Lack of oral tolerance in C3H/HeJ mice

Daily gastric intubation of lipopolysaccharide (LPS)-responsive C3H/HeN, BALB/c, and Swiss mice with SRBC for 2 wk resulted in oral tolerance, whereas similarly treated LPS-nonresponsive C3H/HeJ mice gave splenic anti-SRBC PFC responses, including the IgA isotype, after systemic challenge with antigen. Oral tolerance in LPS-responsive C3H/HeN mice was due to T suppressor (Ts) cells because significant Ts cell activity was demonstrated in both Peyer's patches (PP) and spleens of these animals. On the other hand, T cells from PP and spleens of identically treated C3H/HeJ mice exhibited mainly T helper cell activity. Prior treatment of PP or spleen cell preparations from tolerant C3H/HeN mice with anti-Lyt-2.1 resulted in good in vitro anti- SRBC PFC responses, especially IgA isotype responses in PP cell cultures. These results indicate that oral administration of a thymic- dependent antigen (SRBC) to LPS-responsive mice induced a Ts cell population in PP, which, after migration to peripheral lymphoid tissue (e.g., spleen), suppressed responses to systemically administered antigen. LPS-nonresponsive mice lack this Ts cell pathway and continually respond to oral administration of antigen.     

The importance of a lipopolysaccharide-initiated, cytokine-mediated host defense mechanism in mice against extraintestinally invasive Escherichia coli.

Extraintestinally invasive Escherichia coli (EC) that possess both a complete LPS and K1 capsule evade both complement-mediated bacteriolysis and neutrophil-mediated killing. Since C3H/HeJ mice that are hyporesponsive to LPS were uniquely susceptible to lethal infection with EC of this phenotype, we speculated there was an LPS-initiated host defense mechanism against this pathogenic phenotype. The LPS-normoresponsive C3H/HeN as well as the C3H/HeJ mice cleared these EC from the circulation within 4 h of intravenous administration. Whereas electron micrographs of the liver demonstrated these EC undergoing degeneration within the phagolysosomes of of both macrophages and Kupffer cells of C3H/HeN mice, these EC replicated within these cells of the C3H/HeJ mice. Restoration of anti-EC activity of C3H/HeJ mice occurred with activation of Kupffer cells and peritoneal macrophages in vivo with BCG and in vitro with IFN-gamma, but not with LPS. Pretreatment of C3H/HeJ mice with a combination of recombinant murine IL-1 and TNF-alpha also restored the killing of K1(+)-EC but did not enhance the killing of a K1(-)-EC mutant. These data are consistent with the hypothesis that (a) there is no intrinsic inability of C3H/HeJ phagocytes to kill EC, but (b) an LPS-initiated, cytokine-mediated host defense mechanism is required for such killing. These studies emphasize the importance of bacterial surface characteristics in the interaction with specific host defenses.     

Detection of a mediator derived from endotoxin-stimulated macrohpages that induces the acute phase serum amyloid A response in mice

The mechanism by which LPS stimulates an acute phase serum amyloid A (SAA) response in C3H mice has been studied. A factor (SAA inducer) appears in the blood of C3H/HeN (lipopolysaccharide [LPS]-sensitive) mice approximately 1 h after administration of LPS, which, when passively administered, can induce C3H/HeJ mice to produce SAA although they are resistant to the LPS itself. SAA inducer has been detected in the culture medium of LPS treated C3H/HeN macrophages but not spleen cells. Thus, two stages in the induction of the acute phase SAA response are now recognized: a latent period of 2-3 h during which the SAA concentration remains at baseline values and in which SAA inducer appears, and the period of synthesis of SAA which lasts for approoximately 24 h past induction. It is proposed that a macrophage response to LPS is responsible for production of the serum mediator which induces SAA synthesis.     

Effect of FR167653, a novel inhibitor of tumor necrosis factor-alpha and interleukin-1beta synthesis on lipopolysaccharide-induced hepatic microvascular dysfunction in mice

Tumor necrosis factor-alpha (TNFalpha) and interleukin-1 (IL-1) have been recognized as key proinflammatory mediators in the pathogenesis of lipopolysaccharide (LPS)-induced liver injury. In the present study we examined the effect of FR167653, a novel inhibitor of TNFalpha and IL-1 synthesis, on the hepatic microvascular response to LPS using in vivo microscopy. Significant hepatic microvascular responses comprising leukocyte adhesion to the sinusoidal wall and central venules and reduced sinusoidal perfusion appeared 2 and 4 h after LPS (0.1 mg/kg, i.v.) injection in male C3H/HeN mice (LPS sensitive) when compared with male C3H/HeJ mice (LPS resistant). The serum concentrations of TNFalpha at 1.5 h and IL-1beta at 4 h after injection of LPS, as determined by enzyme-linked immunosorbent assay, were significantly higher in C3H/HeN mice than in C3H/HeJ mice. Administration of murine TNFalpha or IL-1beta (10 microg/kg., i.v., respectively) in both C3H/HeN and C3H/HeJ mice elicited the hepatic microvascular responses that were similar to those produced by LPS injection in C3H/HeN mice. FR167653 (1 and 10 mg/kg, i.v., 0 and 2 h after LPS injection) significantly reduced leukocyte adhesion and restored sinusoidal perfusion in a dose-dependent manner in C3H/HeN mice 4 h after LPS injection. The levels of TNFalpha, IL-1beta, and alanine aminotransferase also were significantly lower in FR167653-treated endotoxemic C3H/HeN mice than those in vehicle-treated endotoxemic animals. The results suggest that the hepatic microvascular response to LPS is partly mediated by TNFalpha and IL-1beta, and that FR167653 prevents LPS-induced hepatic microcirculatory dysfunction by inhibiting the production of TNFalpha and IL-1beta.     

Lethal toxicity of lipopolysaccharide and tumor necrosis factor in normal and D-galactosamine-treated mice

The toxic properties of human recombinant tumor necrosis factor (TNF) were investigated in mice made hypersensitive to endotoxin by treatment with D-galactosamine. C3H/TifF mice treated with D-galactosamine were rendered sensitive to the lethal effects of submicrogram amounts of TNF. In the absence of D-galactosamine, TNF caused approximately 80% lethality with 500 micrograms. The duration of sensitization to TNF lasted up to 8 h after D-galactosamine administration, that towards LPS, up to 4 h. As with LPS, with TNF sensitization could be inhibited by uridine administered up to 2 h after D-galactosamine/TNF, showing that the early biochemical alterations in the liver known to be necessary for sensitization to LPS are also necessary for sensitization to TNF. In contrast to LPS, the toxicity of TNF was expressed also in D-galactosamine-treated endotoxin-resistant C3H/HeJ mice. The susceptibility of these mice to TNF was identical to that of endotoxin sensitive mice. In the absence of D-galactosamine the toxicity of TNF in C3H/HeJ mice was comparable to that obtained in C3H/TifF mice, being lethal with amounts of the order of 500 micrograms. The present results support the hypothesis that TNF is a mediator of lethal toxicity of endotoxin.     

Upregulated expression of S100A8 in mice brain after focal cerebral ischemia reperfusion

BACKGROUND:

Recent studies have showed that S100A8 has been implicated in the pathobiology of inflammatory disorders, and that cerebral ischemia reperfusion (I/R) rapidly activates inflammation responses via Toll-like receptor 4 (TLR4). This study aimed to explore the expression of S100A8 and the relationship between S100A8 and TLR4 in focal cerebral ischemia reperfusion injury.

METHODS:

C3H/HeJ mice (n=30) and C3H/HeN mice (n=30) were divided randomly into a C3H/HeJ model group (n=18), a C3H/HeJ control group (n=12), a C3H/HeN model group (n=18), and a C3H/HeN control group (n=12). Middle cerebral artery I/R model in mice was produced using a thread embolism method. The brains of the mice were collected after ischemia for 1 hour and reperfusion for 12 hours. Stroke outcome was evaluated by determination of infarct volume and assessment of neurological impairment scores. Brain injury after cerebral I/R was observed by an optical microscope after TTC and HE dyeing. The immunofluorescence technique and real time PCR were used to test the expression level of S100A8 in brain damage.

RESULTS:

Compared with C3H/HeN mice, TLR4-deficient mice (C3H/HeJ) had lower infarct volumes and better outcomes in neurological tests. The levels of S100A8 increased sharply in the brains of mice after I/R injury. In addition, mice that lacked TLR4 (C3H/HeJ) had lower expression of I/R-induced S100A8 than C3H/HeN mice in the model group, indicating that a close relationship might exist between the levels of S100A8 and TLR4.

CONCLUSION:

S100A8 interaction with TLR4 might be involved in brain damage and in inflammation triggered by I/R injury.KEY WORDS: S100A8, Toll-like receptor 4, Cerebral ischemia reperfusion, Inflammation     

Isotype-specific immunoregulation. Evidence for a distinct subset of T contrasuppressor cells for IgA responses in murine Peyer''s patches

The ability of murine Peyer's patch (PP) T contrasuppressor cells (Tcs) to reverse oral tolerance to the T cell-dependent (TD) antigen SRBC was studied both in vivo and in vitro. C3H/HeJ mice given SRBC orally for 4 wk are not rendered tolerant to this antigen and were used as a source of PP Tcs cells for adoptive transfer to identically treated, orally tolerized C3H/HeN mice. Transfer of 10(4) or 5 X 10(4) V. villosa-adherent PP T cells resulted in splenic IgM, IgG, and mainly IgA responses in C3H/HeN mice challenged systemically with SRBC. The T cell responsible was Lyt-1+, 2-, L3T4-, I-JK+ and V. villosa lectin-adherent, all characteristics of mature effector Tcs cells. This C3H/HeJ PP Tcs cell subset was also effective when added to in vitro cultures of tolerized spleen cells derived from SRBC-fed, C3H/HeN mice. Interestingly, C3H/HeJ PP Tcs cells restored mainly IgA responses when transferred in vivo or when added to suppressed C3H/HeN splenic cultures. Comparison of the functional activity of Tcs cells derived from spleen or PP of orally immunized C3H/HeJ mice revealed that splenic Tcs cells supported responses of all 3 isotypes; however, PP Tcs cells yielded three-fourfold higher IgA responses, when compared with IgM or IgG anti-SRBC responses. Adherence of C3H/HeJ PP Tcs to an Fc alpha R+ T cell line derived from IgA-specific Th cells resulted in a nonadherent cell fraction that potentiated only IgM and IgG responses, while bound Tcs cells preferentially supported IgA responses. These results suggest that murine PP contain IgA-specific Tcs cells that allow IgA response induction in the presence of Ts cells that mediate oral tolerance.     

Tyrosine phosphorylation of mitogen-activated protein kinases is necessary for activation of murine macrophages by natural and synthetic bacterial products

The purpose of these studies was to determine the intracellular signal transduction pathways of bacterial products in murine macrophages from lipopolysaccharide (LPS)-responder C3H/HeN and LPS-nonresponder C3H/HeJ mice. Both LPS and synthetic lipopeptide CGP 31362 (LPP) induced production of tumor necrosis factor alpha (TNF-alpha) in C3H/HeN macrophages. In C3H/HeJ macrophages, however, TNF-alpha was induced only by incubation with LPP. Both LPS and LPP induced tyrosine phosphorylation on proteins with apparent molecular masses of 39, 41, and 45 kD (p35, p41, and p45) in C3H/HeN macrophages, whereas in C3H/HeJ macrophages, tyrosine phosphorylation was induced only by LPP. 20-h incubation with LPS or LPP downregulated TNF-alpha production/secretion and tyrosine phosphorylation in C3H/HeN macrophages induced by additional LPS or LPP. In C3H/HeJ macrophages, however, the downregulation of TNF-alpha production and tyrosine phosphorylation were observed only with LPP. Protein kinase assays, Western blotting analyses, phenyl-Sepharose chromatography, and immunocomplex kinase assay suggested that p45 and p39 were similar or identical to mitogen-activated protein (MAP) kinase 1 and 2, respectively. Pretreatment of macrophages with LPS or LPP did not change the amount of kinase proteins but inhibited the stimulation of kinase activity by the agents. These data suggest that MAP kinases are among target proteins involved in the transduction of LPS and LPP signals that lead to activation of murine macrophages to produce/secrete TNF.     

Toll-like receptor 4 regulates heme oxygenase-1 expression after hemorrhagic shock induced acute lung injury in mice: requirement of p38 mitogen-activated protein kinase activation

Acute lung injury (ALI) leading to respiratory distress is a common sequela of shock or trauma. The toll-like receptors (TLRs) stand at the interface of innate immune activation in the settings of both infection and sterile injury by responding to a variety of microbial and endogenous ligands alike. This work explored the effects of TLR-4 on hemorrhage-induced ALI and characterizes the signaling pathways and the mechanisms involved in noninfectious ALI. Mice underwent hemorrhagic shock and resuscitation (HSR). Arterial blood gases; expressions of TLR-4, heme oxygenase 1 (HO-1), and p38 mitogen-activated protein kinase (p38MAPK); myeloperoxidase activity; lung wet/dry ratios; and IL-10 levels in lung tissues were obtained at 6, 24, and 48 h after HSR. Hemorrhagic shock and resuscitation induced significant expressions of TLR-4, HO-1, and p38MAPK in C3H/HeN mice. IL-10 and myeloperoxidase were markedly increased at 24 h after HSR, and C3H/HeN mice had ALI with PaO2/fraction of inspired oxygen less than 300 mmHg. The induced amount of each cytokine level and the expressions of TLR-4, HO-1, and p38MAPK of C3H/HeN mice were significantly higher compared with C3H/HeJ mice. This study demonstrated that lung p38MAPK is activated after HSR, and p38MAPK inhibitor FR167653 suppresses HO-1 induction after ALI. We concluded that TLR-4 might induce HO-1 messenger RNA expression, which is probably involved in p38MAPK activation in the development of the lung dysfunction after HSR.     

Pretreatment with recombinant murine tumor necrosis factor alpha/cachectin and murine interleukin 1 alpha protects mice from lethal bacterial infection

Tumor necrosis factor/cachectin (TNF/C) is the principal mediator of bacterial endotoxin-induced shock and death. We found that the C3H/HeJ mouse, which is less able to produce TNF/C in response to endotoxin, has a 1,000-fold greater susceptibility to lethal infection with Escherichia coli than the TNF-responsive congenic mouse, C3H/HeN. This surprising finding suggested that this lethal peptide may also be involved in host protection. To test this hypothesis we pretreated the C3H/HeJ mouse with a combination of recombinant murine TNF/C-alpha and IL-1 alpha. This combination protected these mice against an intraperitoneal bacterial challenge of greater than 20 LD50S (nearly 2 x 10(2) CFU) that grew to a level of greater than 10(7) CFU/ml of blood and per gram of liver in untreated mice. This suggests a significant role for these cytokines in host defenses against invasive infections that require bacterial replication within the host. These protective mechanisms may not be important for less virulent organisms. These findings may have important implications for the proposed use of anti-TNF/C agents in the treatment of septic shock.     

Does Fas ligand or endotoxin contribute to thymic apoptosis during polymicrobial sepsis?

Recent studies have shown that with the onset of sepsis there is an increase in apoptosis (Ao) in the thymus, mediated in part by steroids, which may contribute to a loss of T-cell progenitors, thereby, reducing immune functions. However, reports also suggest that these steroid effects could be mediated by Fas ligand (FasL) and/or by endotoxin (ETX). Thus, our study was to determine: 1) if polymicrobial sepsis (cecal ligation and puncture; CLP) alters thymocyte Fas antigen/receptor (Fas+) expression and 2) if the increase in Ao in septic ETX-sensitive C3H/HeN mice is seen in thymocytes from ETX-tolerant, C3H/HeJ, or the FasL-deficient/ETX-tolerant, C3H/HeJ-FasL(gld), male mouse strains subjected to CLP or sham-CLP (Sham) 12 or 24 h before they were killed. The results of flow cytometric analysis indicated that increased %Ao+ seen in thymocytes of CLP C3H/HeN mice was associated with either no change (12 h) or a decrease in %Fas+ expression at 24 h, although the %Bcl-2+ (an antiapoptotic protein) cells was depressed at both times. Additional studies examining C3H/HeJ or C3H/HeJ-FasL(gld) mice subjected to CLP show that as with the ETX-sensitive mouse, thymocyte Fas and Bcl-2 antigen expression as well as Bcl-2/Bcl-X(L/S) mRNA levels decreased although the %Ao+ increased after CLP in both ETX-tolerant and ETX-tolerant/FasL-deficient mice. Furthermore, if ETX-tolerant/FasL-deficient CLP animals were administered the steroid receptor antagonist RU-38486 (s.c., immediately after CLP) the increase in Ao was markedly attenuated, along with restoration of the percentage of cells expressing Bcl-2 and Fas antigen as well as Bcl-2/Bcl-X(L/S) mRNA levels. Thus, we concluded that increased septic thymocyte Ao is not regulated through either Fas mediated pathway or ETX, but is a result of the release of endogenous steroids possibly acting directly or indirectly on Bcl-2 expression.     

Complement system is involved in anaphylactoid reaction induced by lipopolysaccharides in muramyldipeptide-treated mice

We previously reported that an intravenous injection of specified bacterial lipopolysaccharides (LPS) induced anaphylactoid shock in muramyldipeptide (MDP)-primed mice of various strains, including LPS-resistant C3H/HeJ, accompanied with occasional mortality of mice within 1 h. Prior to shock, rapid accumulation of blood platelets into the lungs and liver followed by degradation of the platelets and tissue destruction were observed. In this report we present the following evidence suggesting that complement activation by LPS is responsible for the anaphylactoid reaction. In C5-deficient DBA/2 mice, the platelet degradation and anaphylactoid reactions did not occur following injection of Prevotella intermedia LPS, although transient platelet accumulation into the lungs and liver was observed. Anti-complement agents K-76 COOH (C5 inhibitor) and cobra venom factor (C5 consumer) protected MDP-primed C3H/HeJ mice from mortality in the anaphylactoid reaction induced by P. intermedia and Salmonella typhimurium LPS, respectively. K-76 COOH also inhibited platelet degradation, but not accumulation, induced by P. intermedia LPS in C3H/HeN mice. LPS specimens carrying mannose-homopolymer (MHP) prepared from wild-type Klebsiella 03 and Escherichia coli 08 and 09 and recombinant E. coli 08 and 09 strains, which have been reported to markedly activate the human complement system probably through the lectin pathway, induced anaphylactoid reactions in MDP-primed C3H/HeJ mice. In contrast, LPS from R-mutant of Klebsiella 03 and the parental strain of the recombinant E. coli strains, which lacked MHP, did not induce anaphylactoid reaction. Based on these findings together with those of our previous studies, we postulated the following mechanism for the anaphylactoid reaction: strong complement activation by specified LPS preparations induced degradation of platelets which have accumulated in the lungs and liver, resulting in acute inflammation accompanied with severe tissue destruction, especially in the lungs, which in turn leads to anaphylactoid reaction. However, the mechanism of platelet accumulation induced by LPS is not yet clear.     

Cellular requirements for lipopolysaccharide adjuvanticity. A role for both T lymphocytes and macrophages for in vitro responses to particulate antigens

By employing primary cultures of purified spleen cells from lipopolysaccharide (LPS) responder (C3H/HeN or C57BL/10Sn) or nonresponder (C3H/HeJ or C57BL/10ScN) mice incubated with particulate antigen and LPS prepared by phenol-water extraction (Ph), we have presented evidence that both T cells and macrophages (MO) are required for LPS-induced adjuvanticity. First, MO derived from C3H/HeN spleen cells, when mixed with responder, C3H/HeN lymphocytes and Ph-LPS, elicited enhanced antibody responses to sheep erythrocytes (SRC) antigen, whereas lymphocytes from the nonresponder, C3H/HeJ mouse strain did not evoke this response. Similarly, purified T cells from C3H/HeN spleens, when cultured with responder, nu/nu spleen cells, and Ph-LPS yielded enhanced anti-TNP PFC responses; whereas, C3H/HeJ T cells did not potentiate immune responses when mixed with optimal concentrations of Ph-LPS. LPS prepared by butanol-water extraction elicited significant adjuvant effects with all cell combinations. Finally, purified responder T cells and MO enabled either responder or nonresponder B cells to elicit LPS potentiation. These data indicate that T cells and MO are controlling LPS-induced augmentation of B-cell responses.     

Viral neuroinvasion and encephalitis induced by lipopolysaccharide and its mediators

The present study was designed to test the effect of bacterial endotoxin on penetration of viruses into the central nervous system (CNS). As a model we used two neurovirulent viruses that lack neuroinvasive capacity: West Nile virus-25 (WN-25) and neuroadapted Sindbis virus (SVN). Administration of lipopolysaccharide (LPS, 100 micrograms/mouse) to CD-1 mice, followed by WN-25 inoculation resulted in 83% encephalitis and death, compared with less than 5% in controls. The results in SVN-inoculated CD-1 mice were quite similar. LPS-treated mice suffered 62% mortality compared with 6% in the nontreated group. No changes in viral neuroinvasiveness were demonstrated in viruses isolated from brains of encephalitic mice, suggesting that neuroinvasion is not due to a selection process for an invasive variant, but to direct penetration of the viruses through the blood-brain barrier (BBB). LPS did not induce WN-25 encephalitis in LPS-insensitive C3H/HeJ mice, compared with 100% neuroinvasion in C3H/HeB mice. Induction of neuroinvasion could be transferred to C3H/HeJ mice by transfusion with serum obtained from LPS-treated, LPS-responsive mice. Passive immunization of CD-1 mice with anti-mTNF antibodies before LPS administration did not prevent LPS-induced WN-25 encephalitis. Furthermore, neutralization of tumor necrosis factor activity in the serum of LPS-treated mice did not abolish its activity, and transfusion-associated encephalitis was observed after the administration of the neutralized serum with WN-25. We suggest that LPS can contribute to virus penetration from the blood into the CNS, a process which turns a mild viral infection into a severe lethal encephalitis. This effect is mediated by soluble factors, and is probably achieved by injury to cerebral microvascular endothelium and modulation of BBB permeability.     

The novel compound NO-1886 increases lipoprotein lipase activity with resulting elevation of high density lipoprotein cholesterol, and long-term administration inhibits atherogenesis in the coronary arteries of rats with experimental atherosclerosis.

We have discovered a novel compound, NO-1886, which possesses a powerful lipoprotein lipase (LPL) activity-increasing action. Administration of NO-1886 increased LPL activity in the postheparin plasma, adipose tissue, and myocardium of rats, and produced a reduction in plasma triglyceride levels with concomitant elevation of HDL cholesterol levels. Administration of NO-1886 increased LPL enzyme mass in postheparin plasma and mRNA activity in epididymal adipose tissue, and it was concluded that the mode of action of this compound is stimulation of tissue LPL synthesis. We also conducted long-term studies to assess the impact of increases in LPL activity and HDL levels on the development of atherosclerotic lesions in rats. Administration of NO-1886 for as long as 90 d significantly decreased the degree of atherosclerotic changes in the coronary arteries of vitamin D2-treated, cholesterol-fed rats. Statistical analysis indicated that increased concentration of HDL is the factor contributing mostly to the prevention of coronary artery sclerosis. In summary, the results of our study indicate that compound NO-1886 increases LPL activity, causing an elevation in HDL levels, and that long-term administration of NO-1886 to rats with experimental atherosclerosis provides significant protection against the development of coronary artery lesions.     

Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes.

Lipoprotein lipase (LPL)-deficient mice have been created by gene targeting in embryonic stem cells. At birth, homozygous knockout pups have threefold higher triglycerides and sevenfold higher VLDL cholesterol levels than controls. When permitted to suckle, LPL-deficient mice become pale, then cyanotic, and finally die at approximately 18 h of age. Before death, triglyceride levels are severely elevated (15,087 +/- 3,805 vs 188 +/- 71 mg/dl in controls). Capillaries in tissues of homozygous knockout mice are engorged with chylomicrons. This is especially significant in the lung where marginated chylomicrons prevent red cell contact with the endothelium, a phenomenon which is presumably the cause of cyanosis and death in these mice. Homozygous knockout mice also have diminished adipose tissue stores as well as decreased intracellular fat droplets. By crossbreeding with transgenic mice expressing human LPL driven by a muscle-specific promoter, mouse lines were generated that express LPL exclusively in muscle but not in any other tissue. This tissue-specific LPL expression rescued the LPL knockout mice and normalized their lipoprotein pattern. This supports the contention that hypertriglyceridemia caused the death of these mice and that LPL expression in a single tissue was sufficient for rescue. Heterozygous LPL knockout mice survive to adulthood and have mild hypertriglyceridemia, with 1.5-2-fold elevated triglyceride levels compared with controls in both the fed and fasted states on chow, Western-type, or 10% sucrose diets. In vivo turnover studies revealed that heterozygous knockout mice had impaired VLDL clearance (fractional catabolic rate) but no increase in transport rate. In summary, total LPL deficiency in the mouse prevents triglyceride removal from plasma, causing death in the neonatal period, and expression of LPL in a single tissue alleviates this problem. Furthermore, half-normal levels of LPL cause a decrease in VLDL fractional catabolic rate and mild hypertriglyceridemia, implying that partial LPL deficiency has physiological consequences.     

The effect of estrogen on the lipoprotein lipase activity of rat adipose tissue.

The effect of 17beta-estradiol or progesterone administration on adipose tissue lipoprotein lipase activity was studied in male and ovariectomized female rats. Lipoprotein lipase activity was measured in acetone-ether-extracted preparations of adipose tissue with doubly labeled (14C-fatty acid, 3H-glyceryl) chylomicron triglyceride as substrate. Administration of 17beta-estradiol to male rats lowered adipose tissue lipoprotein lipase activity from 8.22 plus or minus 1.8 U/g (1 U = 1 mumol triglyceride hydrolyzed per h) to 4.96 plus or minus 0.5 U/g in the treated group. Ovariectomy increased adipose tissue lipoprotein lipase activity from 10.4 plus or minus 1.8 U/g in controls to 22.7 plus or minus 4.3 U/g. 17beta-Estradiol administration to ovariectomized rats cuased a marked fall in adipose tissue lipoprotein lipase activity: 17beta-estradiol (2.5 mug/day) lowered the enzyme activity to 9.00 plus or minus 1.2 U/g, whereas 25 mug/day further decreased lipoprotein lipase activity to 3.2 plus or minus 0.6 U/g. Blood triglyceride levels increased from 0.8 plus or minus 0.05 mumol/ml in ovariectomized rats to 1.4 plus or minus 0.09 mumol/ml in 25 mug/day 17beta-estradiol-treated rats. Progesterone administration did not affect adipose tissue lipoprotein lipase activity in either male or ovariectomized rats. Heart and lung lipoprotein lipase activity was unaffected by hormone treatment. We suggest that the rise in blood triglyceride concentrations, which accompanies high palsma estrogen levels, could be due to the marked inhibition of adipose tissue lipoprotein lipase activity.     

Some Effects of the Administration of Endotoxin in Mice: SPECIFIC CLEAVAGE OF SERUM ALBUMIN BY AN ACID PROTEASE AND THE GENERATION OF AMYLOID SERUM COMPONENT

Endotoxin has been shown to induce amyloidosis in mice and to result in the appearance in serum of large amounts of amyloidrelated protein (SAA). After injection of 300 mug lipopolysaccharide Escherichia coli, SAA behaves as an acute phase reactant with levels reaching a peak of >600 mug/ml at 18-22 h and returning to base line (<50 mug/ml) by 48 h in each of four strains tested; only the endotoxin-resistant C3H/HeJ strain showed a smaller response. Lesser, though significant, elevations were also found after subcutaneous injection of 25 mg of casein, bovine serum albumin, ovalbumin, or monomeric immunoglobulin G, whereas pyrogen-free human serum albumin/U. S. Pharmacopeia failed to raise SAA levels. SAA generation may thus be a result of endotoxin contamination of these protein preparations.Also present in equivalent amounts in acidified serum from endotoxin-treated mice, but barely detectable in control sera, was a 3,000-dalton molecule whose amino acid sequence is identical to the amino terminal 24 residues of mouse albumin. The appearance of SAA and the amino terminal albumin fragment after endotoxin were unaffected by pretreatment with cobra venom factor, and equivalent levels were found in C5-deficient mice. Pretreatment with pepstatin in vivo, or before acidification in vitro, prevented the appearance of the albumin fragment but had no effect on the appearance of SAA, whereas leupeptin and antipain did not affect the appearance of either SAA or the albumin fragment. These studies suggest that the generation of SAA after endotoxin administration does not involve complement activation or intravascular proteolytic activity, whereas the liberation of a specific peptic-like cleavage product of albumin appears to be the consequence of an acid protease.     

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.     

Insulin resistance affects the regulation of lipoprotein lipase in the postprandial period and in an adipose tissue-specific manner

AIMS: Insulin is a potent stimulator of adipose tissue lipoprotein lipase (LPL). Logically, the postprandial period is therefore a privileged time of the day for the regulation of LPL by insulin in this tissue. It is not clear to what extent a defect such as insulin resistance could affect this regulation and contribute to postprandial, as well as fasting, hypertriglyceridaemia. The aim of the present protocol was to study the relationship between insulin resistance and LPL in adipose tissue and in plasma, in the particular context of the postprandial period. METHODS: For this study, 26 adult nondiabetic individuals (12 women and 14 men) with a wide range of whole-body insulin-mediated glucose uptake (as assessed with an insulin suppression test) were studied. An abdominal subcutaneous fat biopsy on one occasion, and post-heparin plasma on another occasion, were obtained 4 h into a standardized meal profile administered in the fasting state. RESULTS: Postprandial triglyceride excursions (evaluated by the incremental area under the curve during the metabolic meal profile) were inversely correlated to adipose tissue LPL mRNA levels (rho = -0.43, P < 0.03) as well as to adipose tissue LPL heparin-releasable activity (rho = -0.58, P < 0.01). Steady-state plasma glucose (SSPG) concentrations during the insulin suppression test, a reflection of the degree of insulin resistance, were also negatively correlated to adipose tissue LPL mRNA (rho = -0.50, P < 0.02) and activity (rho = -0.56, P < 0.01). There was no correlation between plasma post-heparin LPL activity/mass and postprandial triglycerides nor with insulin resistance. CONCLUSION: Regulation of adipose tissue LPL is significantly affected in insulin-resistant individuals in the postprandial period. This presumed impaired effect of insulin on LPL postprandially could be an important contributor to the atherogenic dyslipidaemia described in insulin resistance syndrome.