues and is thought to act locally in an autocrine or paracrine fashion more analogous to cytokines than to classic hormones such as PTH. In addition, we have recently shown that, like cytokines, PTHrP is induced in the spleen during the response to sublethal doses of endotoxin [lipopolysaccharide (LPS)] an effect that is mediated by tumor necrosis factor (TNF). As complex cytokine cascades are induced in response to infectious or inflammatory stimuli, the effects of other prototypical inflammatory [interferon-gamma (IFN gamma)] or antiinflammatory [interleukin-4 (IL-4)] cytokines on PTHrP gene expression were studied. Paradoxically, IFN gamma (50 micrograms), a cytokine that usually synergizes with TNF, inhibited LPS induction of splenic PTHrP messenger RNA (mRNA) levels in LPS-sensitive C3H/OuJ (OuJ) and LPS-resistant C3H/HeJ (HeJ) mice. The stimulation of splenic PTHrP mRNA levels caused by the administration of TNF alpha or interleukin-1 beta was similarly inhibited by IFN gamma, a type II interferon. In contrast, IFN alpha (50 micrograms), a type I interferon, stimulated splenic levels of PTHrP mRNA. IL-4, a prototypical antiinflammatory cytokine, also had a paradoxical effect on LPS induction of splenic PTHrP mRNA levels. Instead of inhibiting LPS induction of splenic PTHrP mRNA levels in OuJ or HeJ mice, IL-4 (200 ng) actually stimulated PTHrP mRNA levels. These complex cytokine interactions suggest that the expression of PTHrP in response to infectious or inflammatory stimuli depends on the counterbalancing effects of the specific cytokine networks induced by each stimulus.

Lipids, lipoproteins, triglyceride clearance, and cytokines in human immunodeficiency virus infection and the acquired immunodeficiency syndrome.

Infection causes disturbances in lipid metabolism that may be mediated by cytokines. Therefore we studied plasma lipids, lipoproteins, triglyceride (TG) metabolism, and serum cytokines in three groups: patients with the acquired immunodeficiency syndrome (AIDS) without active secondary infection, patients with evidence of human immunodeficiency virus infection but without clinical AIDS (HIV+), and controls. Plasma TGs and FFA were increased in AIDS, while plasma cholesterol, high density lipoprotein (HDL) cholesterol, apolipoprotein-A-1 (Apo-A-1), low density lipoprotein (LDL) cholesterol, and Apo-B-100 levels were decreased. Increased TG levels in AIDS were primarily due to increases in very low density lipoprotein of normal composition; in addition, LDL and HDL were TG enriched. In HIV+, TGs and FFA were not increased, but total cholesterol, HDL cholesterol, Apo-A-1, and Apo-B-100 were significantly decreased. Interferon-alpha (IFN alpha) and C-reactive protein levels were increased in AIDS, but tumor necrosis factor and haptoglobin levels were not. There was a significant correlation between plasma TGs and IFN alpha levels (r = 0.477; P less than 0.01), but not between TGs and tumor necrosis factor, C-reactive protein, haptoglobin, or P-24 antigen. In addition, there was no relationship between circulating IFN alpha levels and plasma cholesterol, HDL cholesterol, Apo-A-1, LDL cholesterol, Apo-B-100, or FFA. TG clearance time and postheparin lipase were significantly decreased in AIDS and HIV+. There was a strong correlation between serum IFN alpha levels and TG clearance time in AIDS and HIV+ (r = 0.783; P less than 0.001). In summary, decreases in cholesterol and cholesterol containing lipoproteins (including HDL) in both AIDS and HIV+ precede the appearance of hypertriglyceridemia and are not related to IFN alpha or TG levels. Our data raise the possibility that with development of AIDS, subsequent increases in IFN alpha may contribute to increases in plasma TG levels in part by decreasing the clearance of TG.

Serum leptin levels in the acquired immunodeficiency syndrome.

Leptin, a hormone that is secreted by adipose tissue in proportion to fat stores, regulates energy balance and appetite. Recently, tumor necrosis factor and interleukin-1, cytokines that regulate the host response to infection, have been shown to acutely increase leptin levels, raising the possibility that leptin could mediate the anorexia of some infections. We measured leptin levels in patients with the acquired immunodeficiency syndrome and found that leptin levels were not increased relative to body fat in patients who were anorectic, were losing weight, or had a history of weight loss. Furthermore, leptin levels were not increased during secondary infection, suggesting that elevations in leptin do not play a key role in the anorexia of infections associated with acquired immunodeficiency syndrome.

Resting energy expenditure, caloric intake, and short-term weight change in human immunodeficiency virus infection and the acquired immunodeficiency syndrome.

To assess the causes of short-term weight loss in patients with acquired immunodeficiency syndrome (AIDS), we measured resting energy expenditure (REE), caloric intake, and the 28-d weight trend in control subjects, human immunodeficiency virus (HIV)+ subjects, AIDS patients, and AIDS patients during secondary infection (AIDS-SI). REE was increased in HIV+ (11%), AIDS (25%), and AIDS-SI (29%). Caloric intake was similar in control subjects, HIV+, and AIDS but reduced 36% in AIDS-SI, who consumed 17% fewer calories than their REE. Average short-term weight was stable for HIV+ and AIDS but decreased 5% in AIDS-SI. Weight trend correlated with caloric intake but not with REE. Thus HIV+ and AIDS are able to partially compensate for increased REE because they do not show short-term weight loss. Decreased caloric intake is critical for short-term weight loss and is seen during secondary infection. Inability of decreased caloric intake to decrease REE during infection accelerates short-term weight loss. Rapid weight loss with anorexia may be a harbinger of secondary infection in AIDS.

Endotoxin and interleukin-1 decrease hepatic lipase mRNA levels.

The acute phase response induces a multitude of changes in lipoprotein metabolism including hypertriglyceridemia, triglyceride enriched LDL, and decreased HDL levels accompanied by changes in HDL composition including increased free cholesterol and triglycerides and a decrease in esterified cholesterol. Here we demonstrate that endotoxin (LPS) induces a 56% decrease in hepatic lipase activity in liver and a 45% decrease in hepatic lipase activity in post heparin plasma in Syrian hamsters. LPS treatment also produces a marked decrease in hepatic lipase mRNA levels in the liver. Half maximal reduction in hepatic lipase mRNA levels occurred at approximately 0.2 microg LPS/100 g BW with a maximal decrease at 1.0 microg/100 g BW ( > 90% decrease), indicating that inhibition of hepatic lipase is a sensitive host response to LPS. Additionally, IL-1 produced a marked decrease in hepatic lipase mRNA levels while TNF had no effect. Moreover, IL-1 treatment of HepG2 cells in vitro also decreased hepatic lipase mRNA levels suggesting that IL-1 can directly regulate hepatic lipase expression in liver cells. LPS decreased hepatic lipase mRNA levels in control as well as IL-1 type 1 receptor deficient mice indicating that IL-1 action is not absolutely essential and that several cytokines and/or small molecular mediators can regulate hepatic lipase during the acute phase response. The LPS and IL-1 induced decrease in hepatic lipase could have several consequences including decreasing the clearance of triglyceride rich lipoprotein particles and producing an increase in triglyceride rich HDL. The decrease in hepatic lipase activity and mRNA levels may be part of a series of coordinated changes in lipoprotein metabolism that occur during the acute phase response. These changes may be initially beneficial to the host but if present for an extended period may be proatherogenic.

Circulating interferon-alpha levels and hypertriglyceridemia in the acquired immunodeficiency syndrome.

PURPOSE: The metabolic disturbances seen during infection are thought to be due to cytokines, modulators of the immune response. The acquired immunodeficiency syndrome (AIDS) is characterized by a high prevalence of hypertriglyceridemia and at times depletion of body cell mass (wasting). Elevated circulating levels of cytokines have also been reported in AIDS. Therefore, we determined the relationship between circulating cytokine levels and lipid levels and between circulating cytokine levels and wasting in AIDS and human immunodeficiency virus (HIV) infection. PATIENTS AND METHODS: Serum samples from 45 patients with AIDS, 13 subjects with evidence of HIV infection by presence of antibody but without AIDS (HIV positive), and 17 seronegative control subjects who had previously undergone body composition analysis were analyzed for triglyceride, cholesterol, interferon, tumor necrosis factor (TNF), and interleukin-1 levels. Eleven subjects with AIDS or HIV infection had sequential measurements. Interferon was analyzed by bioassay with identification using specific antibodies. TNF and interleukin-1 were assayed by enzyme-linked immunosorbent assay. Lean body mass was assessed by total body potassium. RESULTS: Serum interferon-alpha levels were significantly elevated in patients with AIDS (p less than 0.001 compared to controls), with detectable levels in 84% of AIDS patients. Interferon-alpha was not detectable in serum from controls, while three of 13 HIV-positive subjects had detectable interferon-alpha levels. There was a significant correlation between interferon-alpha levels and serum triglyceride levels in AIDS and HIV-positive patients (R = 0.446, p less than 0.002). There was no relationship between interferon-alpha and serum cholesterol levels (R = -0.039, NS). In contrast only 11% of AIDS patients had detectable circulating TNF levels; the mean value for and the prevalence of detectable serum TNF levels were not significantly different from those of control subjects. Interleukin-1 was not detected in the circulation. There was no correlation between the presence of circulating TNF and serum triglycerides. There was no relationship between circulating interferon-alpha or TNF levels and the presence of wasting as measured by total body potassium. CONCLUSION: These studies suggest that interferon-alpha, which has previously been shown to modulate lipid metabolism in vivo and in vitro, may be responsible for the hypertriglyceridemia found in AIDS.

Plasma platelet-activating factor acetylhydrolase activity in human immunodeficiency virus infection and the acquired immunodeficiency syndrome.

Platelet-activating factor (PAF) acetylhydrolase (PAF-AH) catalyzes the hydrolysis of PAF, a mediator of inflammation, as well as other biologically active oxidized phospholipids. In humans, plasma PAF-AH activity is bound to low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Higher levels of plasma PAF-AH activity have been found in a variety of diseases, and are thought to be a defense mechanism against the toxic effects of PAF and oxidized phospholipids. We studied plasma PAF-AH activity in patients with human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS), a disease characterized by chronic HIV infection and a systemic host response. Plasma PAF-AH activity was significantly greater in AIDS patients compared with control subjects (25.2 +/- 2.0 v 17.0 +/- 0.8 nmol/min/mL, P < .001). The higher levels of plasma PAF-AH activity were found in LDL (28.2 +/- 2.2 v 18.3 +/- 1.0 nmol/min/mL for AIDS v controls, respectively, P = .0005), but not in HDL. Plasma PAF-AH activity in AIDS correlated with circulating interferon alfa (r = .575, P = .005) and plasma triglycerides (r = .556, P < .0025). The presence of secondary infection in AIDS did not significantly change plasma PAF-AH activity. The initiation of a new antiretroviral regimen with either a protease inhibitor or the nucleoside analog lamivudine did not significantly decrease plasma PAF-AH activity, despite successful suppression of HIV RNA levels. Plasma PAF-AH activity may be a sensitive marker of the host response to infection, and the higher levels of plasma and LDL-associated PAF-AH activity in patients with HIV infection and AIDS may be a physiological response to protect the host against oxidative injury from PAF and oxidized phospholipids.

Nicotinamide and other inhibitors of ADP-ribosylation block deoxyglucose uptake in cultured cells.

Nicotinamide was shown to inhibit deoxyglucose uptake in three diverse differentiated cell lines. In 3T3-L1 fat cells, nicotinamide equally inhibited basal and insulin stimulated deoxyglucose uptake. Inhibition by nicotinamide was non-competitive. A variety of inhibitors of ADP-ribosylation blocked deoxyglucose uptake while some analogs with no activity against ADP-ribose synthetase also had little effect on deoxyglucose uptake. These findings should be taken into account when inhibitors of ADP-ribosylation are used with intact cells.

Urea treatment allows dithiothreitol to release the binding subunit of the insulin receptor from the cell membrane: implications for the structural organization of the insulin receptor.

The sequence of the human insulin receptor has only one identifiable transmembrane region which is located in the beta subunit. The structure predicts that the alpha subunit, which binds insulin, is attached to the cell only by disulfide bonds to the beta subunit. However, treatment of membranes with dithiothreitol is ineffective at releasing the alpha subunit. If the receptor structure is unfolded with urea, dithiothreitol is able to release the alpha subunit. These data provided confirmatory evidence that the alpha subunit is not a transmembrane protein.

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 alpha (PPARalpha) 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 RXRalpha, RXRbeta, and RXRgamma proteins in hamster liver. Maximum inhibition was observed at 4 h for RXRalpha (62%) and RXRbeta (50%) and at 2 h for RXRgamma (61%). These decreases were associated with a marked reduction in RXRalpha, RXRbeta, and RXRgamma mRNA levels. Increased RNA degradation is likely responsible for the repression of RXR, because LPS did not decrease RXRbeta and RXRgamma transcription and only marginally inhibited (38%) RXRalpha transcription. RXR repression was associated with decreased LXRalpha and PPARalpha mRNA levels and reduced RXR x RXR, RXR x PPAR and RXR x 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 PPARalpha 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.

Discordant regulation of proteins of cholesterol metabolism during the acute phase response.

Recent studies have shown that the administration of endotoxin (LPS) and cytokines to Syrian hamsters increases serum cholesterol levels, hepatic cholesterol synthesis, and the activity, protein levels, and mRNA levels of hepatic HMG-CoA reductase. Despite the greater than 10-fold increase in HMG-CoA reductase mRNA levels, LPS had only minimal effects on hepatic LDL receptor mRNA levels. In the present study, we demonstrate that LPS increases the transcription rate in the liver of HMG-CoA reductase mRNA approximately 4- to 5-fold without affecting LDL receptor mRNA transcription. Most stimuli that regulate HMG-CoA reductase and LDL receptor mRNA levels also regulate, in parallel, HMG-CoA synthase and farnesyl pyrophosphate (FPP) synthetase. However, in chow-fed animals, LPS and cytokines (TNF, IL-1, TNF + IL-1) increased hepatic HMG-CoA reductase mRNA levels without increasing LDL receptor, HMG-CoA synthase, or FPP synthetase mRNA levels. The feeding of cholesterol or bile resin binders regulates the mRNA levels of HMG-CoA reductase, LDL receptor, HMG-CoA synthase, and FPP synthetase. In both cholesterol- and colestipol-fed animals, LPS increased HMG-CoA reductase mRNA levels while either decreasing or causing minimal increases in the mRNA levels of the other proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol efflux by acute-phase high density lipoprotein: role of lecithin: cholesterol acyltransferase.

HDL plays an initial role in reverse cholesterol transport by mediating cholesterol removal from cells. During infection and inflammation, several changes in HDL composition occur that may affect the function of HDL; therefore, we determined the ability of acute-phase HDL to promote cholesterol removal from cells. Acute-phase HDL was isolated from plasma of Syrian hamsters injected with lipopolysaccharide. Cholesterol removal from J 774 murine macrophages by acute-phase HDL was less efficient than that by control HDL because of both a decrease in cholesterol efflux and an increase in cholesterol influx. LCAT activity of acute-phase HDL was significantly lower than that of control HDL. When LCAT activity of control HDL was inactivated, cholesterol efflux decreased and cholesterol influx increased to the level observed in acute-phase HDL. Inactivation of LCAT had little effect on acute-phase HDL. In GM 3468A human fibroblasts, the ability of acute-phase HDL to remove cholesterol from cells was also lower than that of normal HDL. The impaired cholesterol removal, however, was primarily a result of an increase in cholesterol influx without changes in cholesterol efflux. When control HDL in which LCAT had been inactivated was incubated with fibroblasts, cholesterol influx increased to a level comparable to that of acute-phase HDL, without any change in cholesterol efflux. These results suggest that the ability of acute-phase HDL to mediate cholesterol removal was impaired compared with that of control HDL and the lower LCAT activity in acute-phase HDL may be responsible for this impairment. The decreased ability of acute-phase HDL to remove cholesterol from cells may be one of the mechanisms that account for the well-known relationship between infection/inflammation and atherosclerosis.

In vivo and in vitro regulation of sterol 27-hydroxylase in the liver during the acute phase response. potential role of hepatocyte nuclear factor-1.

The host response to infection is associated with several alterations in lipid metabolism that promote lipoprotein production. These changes can be reproduced by lipopolysaccharide (LPS) administration. LPS stimulates hepatic cholesterol synthesis and suppresses the conversion of cholesterol to bile acids. LPS down-regulates hepatic cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in the classic pathway of bile acid synthesis. We now demonstrate that LPS markedly decreases the activity of sterol 27-hydroxylase, the rate-limiting enzyme in the alternate pathway of bile acid synthesis, in the liver of Syrian hamsters. Moreover, LPS progressively decreases hepatic sterol 27-hydroxylase mRNA levels by 75% compared with controls over a 24-h treatment period. LPS also decreases oxysterol 7alpha-hydroxylase mRNA levels in mouse liver. In vitro studies in HepG2 cells demonstrate that tumor necrosis factor and interleukin (IL)-1 decrease sterol 27-hydroxylase mRNA levels by 48 and 80%, respectively, whereas IL-6 has no such effect. The IL-1-induced decrease in sterol 27-hydroxylase mRNA expression occurs early, is sustained for 48 h, and requires very low doses. In vivo IL-1 treatment also lowers hepatic sterol 27-hydroxylase mRNA levels in Syrian hamsters. Studies investigating the molecular mechanisms of LPS-induced decrease in sterol 27-hydroxylase show that LPS markedly decreases mRNA and protein levels of hepatocyte nuclear factor-1 (HNF-1), a transcription factor that regulates sterol 27-hydroxylase, in the liver. Moreover, LPS decreases the binding activity of HNF-1 by 70% in nuclear extracts in hamster liver, suggesting that LPS may down-regulate sterol 27-hydroxylase by decreasing the binding of HNF-1 to its promoter. Coupled with our earlier studies on cholesterol 7alpha-hydroxylase, these data indicate that LPS suppresses both the classic and alternate pathways of bile acid synthesis. A decrease in bile acid synthesis in liver would reduce cholesterol catabolism and thereby contribute to the increase in hepatic lipoprotein production that is induced by LPS and cytokines.

Regulation of scavenger receptor class B type I in hamster liver and Hep3B cells by endotoxin and cytokines.

Multiple changes in HDL metabolism occur during infection and inflammation that could potentially impair the antiatherogenic functions of HDL. Scavenger receptor class B type I (SR-BI) promotes cholesterol efflux from peripheral cells and mediates selective uptake of cholesteryl ester into hepatocytes, thereby playing a pivotal role in reverse cholesterol transport. We studied the effect of endotoxin (lipopolysaccharide, LPS) and cytokines [tumor necrosis factor (TNF) and interleukin 1 (IL-1)] on hepatic SR-BI mRNA and protein levels in Syrian hamsters. LPS significantly decreased SR-BI mRNA levels in hamster liver. This effect was rapid and sustained, and was associated with a decrease in hepatic SR-BI protein levels. High cholesterol diet did not change hepatic SR-BI mRNA levels, and LPS was able to decrease SR-BI mRNA levels during high cholesterol feeding. TNF and IL-1 decreased SR-BI mRNA levels in the liver, and the effects of TNF and IL-1 were additive. TNF and IL-1 also decreased SR-BI levels in Hep3B hepatoma cells. More importantly, TNF and IL-1 decreased the uptake of HDL cholesteryl ester into Hep3B cells. In addition, we studied the effect of LPS on SR-BI mRNA in RAW 264.7 cells, a macrophage cell line. LPS rapidly decreased SR-BI mRNA levels in RAW 264.7 cells, but the effect was not sustained and did not lead to a reduction in SR-BI protein levels. Our results suggest that the decrease in hepatic SR-BI levels due to LPS and cytokines during infection and inflammation may decrease selective uptake of cholesteryl ester into the liver and result in impaired reverse cholesterol transport.

Role for circulating lipoproteins in protection from endotoxin toxicity.

Previous studies have shown that endotoxin (lipopolysaccharide [LPS])-induced death can be prevented by preincubating LPS with lipoproteins in vitro or by infusing large quantities of lipids into animals prior to LPS administration. In the present study we determined whether physiological levels of lipids also provide protection. Serum lipid levels were decreased by two different mechanisms: administration of 4-aminopyrolo-(3,4-D)pyrimide, which prevents the hepatic secretion of lipoproteins, and administration of pharmacological doses of estradiol, which increases the number of hepatic low-density lipoprotein receptors, leading to increased lipoprotein clearance. In both hypolipidemic models, LPS-induced mortality is markedly increased compared with that of controls with normal serum lipid levels. In both hypolipidemic models, administration of exogenous lipoproteins, which increase levels of serum lipids into the physiological range, reduces the increased mortality to levels similar to that seen in normal animals. In normal lipidemic animals, 63% of 125I-LPS in plasma is associated with lipoproteins, where it would not be capable of stimulating cytokine production. In contrast, in hypolipidemic animals, very little LPS (12 to 17%) is associated with lipoproteins. Rather, more LPS is in the lipoprotein-free plasma compartment, where it could exert biological effects. In both hypolipidemic models, LPS produces a greater increase in serum tumor necrosis factor levels than it does in controls (three- to fivefold increase), and administration of exogenous lipoproteins prevents this increase. Cytokines, in particular tumor necrosis factor, are responsible for most of the toxic effects of LPS. These data provide evidence that physiological levels of serum lipids protect animals from LPS toxicity. Thus, lipoproteins, in addition to playing a role in lipid transport, may have protective functions. Moreover, as part of the immune response, cytokine-induced increases in serum lipid levels may play a role in host defense by decreasing the toxicities of biological and chemical agents.