Augmented resistance to oxidative stress in fatty rat livers induced by a short-term sucrose-rich diet.

Hepatic steatosis and the accompanying oxidative stress have been associated with a variety of liver diseases. It is not known if fat accumulation per se plays a direct role in the oxidative stress of the organ. This study tested if steatosis induced by a short-term carbohydrate-rich diet results in an increased hepatic sensitivity to oxidative stress. Antioxidant status was determined in a liver perfusion system and in isolated parenchymal, endothelial and Kupffer cells from rats kept on sucrose-rich diet or on regular diet for 48 h. t-Butyl hydroperoxide addition (2 mM) to the perfusion fluid resulted in a release of alanine aminotransferase (ALT) in livers from controls, whereas no ALT release was observed in fatty livers. After t-butyl hydroperoxide addition, oxidized glutathione release was 40% less in fatty than in control livers, whereas reduced glutathione (GSH) release was not different. Sinusoidal oxidant stress was mimicked by the addition of lipopolysaccharide (LPS) from Escherichia coli (10 microg/ml) followed by the addition of opsonized zymosan (8 mg/ml) to the perfusion medium. LPS plus zymosan treatments resulted in the release of ALT in control but not in fatty livers. At the end of perfusion, liver glutathione content was 3-fold elevated, and the tissue content of lipid peroxidation products was approx. 40% less in fatty livers compared to controls. GSH content was doubled and glucose-6-phosphate dehydrogenase (G6PD) expression was elevated by 3- and 10-fold in sinusoidal endothelial and parenchymal cells form fatty livers compared to cells from control animals. Following H(2)O(2) administration in vitro (0.2-1 mM), GSH remained elevated in endothelial and parenchymal cells from fatty livers compared to cells from controls. In contrast, G6PD activity and GSH content were similar in Kupffer cells isolated from fatty or control livers. The study shows that hepatic fat accumulation caused by a short-term sucrose diet is not accompanied by elevated hepatic lipid peroxidation, and an elevated hepatic antioxidant activity can be manifested in the presence of prominent steatosis. The diet-induced increase in G6PD expression and, thus, the efficient maintenance of reduced glutathione in endothelial and parenchymal cells are a supportive mechanism in the observed hepatic resistance against intracellular or sinusoidal oxidative stress.

Primed pentose cycle activity supports production and elimination of superoxide anion in Kupffer cells from rats treated with endotoxin in vivo.

Glucose use and pentose cycle activity were determined in freshly isolated rat Kupffer cells 3 h after an i.v. injection of Escherichia coli endotoxin (0.1 mg/kg body weight), by using [1-14C], [6-14C] and [2-3H]glucose. Endotoxin treatment in vivo caused a 5-fold increase in the basal glucose uptake in Kupffer cells. Pentose cycle activity was elevated from 8.7 to 13.6 nmol/h per 10(7) cells after endotoxin. In vitro treatment of the cells from saline- and endotoxin-treated animals with phorbol ester (10(-6) M) increased pentose cycle activity 2-fold and 8-fold, respectively. Phorbol ester caused a 50% increase in glucose uptake in both groups. t-Butyl hydroperoxide (0.5 mM) caused a similar increase in pentose cycle activity as phorbol ester. Glucose oxidation in the Krebs cycle was also doubled after endotoxin. KC from endotoxin-treated animals produced O2- spontaneously, and were primed to produce additional large amounts of O2- upon phorbol ester treatment. Addition of t-butyl hydroperoxide inhibited O2- production by Kupffer cells. Depletion of glutathione by N-ethylmaleimide (0.1 mM), or inhibition of NADPH oxidase by diphenyliodonium (0.1 mM) inhibited both the pentose cycle activity and the O2- production. Increasing the concentration of exogenous glucose in the cell medium elevated the glycolytic rate, while pentose cycle flux was not affected either under basal conditions or following subsequent challenges by phorbol ester or t-butyl hydroperoxide. Our data suggest that the endotoxin-induced elevated glucose use in Kupffer cells is accompanied by a primed state of the pentose cycle. This condition supports superoxide and macromolecule synthesis and could also represent a potentiated protective mechanism against oxidative cellular injury during bacterial infections.

Association of thrombin, plasmin, thrombin-antithrombin III complex and plasmin-antithrombin III complex with isolated hepatocytes.

The interaction of thrombin, plasmin or their antithrombin III complexes with isolated mouse hepatocytes was studied. Plasmin bound to hepatocytes in a concentration-dependent manner with an apparent Kd of 6.4.10(-8) M, attaining equilibrium within 10 min, and the interaction was inhibited by 6-amino-n-hexanoic acid. Plasmin treated with diisopropylfluorophosphate (DFP) bound to the cells in similar way as the untreated form of the enzyme. Thrombin bound also to hepatocytes, in a concentration-dependent manner, with a Kd of 5.4.10(-8) M reaching a steady state after 180 min. Thrombin inactivated with DFP, however, was inhibited in its binding to these cells. These data suggest that, whereas the kringle domains of plasmin are responsible for the enzyme-cell interaction, the active center of thrombin may be involved in the binding of this enzyme to hepatocytes. Plasmin-antithrombin III and thrombin-antithrombin III complexes were also associated with hepatocytes in a time-dependent manner, reaching a plateau after 180 min, and the two complexes competed in the interaction. While the interaction of active proteinases plasmin or thrombin with hepatocytes did not result in their internalization, the antithrombin III complexes were taken up by the cells, and thrombin-antithrombin III complex was degraded. These results indicate that hepatocytes may participate in the elimination of proteinase-antithrombin III complexes from the plasma, while the association of plasmin and thrombin with hepatocytes could imply distinct biological importance.

Association of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin-plasmin complexes with isolated hepatocytes.

125I-labelled alpha 2-macroglobulin complexed with thrombin or plasmin bound to hepatocytes in a concentration- and time-dependent manner. The apparent Kd values calculated from displacement experiments were 7.9 X 10(-8) M for alpha 2-macroglobulin-thrombin and 8.5 X 10(-8) M for alpha 2-macroglobulin-plasmin. Association of these complexes was only partially reversible; after a 180 min incubation period, 50-60% of the bound radioactivity was internalized by the cells. alpha 2-Macroglobulin itself bound also to hepatocytes, but the affinity of the alpha 2-macroglobulin complexes was higher than that of the inhibitor alone, and alpha 2-macroglobulin was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their alpha 2-macroglobulin complexes. The alpha 2-macroglobulin-thrombin complex competed with the alpha 2-macroglobulin-plasmin complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for alpha 2-macroglobulin-proteinase and antithrombin III-proteinase complexes are different.

Endotoxemia, pentose cycle, and the oxidant/antioxidant balance in the hepatic sinusoid.

During the innate immune response, excessive release of reactive oxygen species (ROS) from sequestered phagocytes and activated resident macrophages represents the predominant component of oxidative stress in the liver and other tissues. The consequence of oxidative stress is determined by the status and adaptive changes of antioxidant pathways. In this review, we present evidence that the synchronized response of hepatic sinusoidal endothelial cells, the primary sites of phagocyte attachment, plays an important role in defense against phagocyte-derived ROS. An essential component of the metabolic adaptation of hepatic sinusoidal cells to lipopolysaccharide (LPS)-induced oxidative stress is the stimulated expression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the pentose cycle (hexose monophosphate shunt, HMS). All major ROS-metabolic enzymes, i.e., glutathione peroxidase, glutathione reductase, catalase, superoxide dismutases, NADPH oxidase, and nitric oxide synthase, directly or indirectly depend on NADPH, which is produced in the HMS in these cells. The functional significance of up-regulated HMS within a particular cell type depends on the accompanying adaptive changes in ROS-metabolizing enzymes. In LPS-activated Kupffer cells, the elevated expression of glucose transporter GLUT1 and G6PD mainly serves primed production of superoxide anion, hydrogen peroxide, and nitric oxide. In sinusoidal endothelial cells, the LPS-induced response pattern of glucose- and ROS-metabolizing enzymes results in elevated ROS detoxifying capacity. The described studies also suggest the existence of an intercellular oxidant balance between pro-oxidant Kupffer cells and antioxidant endothelial cells in the hepatic micro-environment. Maintenance of the intercellular oxidant/antioxidant balance between phagocytes and endothelial cells may represent an important mechanism protecting the hepatic parenchyma against exogenous oxidative stress during the inflammatory response.

Endotoxin stimulates the expression of glucose-6-phosphate dehydrogenase in Kupffer and hepatic endothelial cells.

The aim of the study was to elucidate the effect of lipopolysaccharide (LPS) administration in vivo (Escherichia coli endotoxin, 1 mg/kg body weight) on the expression and cellular activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), the rate-limiting enzyme of the hexose monophosphate shunt in hepatic cells. Under basal conditions, Kupffer cells displayed higher activity of G6PDH than endothelial or parenchymal cells. In vivo LPS treatments for 7 and 22 h resulted in 40 and 60% increases, respectively, in the cellular activity of G6PDH in Kupffer cells. G6PDH activity was increased by 140 and 90% after 7- and 22-h LPS treatments in endothelial cells. G6PDH activity in parenchymal cells prepared from animals after 22 h of LPS treatment was decreased by approximately 60% compared with that in cells from saline-injected animals. Total cellular RNA or protein extracts from these cells were analyzed by Northern or Western blots. Under basal conditions, G6PDH mRNA levels relative to total cellular RNA were higher in Kupffer than in endothelial cells and were not detectable in parenchyma cells. LPS injection caused a time-dependent increase in G6PDH mRNA expression in Kupffer and endothelial cells. Western blot analysis of Kupffer cell extracts also showed that LPS treatments caused markedly elevated expression of protein in these cells. These results show that endotoxemia results in marked induction of G6PDH in Kupffer and hepatic endothelial cells but has no such effect in the parenchymal cells. These findings also suggest that the elevated cellular expression of G6PDH is an important regulatory event in the adaptive responses of hepatic nonparenchymal cells to infections. The elevated expression of G6PDH may be important for support of the upregulated NADPH-dependent pathways, such as superoxide anion and nitric oxide production, macromolecular synthesis, or the maintenance of cellular glutathione status.

Superoxide generation by neutrophils and Kupffer cells during in vivo reperfusion after hepatic ischemia in rats.

Kupffer cells and polymorphonuclear leukocytes (PMNs) contribute to the severe reperfusion injury of the liver after ischemia at different time points. The objective of this study was to identify the cellular source(s) of reactive oxygen formation during the PMN-induced injury phase. Kupffer cells and PMNs were isolated from the liver after 45 min of ischemia and 5 h or 24 h of reperfusion using collagenase-pronase digestion and a centrifugal elutriation method. Spontaneous superoxide anion (O2-) formation by large Kupffer cells (basal value 0.65 +/- 0.16 nmol/h/10(6) cells) was increased (up to 550%) during the entire reperfusion period. No enhanced O2- generation by the small Kupffer cell fraction was observed at any time. Control PMNs generated only small amounts of O2- spontaneously (0.25 +/- 0.05 nmol O2-/h/10(6) cells), but hepatic PMNs generated significantly more superoxide: 1.90 +/- 0.58 nmol O2-/h/10(6) cells at 5 h and similarly at 24 h of reperfusion. All cell types were significantly primed for enhanced O2- formation during reperfusion; the priming effect was consistently higher for stimulation with opsonized zymosan (receptor-mediated signal transduction pathway) compared to phorbol myristate acetate (protein kinase C activation). Our data support the hypothesis that PMNs and large Kupffer cells are predominantly responsible for the postischemic oxidant stress during the later reperfusion injury phase after hepatic ischemia in vivo.

In vivo latex phagocytosis primes the Kupffer cells and hepatic neutrophils to generate superoxide anion.

Activation of liver macrophages during clearance of endotoxins, bacteria, or other particulate materials may be accompanied by the migration of polymorphonuclear neutrophils (PMNs) into the liver and priming of the hepatic phagocytes to release toxic oxygen metabolites. In the present study we investigated the effect of in vivo administration of latex particles on the hepatic sequestration of PMNs and the release of superoxide anion (O2-) by the in situ perfused rat liver and isolated hepatic phagocytes. One hour after an intravenous injection of latex beads, a significant amount of O2- (0.7 nmol/min/g) was produced by the in situ perfused liver. Administration of latex particles into the perfused liver also elicited O2- production. Hepatic phagocytes from latex-treated rats generated large amounts of O2- (2-14 nmol/60 min/10(6) cells) when these cells were stimulated in vitro with opsonized zymosan or phorbol myristate acetate (PMA), whereas phagocytes from saline-treated rats released less than 0.8 nmol O2-. Intravenous infusion of superoxide dismutase or ibuprofen did not prevent the immigration of PMNs to the liver. However, ibuprofen inhibited the production of O2- by the perfused liver. Also, after addition of ibuprofen in vitro to isolated cells, there was more than 50% inhibition of O2- generation by Kupffer cells and hepatic PMNs treated with either zymosan or PMA. These observations suggest that arachidonic acid metabolites play a role in O2- release under these conditions. Thus, activation of the reticuloendothelial system by latex phagocytosis induces the migration of PMNs into the liver and enhances the production of toxic oxygen-derived radicals by these cells and the resident Kupffer cells. The toxic oxygen radicals may also contribute to hepatic injury.

Antineutrophil monoclonal antibody (1F12) alters superoxide anion release by neutrophils and Kupffer cells.

The formation of oxygen-derived radicals by phagocytes is regulated by chemotactic agents, cytokines, and adhesion molecules, such as CD11b/CD18 (Mac-1). In the rat system, we investigated the effect of monoclonal antibody 1F12 against rat neutrophils on hepatic sequestration of neutrophils and superoxide release by hepatic phagocytes. Within 15 min after 1F12 injection, there was profound neutropenia, which persisted for 24 h. The majority of the "lost" neutrophils were sequestered in the liver 4 h after treatment. Zymosan-induced superoxide release in vitro by isolated hepatic neutrophils from 1F12-treated rats was significantly attenuated at 4 and 24 h. The phorbol myristate acetate mediated superoxide release was inhibited 24 h after treatment. Superoxide anion release by normal adherent neutrophils in the presence of agonists was also inhibited by 1F12 in vitro. The in vivo administration of 1F12 primed the Kupffer cells to release superoxide. In vitro treatment of Kupffer cells with 1F12 also stimulated superoxide release. Monoclonal antibody WT.3 (also directed against rat neutrophils), which does not cause neutropenia, did not alter superoxide generation by neutrophils and Kupffer cells. These results indicate that 1F12 may be useful in attenuating inflammation and tissue injury associated with neutrophil activation. However, the activation of Kupffer cells to release toxic oxygen-derived metabolites may predispose the liver to injury in certain pathological conditions.

Tumor necrosis factor increases in vivo glucose uptake in hepatic nonparenchymal cells.

This study aims to elucidate the in vivo metabolic response of different liver cells following a short-term (30 min) infusion of a nonlethal dose of human recombinant tumor necrosis factor (TNF). In vivo glucose uptake of different tissues and isolated liver cells was determined by a sequential double-labeling version of the tracer 2-deoxyglucose technique. Following TNF administration glucose uptake was increased in the liver, lung, spleen, and skin while it was not changed in muscle and testis. In response to TNF infusion neutropenia developed which was sustained for 40 min. The number of lymphocytes in the blood was also decreased after the termination of TNF infusion. This short-term infusion of TNF, however, was not accompanied by marked sequestration of leukocytes into the liver. In vivo glucose uptake in response to TNF was doubled in the Kupffer cells and increased by 56% in hepatic endothelial cells. Glucose uptake of parenchymal cells was not significantly affected. The prompt increase of glucose uptake in the reticuloendothelial cells of the liver, primarily in the Kupffer cells, following TNF administration suggests that a similar metabolic response of these cells to sepsis may be mediated at least in part by TNF. It is suggested that the increased glucose uptake by the hepatic nonparenchymal cells is a reflection of the immunomodulatory effect of TNF.

In vivo metabolic response of hepatic nonparenchymal cells and leukocytes to granulocyte-macrophage colony-stimulating factor.

This study investigates the in vivo glucose utilization of various immune-competent cells after an intra-arterial injection of a nonlethal dose (30 micrograms/kg body weight) of murine recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Injection of GM-CSF resulted in a rapid but transient reduction in the number of circulating neutrophils. After 20 min the number of neutrophils returned to normal values, and by 4 h it was about 80% greater than in time-matched saline-injected controls. One hour after the treatment, neutrophils were accumulated in the livers of GM-CSF-injected animals but not in control livers. In vivo glucose utilization by circulating neutrophils and mononuclear cells and various liver cell types was investigated by combining the 2-deoxyglucose tracer technique with cell isolation procedures. GM-CSF increased the in vivo glucose utilization of circulating and infiltrating neutrophils by more than 200%. Glucose utilization by circulating mononuclear cells was also doubled. After GM-CSF injection, glucose utilization by Kupffer cells was increased by 130% and by hepatic endothelial cells was increased by 60%. Indomethacin pretreatment blunted the hyperglycemia caused by GM-CSF injection; however, it did not inhibit the increased glucose utilization by immune-competent cells. This suggests that the effect of GM-CSF on glucose utilization by these cells is not mediated by prostanoids and is at least partially independent of the mass action of elevated glucose concentration. These findings indicate that GM-CSF may be an important member of the cytokine cascade that mediates the acute in vivo metabolic response of immune-competent cells in sepsis or endotoxemia.

Augmented TNF-alpha and IL-10 production by primed human monocytes following interaction with oxidatively modified autologous erythrocytes.

The presence of dysfunctional/damaged red blood cells (RBCs) has been associated with adverse clinical effects during the inflammatory response. The aim of this study was to elucidate whether oxidatively modified, autologous RBCs modulate monocyte cytokine responses in humans. Monocyte tumor necrosis factor alpha (TNF-alpha) and IL-10 production was measured in whole blood from healthy volunteers using ELISA and flow cytometry. Oxidatively modified RBCs (15 mM phenylhydrazine, 1 h, OX-RBC) or vehicle-treated RBCs (VT-RBC) opsonized by autologous serum were administered alone or in combination with one of three priming agents: E. coli lipopolysaccharide (LPS, 0.2 ng/ml), zymosan A (1 mg/ml), or phorbol 12-myristate 13-acetate (PMA, 50 ng/ml). OX-RBC or VT-RBC alone did not result in the release of TNF-alpha or IL-10. LPS, zymosan, and PMA caused marked and dose-dependent increases in TNF-alpha and IL-10 production. Addition of OX-RBC augmented the LPS-, zymosan-, and PMA-induced TNF-alpha release by approximately 100%. OX-RBC augmented LPS- and zymosan-induced IL-10 release by 400-600%. Flow cytometry analyses showed that monocytes were responsible for TNF-alpha and IL-10 production in whole blood. The presence of OX-RBC alone increased the complexity of CD14+ monocytes but caused no cytokine production. LPS alone induced cytokine production without altering cell complexity. After the combined (OX-RBC+LPS) treatment, monocytes of high complexity were responsible for TNF-alpha production. The presence of mannose or galactose (at 10-50 mM) did not alter the observed augmentation of cytokine production by OX-RBC, suggesting that lectin receptors are not involved in the response. These studies indicate that the interaction between damaged autologous erythrocytes and monocytes has a major impact on the cytokine responses in humans. An augmented cytokine production by the mononuclear phagocyte system may adversely affect the clinical course of injury and infections especially in genetic or acquired RBC diseases or after transfusions.

Effect of high-dose endotoxin on glucose production and utilization.

The purpose of the present study was to determine how a high dose of endotoxin (lipopolysaccharide [LPS]), which produces hypoglycemia, alters in vivo glucose uptake by individual tissues. Catheterized conscious fasted rats were injected intravenously (i.v.) with either saline, LPS (1 mg/100 g body weight [BW], lethal dose [LD] 100), or 3-mercaptopicolinic acid (3-MP), an inhibitor of gluconeogenesis. In the latter two groups, blood glucose levels were clamped at either 6 mmol/L (euglycemia) or 3 mmol/L (hypoglycemia). In the first series of experiments, whole-body glucose flux was determined using [3-3H]glucose, and in the second study in vivo glucose uptake (Rg) by individual tissues was estimated by the tracer [U-14C]-2-deoxyglucose technique. The relative contribution of hypoglycemia per se to the LPS effect was determined by comparing the values from LPS- versus 3-MP-treated animals. There was no difference in the rate of whole-body glucose utilization (Rd) between saline-infused control rats and LPS-treated animals that were hypoglycemic. However, Rg by diaphragm, spleen, liver, and lung was increased in hypoglycemic LPS-treated rats. The increased Rg in these tissues was not observed in 3-MP-treated rats with a comparable hypoglycemia. Only the gastrocnemius muscle showed a reduction in Rg under hypoglycemic conditions, and the decrease was similar in both LPS- and 3-MP-treated animals. When sufficient glucose was infused into LPS-injected rats to maintain euglycemia, whole-body glucose Rd was increased compared with that in hypoglycemic LPS-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes of prostacyclin and thromboxane synthesis in the course of mouse liver perfusion. Stimulated thromboxane A2 synthesis of freshly prepared isolated mouse hepatocytes.

The formation of prostacyclin and thromboxane A2 (measured as 6-keto PGF1 alpha and TXB2 by radioimmunoassay) was investigated during a 30 min perfusion of mouse liver in a recirculation system. After cannulation of the portal vein an immediate increase of de novo synthesis and secretion of PGI2 occurred followed by a sharp decrease. Increased PGI2 synthesis was also followed by a continuous increase of TXA2 synthesis and secretion reaching a maximum at the end of the 30 min perfusion. Elevated TXA2 synthesis was also shown in freshly isolated hepatocytes investigated in the course of a 20 min incubation period immediately after the perfusion. However, the elevated TXA2 formation was not observed when it was measured after a 120 min preincubation of the cells. Both PGI2 and TXA2 production could be provoked to a similar extent by the addition of arachidonate and A 23187 immediately after the perfusion or after a 120 min preincubation.

Uptake of arachidonic acid, arachidic acid, oleic acid and their incorporation into phospholipids and triacylglycerols of isolated murine hepatocytes. Effect of thrombin-antithrombin III complex.

Uptake and metabolism of arachidonic acid, arachidic acid and oleic acid were investigated in isolated hepatocytes prepared from mouse liver with the collagenase perfusion method. The rate of uptake of arachidonic acid was time- and concentration- dependent. 94-98% of the arachidonic acid was incorporated into the phospholipid and triacylglycerol fractions following a 60 min incubation period at 37 degrees C. In the presence of thrombin-anti-thrombin III complex a change in the distribution of arachidonic acid incorporated into lipid fractions was found, i.e. increased incorporation into phosphatidyl-serine and phosphatidylethanolamine, whereas the uptake was not altered. There was no change in the uptake and incorporation of arachidic acid and oleic acid.

Tumor necrosis factor alpha augments the expression of glucose-6-phosphate dehydrogenase in rat hepatic endothelial and Kupffer cells.

Cellular activity of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the hexose monophosphate shunt, supports several pathways involved in the nonspecific immune response. In the present study, we investigated the in vivo effects of selected pro-inflammatory cytokines on the expression of G6PD in Kupffer and hepatic endothelial cells. Murine recombinant TNF alpha, IL-1 beta, or IL-6 (1.5 x 10(5) U/kg) was injected and cellular G6PD mRNA level determined using a quantitative reverse transcription and polymerase chain reaction method. G6PD mRNA was elevated two- to threefold seven hours after the injection of TNF alpha in Kupffer and endothelial cells as compared to cells from saline-injected animals. The elevated G6PD mRNA was accompanied by increased cellular enzyme activity in both cells. The cellular activity of 6-phosphogluconate dehydrogenase (6PGD) was also increased seven hours after TNF alpha treatment in these cells. G6PD mRNA and enzyme activity returned to control levels 22h after TNF alpha administration. In contrast to the marked effects of TNF alpha, no significant alterations were found on G6PD expression following IL-1 beta or IL-6 injections in these cells. None of these cytokines caused changes in G6PD or 6PGD expression in parenchymal cells. These data indicate that the proinflammatory cytokine TNF alpha plays an important role in the regulation of cellular G6PD expression in hepatic immune competent cells.

Upregulation of glucose metabolism by granulocyte-monocyte colony-stimulating factor.

Alterations of glucose metabolism were investigated for 6 hours following an intraarterial injection of murine recombinant granulocyte-monocyte colony-stimulating factor (GM-CSF) (30 micrograms/kg body weight). GM-CSF resulted in a transient elevation of plasma glucose. The rate of whole body glucose appearance, as measured by infusion of [6-3H] glucose, was increased by about 10% between 0.5 and 3 hours following GM-CSF injection. In vivo glucose utilization of individual tissues was investigated by the tracer 2-deoxyglucose technique. At 30 min, GM-CSF increased glucose utilization by 80-90% in liver and lung, and 50-60% in skin and spleen. At 3 and 6 hours, glucose utilization by these tissues returned toward control levels except for lung. There was a 40-50% increase in glucose utilization by skeletal muscle 30 min after GM-CSF which was sustained for 6 hours. Glucose utilization of testis, ileum and kidney did not change significantly. Plasma concentrations of insulin, glucagon and tumor necrosis factor were not altered in response to GM-CSF. These findings indicate that some of the acute metabolic effects of a short-term administration of GM-CSF are observed in macrophage-rich tissues, and suggest that GM-CSF may be involved in the metabolic upregulation of immunologically active tissues.

Endotoxin stimulates gene expression of ROS-eliminating pathways in rat hepatic endothelial and Kupffer cells.

Reactive oxygen species (ROS) are mediators of cellular injury and play a putative role in the onset of hepatic damage during endotoxemia or sepsis. It has been suggested that induction of glucose-6-phosphate (G-6-P) dehydrogenase, the key enzyme of the hexose monophosphate shunt (HMS), may support ROS-producing or ROS-eliminating pathways in hepatic endothelial and Kupffer cells during endotoxemia. The aim of the study was to assess in vivo lipopolysaccharide (LPS)-induced alterations in rat gene expression of selected enzymes that are in functional relationship with the HMS. mRNA levels and activities of glucose transporter GLUT-1, Mn- and CuZn-dependent superoxide dismutases (Mn-SOD and CuZn-SOD), and Se-dependent glutathione peroxidase (Se-GPX) were determined. Cellular extracts were analyzed 7 or 22 h after injection of LPS (Escherichia coli, 2 mg/kg ip) or injection of saline. Exposure to LPS for 7 or 22 h caused a 10- to 25-fold increase in GLUT-1 mRNA levels in endothelial and Kupffer cells. In parenchymal cells, GLUT-1 mRNA expression was low, and LPS caused no marked changes. Cellular levels of Mn-SOD mRNA were 20-40 times greater in all hepatic cells from LPS-treated animals than in cells from control rats. LPS at 22 h increased Mn-SOD activity by 45% in endothelial cells but caused no significant changes in Kupffer or parenchymal cells. Message levels and enzyme activities of CuZn-SOD and Se-GPX were significantly elevated 22 h after LPS injection in endothelial cells only. Thus LPS results in marked upregulation of functionally related genes in hepatic cells. In endothelial cells, the simultaneous upregulation of GLUT-1, G-6-P dehydrogenase, Mn-SOD, CuZn-SOD, and Se-GPX may represent an important mechanism for accelerated elimination of ROS released from activated sinusoidal phagocytes. In Kupffer cells, upregulated GLUT-1 and G-6-P dehydrogenase, together with constitutively present SOD and lack of upregulated Se-GPX, suggest an elevated capacity to produce O2- and H2O2 that is consistent with primed bacterial killing.

A carbohydrate-rich diet stimulates glucose-6-phosphate dehydrogenase expression in rat hepatic sinusoidal endothelial cells.

A carbohydrate-rich diet induces glucose-6-phosphate dehydrogenase (G6PD) in liver parenchymal cells, which supports fatty acid synthesis de novo. Bacterial endotoxins stimulate G6PD expression in hepatic sinusoidal endothelial and Kupffer cells but not in parenchymal cells. This study was designed to elucidate whether G6PD expression is regulated uniformly by dietary carbohydrates in hepatic sinusoidal and parenchymal cells. Freshly isolated cells from five groups of Sprague-Dawley rats were analyzed for G6PD activity and mRNA abundance. The rats were grouped as follows: 1) food deprived for 24 h; 2) food deprived for 24 h followed by consumption of the standard diet for 48 h; 3) food deprived for 24 h followed by consumption of a carbohydrate-rich diet for 48 h; 4) fed standard diet; and 5) fed standard diet followed by consumption of a carbohydrate-rich diet for 48 h. In endothelial cells, G6PD activity was 150% greater in group 3 than in group 1 and 125% greater in group 5 than in group 4. Steady-state G6PD mRNA levels were elevated by 300% in endothelial cells from group 3 compared with those from group 1. In Kupffer cells, G6PD activity and mRNA abundance were not different among the groups. As expected, G6PD expression was 700-1200% greater in parenchymal cells from rats fed a carbohydrate diet (groups 3 and 5) than from controls. Our results indicate that short-term consumption of a carbohydrate-rich diet stimulates G6PD expression in endothelial and parenchymal cells. Because G6PD supports reactive oxygen metabolism, the response may represent a preconditioning of antioxidant pathways in the hepatic cell populations that are targets of sinusoid-born reactive oxygen species during infections.