Monokines mediate decreased hepatic glucocorticoid binding in endotoxemia.

The purpose of this study was to determine whether endotoxin decreased hepatic glucocorticoid binding by the action of mediator(s). Steroid binding in liver cytosol, plasma glucose levels, and plasma corticosterone levels were assayed in C3HeB/FeJ LPS normoresponsive and C3H/HeJ LPS hyporesponsive mice. In C3HeB/FeJ mice, endotoxin significantly depressed the maximum number of steroid binding sites (Bmax) to 30% of control. Plasma glucose levels were decreased to 50% of control, and plasma corticosterone levels increased 4-fold. No changes in these parameters were seen in C3H/HeJ mice given endotoxin, except for decreased plasma glucose levels at the highest dose of endotoxin. Decreased steroid binding was observed in C3H/HeJ mice 4-6 hours after receiving C3HeB/FeJ peritoneal exudate cells (elicited with thioglycolate) and endotoxin. No change in steroid binding was observed in C3H/HeJ mice that received C3H/HeJ peritoneal exudate cells and endotoxin. Mediator-rich plasma was produced in CF-1 mice by infecting them with 1 X 10(7) BCG and by challenging them with endotoxin (2 micrograms) 2 weeks later for 2 h. Transfer of BCG-endotoxin plasma to C3H/HeJ mice also resulted in decreased steroid binding and plasma glucose. These results indicate that perturbation of glucocorticoid action during endotoxin shock is mediated by soluble factor(s) other than endotoxin. A likely source of mediator(s) is the mononuclear phagocyte.

Effects of aging and endotoxin on hepatic glucocorticoid action and glucose metabolism in mice.

Hepatic binding of [3H] dexamethasone, phosphoenolpyruvate carboxykinase activity, glycogen levels, and plasma glucose and corticosterone concentrations were measured in young, mature, and old mice given either endotoxin or vehicle. Endotoxin treatment differentially lowered plasma glucose concentration, hepatic glycogen content, PEPCK activity and specifically bound [3H] dexamethasone, but increased plasma corticosterone concentration by a magnitude dependent upon the age of the animal.

The removal of 14C labeled endotoxin by activated charcoal.

Endotoxin shock due to Gram-negative enteric bacteria is of major medical concern with an estimated 100,000 fatalities in the United States per year. An effective therapy for endotoxin shock, particularly in combination with significant liver damage, has not been available to date. Since activated charcoal is known as a universal sorbent, the use of activated charcoal in a hemoperfusion apparatus to remove endotoxin has interesting possibilities. Current assays for endotoxin are inadequate. The Limulus Amoebocyte Lysate (LAL) assay was found to give nonreproducible results within our range of requirements for accuracy. We, therefore, grew Salmonella typhimurium in 14C-labeled glucose to obtain 14C labeled endotoxin. Radiolabeled endotoxin was used to measure the rate of adsorption on activated charcoal. The rates of removal of endotoxin from normal saline, plasma, and whole blood will be presented in graphical form for use in design calculations. This work provides a foundation for encouraging in vivo hemoperfusion experimentation now underway at the University of Oklahoma and the Veteran's Administration Hospital in Oklahoma City.

Failure of glucagon to induce hepatic phosphoenolpyruvate carboxykinase in endotoxic shock.

We have determined that one reason for diminished PEPCK activity during endotoxemia is the inhibition of glucocorticoid action in hepatic cells. Since glucocorticoid and glucagon hormones act cooperatively to regulate the expression of PEPCK mRNA, we examined whether endotoxin also inhibits the action of glucagon to induce this enzyme. Treated mice were injected intraperitoneally with endotoxin and glucose after a 24 hr fast and given ad libitum access to food and water. Control mice received the same amount of glucose and access to food and water. All mice were given intravenous injections of glucagon for 3 consecutive hours before euthanasia. Blood was analyzed for glucose concentrations, and the liver was assayed for PEPCK activity. Refeeding control mice after a 24 hr fast increased plasma glucose levels to 173 +/- 14 mg/dL and decreased PEPCK activity to 20.6 +/- 2.0 units/mg liver. Subsequent administration of exogenous glucagon further increased plasma glucose to 224 +/- 17 mg/dL and hepatic PEPCK to 31.4 +/- 1.4 units/mg liver. Refeeding endotoxin-treated mice after a 24 hr fast slightly increased plasma glucose levels to 75 +/- 4 mg/dL but had no effect on PEPCK activity. Subsequent glucagon administration had no effect on plasma glucose levels (75 +/- 1.0 mg/dL) or hepatic PEPCK activities (18.8 +/- 5.0 units/mg liver). Therefore, glucagon action to increase liver PEPCK activity and plasma glucose levels was inhibited in endotoxin-treated mice.

Studies on bacterial synergism in mice infected with Bacteroides intermedius and Fusobacterium necrophorum.

This study was undertaken to characterize the kinetics of possible bacterial synergy using a mouse model of mixed intraabdominal infection with Bacteroides intermedius and Fusobacterium necrophorum. Female CD-1 mice were injected intraperitoneally with B. intermedius, F. necrophorum or mixtures of both organisms. Generalized septic peritonitis developed within 24 hr, with abscess formation occurring after one to two wk in survivors with the mixed infection. Involvement of the reticuloendothelial system was evidenced by dose-dependent hepatosplenomegaly, which appeared during the first wk postinfection and progressed throughout the course of the experiment. Indirect immunofluorescence confirmed the presence of both species of bacteria in frozen sections of liver tissue. The median lethal dose (LD50) was 2.11 x 10(9) for the mixture, 3.03 X 10(9) for B. intermedius alone, and 1.07 X 10(9) for F. necrophorum alone. The median abscess-producing dose (AD50), the dose required to produce abscesses in fifty percent of the surviving mice at two wk, was approx. 1/100 of the LD50 dose. The AD50 for intrahepatic abscesses was 2.8 x 10(8) for the mixture, whereas the AD50 for intraabdominal abscesses occurring in any site was 5.14 X 10(7). Both Bacteroides and Fusobacterium persisted in tissue for at least 22 wk following mixed infection. The persistence of the Bacteroides in tissue represents a synergistic result of mixed infection with Fusobacterium and contributed to the chronicity of intraabdominal abscess formation. Bacteroides, injected alone, did not produce abscesses at any of the doses tested. However, when passaged (isolated from mixed infection hepatic abscesses) B. intermedius was used, the bacteria did induce abscesses.

Down regulation of hepatic glucocorticoid receptors after endotoxin treatment.

The mechanism by which endotoxin administration results in hypoglycemia was evaluated by characterizing [3H]dexamethasone binding and phosphoenolpyruvate carboxykinase activity in hepatic cytosol preparations from treated and control mice. Starved mice were given Escherichia coli O111:B4 endotoxin or saline intraperitoneally on day 3 after bilateral adrenalectomy. [3H]dexamethasone binding was measured by the charcoal method after the incubation of cytosol preparations with [3H]dexamethasone in the presence or absence of unlabeled dexamethasone. Changes in [3H]dexamethasone binding were found to be time and dose dependent in treated mice. When mice given different doses of endotoxin reached the same stage of morbidity, as indicated by the average time of death, significantly lower glucocorticoid binding was measured. Scatchard analysis of binding isotherms defined a single class of binding sites. Association and dissociation rate constants and the equilibrium dissociation constant (Kd) were not altered, but the maximum number of binding sites was depressed by endotoxin. The rank order of potency of competitors for [3H]dexamethasone binding, dexamethasone greater than hydrocortisone = corticosterone greater than deoxycorticosterone greater than progesterone greater than testosterone = estradiol, was consistent with a glucocorticoid receptor, although the competition was not altered by endotoxin. Endotoxin treatment prevented the glucocorticoid-induced increase in hepatic phosphoenolpyruvate carboxykinase activity. We conclude that the hypoglycemia of endotoxin poisoning is effected, in part, by the inhibition of the glucocorticoid-mediated induction of phosphoenolpyruvate carboxykinase via the down regulation of hepatic glucocorticoid receptors.

Growth yields and fermentation balance of Bacteroides fragilis cultured in glucose-enriched medium.

Bacteroides fragilis is an obligate anaerobic bacterium classified with the gram-negative, non-sporeforming bacilli and is the Bacteroides species most frequently isolated from human infections. In the present study, experiments were designed to investigate growth characteristics of B. fragilis in a complex medium. In a minimal defined medium, which was employed for comparison purposes, B. fragilis grew with a generation time of 2 h. Growth of the organism in glucose-enriched medium used in the present study was superior. Maximum generation time was 60 min. Total and viable cells (colony-forming units) were 8.9 x 10(9) and 2.1 x 10(9), respectively, at maximum measurable growth. The molar growth yield (Ym) was 51.5. Growth yields were found to reach a maximum 2 to 3 h before maximum growth and to vary with respect to the phase of growth. Estimates of the fermentation products indicated that glucose was the sole energy substrate. Major products included acetic acid, propionic acid, lactic acid, and succinic acid. Other products included ethyl alcohol, pyruvic acid, and fumaric acid. No attempt was made to recover CO2 or formic acid. The OR balances from two experiments were 0.013 and -0.093 and the respective carbon recoveries were 6.268 and 6.241. The results of the present study show that B. fragilis is capable of rapid rates of growth in vitro by using glucose as the sole energy source.

Carbohydrate metabolism in C3H/HeJ (nonresponder) mice during endotoxic shock.

This study was undertaken to evaluate the hypoglycemic effects of endotoxin in C3H/HeJ (nonresponder) mice. Endotoxin from Salmonella enteritidis ser Typhimurium strain SR-11 was used and the median lethal dosed (LD50) for random-outbred Swiss-Webster mice and C3H/HeJ mice were 450 microgram and 3,000 microgram, respectively. At intervals after intraperitoneal injection of endotoxin (1 LD50) animals were killed, and blood glucose, liver glucose, and liver glycogen levels were killed, and blood glucose, liver glucose, and liver glycogen levels were measured. The time course of carbohydrate depletion in both strains of mice was almost identical. Little change from controls was noted, however, in nonresponder mice given the LD50 dose for normal responder mice. Passive transfer of plasma from C3H/HeJ mice appeared to protect conventional responder mice from the carbohydrate-depleting effects of endotoxin; whereas, passive transfer of peritoneal cells from C3HeB/FeJ responder mice to nonresponders appeared to sensitize C3H/HeJ mice to this effect. In order to evaluate clearance and detoxification of endotoxin in non-responder mice, 14 C-labeled lipopolysaccharide was prepared from bacteria grown in broth containing D-glucose-14 C(U). Mice were injected intravenously with labeled endotoxin, and blood, liver, spleen, kidney, heart, lung, and brain were counted for radioactivity at intervals after injection. Results from these tracer studies indicate that the clearance of lipopolysaccharide in nonresponder mice is slower than that seen in conventional animals. The results of this study further support the suggestion that endotoxin exerts its effects on carbohydrate metabolism via mediators resulting from endotoxin-cell surface interactions.

Lipopolysaccharide-tumor necrosis factor-glucocorticoid interactions during cecal ligation and puncture-induced sepsis in mature versus senescent mice.

Previous work in our laboratory demonstrated increased sensitivity of senescent (24-month-old) mice to cecal ligation and puncture (CLP) sepsis compared with that of mature (12-month-old) mice. In this study the median lethal dose of the strain of Escherichia coli most frequently isolated during CLP sepsis was determined. No significant age-associated difference in the mean lethal dose or the mean survival time was noted; however, sham surgery before injection of E. coli decreased the mean lethal dose by at least 100-fold. With surgical manipulation, the average time to death after bacterial injection simulated more closely that observed after CLP surgery. Host responses to CLP sepsis were investigated by measuring the levels of corticosterone, glucose, and tumor necrosis factor (TNF) in the sera of mature and senescent mice at 2-h intervals after surgery. Corticosterone levels increased gradually during the course of sepsis in mature mice; however, senescent mice demonstrated a pronounced elevation in hormone levels at 2 and 4 h after surgery. At subsequent sampling intervals the corticosterone levels remained elevated, although they were similar for both ages. At all sampling intervals, the glucose levels in serum were lower in senescent mice than in mature mice. Pronounced hypoglycemia (less than 80 mg/dl) was observed in senescent mice at 8 h postsurgery. TNF was detected in serum within a narrow time frame in both age groups at 6, 8, and 10 h postsurgery. Although elevated TNF levels in serum were not seen in every mouse in each group (approximately 50%), the data hinted that senescent animals produced larger quantities of TNF during CLP sepsis than did mature animals. E. coli lipopolysaccharide (1 mg/kg) was injected intraperitoneally, and the TNF levels in serum and peritoneal lavage fluid were measured at 30, 60, and 90 min. Senescent mice demonstrated a level of TNF in serum at 90 min after lipopolysaccharide treatment that was 20-fold higher than that of mature mice (299,877 pg/ml versus 15,594 pg/ml). The amount of TNF produced locally in the peritoneum was also substantially higher in senescent mice than in mature animals (1,716 pg/ml versus 776 pg/ml). The increased production of TNF in senescent animals, despite elevated circulating corticosterone levels, suggested an age-related defect in glucocorticoid-directed downregulation of TNF production. This was confirmed in lipopolysaccharide-treated animals given exogenous dexamethasone.(ABSTRACT TRUNCATED AT 400 WORDS)

General effect of endotoxin on glucocorticoid receptors in mammalian tissues.

Considering the ubiquitous nature of glucocorticoid actions and the fact that endotoxin inhibits glucocorticoid action in the liver, we proposed to examine whether endotoxin affected extrahepatic actions of glucocorticoids. Fasted C57BL/6J mice were injected intraperitoneally with endotoxin (LD50) at 0800 and were killed 6 h later. Control mice were injected with an equal volume of saline. 3H-dexamethasone binding, measured by a new cytosol exchange assay utilizing molybdate plus dithiothreitol, in liver, kidney, skeletal muscle, spleen, lung, and heart tissue was significantly lower in treated than in control mice. The equilibrium dissociation constants were not significantly different, but the number of available binding sites in each tissue was reduced by endotoxin treatment. Phosphoenolpyruvate carboxykinase activity was significantly reduced in liver but not in kidney. Endotoxin treatment lowered glycogen content in liver but not in skeletal muscle. The reduction observed in the "a" form of liver glycogen synthase due to endotoxin was not seen in skeletal muscle glycogen synthase "a." These data support the proposal that endotoxin or a mediator of its action inhibits systemic glucocorticoid action. The results also emphasize the central role of the liver in the metabolic disturbances of the endotoxin-treated mouse.

Enhancement of Bacteroides intermedius growth by Fusobacterium necrophorum.

Previous work from this laboratory has demonstrated the persistence of Bacteroides intermedius in the livers of mice receiving an intraperitoneal inoculum of B. intermedius and Fusobacterium necrophorum. This study was undertaken to determine whether F. necrophorum enhanced the in vitro growth of B. intermedius. Tryptose phosphate broth did not support the growth of B. intermedius alone, but the bacterium did survive in a tryptose phosphate broth culture of F. necrophorum. B. intermedius cultured in F. necrophorum-conditioned tryptose phosphate broth grew impressively, reaching maximal absorbance at 24 h after inoculation. The growth of B. intermedius in F. necrophorum-conditioned tryptose phosphate broth was proportional to the amount of conditioned medium present. The B. intermedius growth-stimulating factor was detectable in conditioned medium 8 h after inoculation with F. necrophorum and could be detected throughout the 96-h incubation period. Growth-factor-active fractions eluted from a Sephadex G-100 column did not absorb at 280 nm and were retained on the column until 4 column volumes were eluted. The growth factor was nondialyzable and stable to boiling, lyophilization, extraction with hot aqueous phenol, and trypsin digestion. The factor was inactivated by exposure to pH 2.0 in the pepsin digestion protocol. Significant amounts of hexose, methyl pentose, and 2-keto-3-deoxyoctonate were detected in pooled growth-factor-active fractions eluted from the Sephadex column. This pool was also active in the Limulus lysate endotoxin assay. These results suggest that the B. intermedius growth-stimulating factor produced by F. necrophorum is a lipopolysaccharide.

Identification of tumor necrosis factor as a transcriptional regulator of the phosphoenolpyruvate carboxykinase gene following endotoxin treatment of mice.

The decreased synthesis of hepatic phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme of gluconeogenesis, that occurs during endotoxemia was shown previously in rats to occur at the transcriptional level. In the current study, the exogenous administration of human recombinant tumor necrosis factor (TNF), a proximal mediator of endotoxic shock, reduced the PEPCK transcription rate, mRNAPEPCK levels, and PEPCK enzyme activity in a time- and dose-dependent manner in CD-1 mice. Comparable amounts of circulating TNF were measured in mice 2 h after injection of human recombinant TNF (10(5) U) or a 50% lethal dose of Escherichia coli endotoxin (20 mg/kg). Direct action of TNF to decrease the PEPCK transcription rate was confirmed in vitro with H-4-II-E Reuber hepatoma cells, in which a dose-dependent inhibition of PEPCK transcription was observed with 1 to 100 U of TNF per ml. A role for TNF-elicited changes in PEPCK gene expression during endotoxemia was confirmed by the protective effect of rabbit polyclonal antibodies to recombinant murine TNF. C57BL/6 mice passively immunized with anti-TNF 4 h prior to endotoxin challenge exhibited normal PEPCK enzyme activity. Neutralization of circulating TNF with anti-TNF failed, however, to prevent the hypoglycemia commonly observed during endotoxemia, suggesting the participation of other mediators. Anti-TNF treatment reduced circulating interleukins 1 and 6 at 3 and 6 h after endotoxin treatment, respectively. These results suggest that during endotoxemia, the development of hypoglycemia is multifaceted and that several cytokines are most likely involved. The findings from the Reuber hepatoma cell model afford an opportunity in future work to map putative cytokine response elements in the PEPCK promoter responsible for perturbed hormonal regulation of the gene during endotoxemia.

Mechanisms of Pathogenesis in Listeria monocytogenes Infection IV. Hepatic Carbohydrate Metabolism and Function in Experimental Listeriosis.

Previous reports have shown alterations in carbohydrate metabolism in mice infected with Listeria monocytogenes. This study was undertaken to elucidate mechanisms involved in these changes. Female CD-1 mice were injected intraperitoneally with 10(6)L. monocytogenes strain A4413. Animals were fasted 12 hr prior to infection, and pooled tissue from several mice was observed at intervals after infection. Blood glucose, liver glucose, and liver glycogen decreased within 10 hr after infection. Sustained treatment with gluconeogenic precursors, including glucose-6-phosphate, fructose-1, 6-biphosphate, phosphoenolpyruvate, alpha-glycerophosphate, pyruvate, and amino acids, did not restore and maintain glucose and glycogen at normal levels and did not affect survival. Administration of hydrocortisone induced restoration of liver glycogen early in the infection but did not maintain normal levels as the infection progressed. Activities of succinic dehydrogenase and cytochrome oxidase in liver homogenates from infected mice were elevated as early as 10 hr after infection. Liver function tests using rose bengal sodium-(131)I showed no significant differences in plasma clearance or liver uptake between normal and infected mice except in terminal infections (60 hr after infection).

Endotoxin-induced inhibition of steroid binding by mouse liver cytosol.

Glucocorticoids have been reported to ameliorate the lethal effects of endotoxin in a wide variety of animals, including man. Restoration of hepatic gluconeogenic enzymes and nucleotides may be involved in this effect. In this study decreased binding of 3H-dexamethasone was observed in liver cytosol preparations obtained from adrenalectomized C3HeB/FeJ mice treated with 100 micrograms of Escherichia coli 0111:B4 endotoxin (Boivin). Adrenalectomy significantly reduced th endotoxin LD50 value from 4.7 mg/kg to 2.3 micrograms/kg. The amount of labeled steroid bound by endotoxin-treated mice was 13,606 +/- 2,027 dpm/mg cytosol protein compared with 17,247 +/- 2,084 dpm/mg cytosol protein in adrenalectomized controls. Results from studies on the distribution of 14C-endotoxin suggested the inhibition of steroid binding seen in liver may be a mediated event. It is likely perturbations in steroid action at the subcellular/molecular level are involved.

Changes in macromolecular composition and morphology of Bacteroides fragilis cultured in a complex medium.

Changes in cell macromolecular composition during different phases of growth correlated with alterations in morphology of the obligately anaerobic bacterium, Bacteroides fragilis, grown in a complex medium.

Response of mouse liver glycogen cycle enzymes to endotoxin treatment.

The present study was undertaken to characterize endotoxin-induced changes in carbohydrate metabolism and more specifically, to determine the contribution of glycogenolysis to the loss of liver glycogen. Female ICR mice, fasted overnight, were injected with a median lethal dose (LD50, 9 mg/kg) of endotoxin extracted from Salmonella typhimurium strain SR-11. Glycogen synthase and glycogen phosphorylase activities were measured at 0.5 and 6 h after treatment. Endotoxin treatment did not alter total glycogen synthase activity, but the amount of enzyme present in the active form was significantly lower in endotoxic mice. There was no significant increase in glycogen phosphorylase activity in endotoxin-treated mice. Glycogen phosphorylase was activated to the same extent in control and endotoxic mice by decapitation or intravenous epinephrine (25 or 1 mug/kg). The results of this study indicate no significant increase in glycogen phosphorylase activity in endotoxic mice, contraindicating enhanced glycogenolysis as a mechanism for depletion of carbohydrate following endotoxin injection. Altered activation of glycogen synthase, however, may contribute to the loss of glycogen during endotoxemia.

Mechanisms of pathogenesis in Listeria monocytogenes infection. V. Early imbalance in host energy metabolism during experimental listeriosis.

Early changes in hepatic carbohydrate metabolism without apparent hepatocyte dysfunction were reported previously in mice infected with Listeria monocytogenes. This study was undertaken to examine possible imbalance in host regulatory mechanisms which might be responsible for these changes. Female CD-1 mice fasted 12 hr prior to the experiments were injected intraperitoneally with 10(5), 10(6), or 10(7)Listeria. Control mice received either 10(9) heat-killed Listeria or 150 mug of Salmonella typhimurium lipopolysaccharide. Hepatic glycogen, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and nicotinamide adenine dinucleotide (NAD) (NAD(+), NADH, NADP(+), and NADPH) levels were assayed periodically. Activities of ATP hydrolyzing enzyme and NAD glycohydrolase were measured at various intervals after infection. Decreases in glycogen occurred as early as 10 hr after infection. Responses in the controls differed from those in infected mice. Hepatic ATP levels decreased as early as 10 hr after infection, with concomitant increases noted in ADP. Hepatic ATP hydrolyzing enzyme activity increased as the infection progressed. Decreases were noted in hepatic NAD levels, with the greatest reduction in the reduced form of NAD. Slight changes were observed after 10 hr, and greater differences were noted 20 hr after infection. The magnitude of these biochemical changes appeared to be dose-dependent. Significant increases in hepatic NAD glycohydrolase activity were noted as the infection progressed. Small but significant increases in serum inorganic phosphate were noted 10 and 20 hr after infection, with a larger increase observed 30 hr after infection. The results indicate impairment of host energy metabolism early in the course of experimental listeriosis.

Mouse liver fructose-1, 6-diphosphatase and glucose-6-phosphatase activities after endotoxin poisoning.

Hepatic fructose-1, 6-diphosphatase and glucose-6-phosphatase were significantly decreased in fasted mice 17 hr after the intraperitioneal injection of a median lethal dose of Salmonella typhimurium endotoxin. Liver glycogen levels were essentially depleted at 17 hr.