BRUNO CHRIST, EMINE YAZICI, AND ANNEGRET NATH

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1 Phosphatidylinositol 3 Kinase and Protein Kinase C Contribute to the Inhibition by Interleukin 6 of Phosphoenolpyruvate Carboxykinase Gene Expression in Cultured Rat Hepatocytes BRUNO CHRIST, EMINE YAZICI, AND ANNEGRET NATH The participation of phosphatidylinositol 3 kinase (PI3- kinase), protein kinase C, and mitogen-activated protein kinase (MAP-kinase) in the inhibition by interleukin 6 (IL-6) and insulin of phosphoenolpyruvate carboxykinase (PCK) gene expression was investigated in cultured rat hepatocytes. IL-6 or insulin inhibited the glucagonstimulated increase in PCK messenger RNA (mrna) by about 70%. In the presence of either the PI3-kinase inhibitor, wortmannin, or the protein kinase C inhibitor, GF109203x, the inhibition by IL-6 was only about 40%, although it was abolished with both inhibitors in combination. Wortmannin alone but not GF109203x prevented the inhibition by insulin of glucagon-stimulated PCK gene expression. The MAP-kinase pathway inhibitor, PD98059, did not affect IL-6 or insulin inhibition of PCK mrna increase. When chlorophenylthio cyclic 3,5 adenosine monophosphate (CPT-cAMP) was used instead of glucagon, IL-6 or insulin inhibited the increase in PCK mrna by 75% and 85%, respectively. The inhibition by IL-6 was only about 50% in the presence of either wortmannin or GF109203x alone but was abolished with the combination of both inhibitors. The inhibition by insulin was only about 50% in the presence of GF109203x and was abolished by wortmannin. The inhibitors did not affect the inhibition by IL-6 or insulin of the glucagon-stimulated increase in camp. It is concluded that the inhibition by IL-6 of PCK gene expression involved both PI3-kinase and protein kinase C, whereas the inhibition by insulin required only PI3-kinase. The inhibition occurred downstream from camp formation. Hence, IL-6 and insulin may share, in part, common signal transduction pathways in the inhibition of PCK gene expression. (HEPATOLOGY 2000;31: ) Abbreviations: PCK, phosphoenolpyruvate carboxykinase; camp, cyclic 3,5 adenosine monophosphate; PI3-kinase, phosphatidylinositol 3 kinase; MAP-kinase, mitogen activated protein kinase; IL-6, interleukin-6; TNF-, tumor necrosis factor ; mrna, messenger RNA; CPT, chlorophenylthio. From the Institute of Biochemistry and Molecular Cell Biology, Georg-August University, Göttingen, Germany. Received August 16, 1999; accepted November 23, Supported by grants of the Deutsche Forschungsgemeinschaft (Bonn) through the SFB 402, Teilprojekt A4, and the Fonds der Chemischen Industrie (Frankfurt). B.C. s present address is the Institute of Medical Biochemistry and Genetics, The Panum Institute, Biochemistry Department A, University of Copenhagen, Denmark. Address reprint requests to: Bruno Christ, Ph.D., Institute of Medical Biochemistry and Genetics, The Panum Institute, Biochemistry Dept. A, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark. bchrist@imbg.ku.dk; fax: (45) Copyright 2000 by the American Association for the Study of Liver Diseases /00/ $3.00/0 Glucagon stimulated the expression of the rate controlling enzyme of gluconeogenesis in the liver, phosphoenolpyruvate carboxykinase (PCK), via the increase in intracellular cyclic 3,5 adenosine monophosphate (camp), and subsequent camp-dependent transcriptional activation of the PCK gene, which was antagonized by insulin (reviewed in Hanson and Reshef 1 and O Brien and Granner 2 ). The insulin inhibition of PCK gene expression involved phosphatidylinositol 3 kinase (PI3-kinase) 3-5 but not mitogen-activated protein kinase (MAP-kinase). 5-7 Phorbol esters inhibited camp-stimulated PCK gene expression in H4IIE rat hepatoma cells but protein kinase C was not involved in this inhibition. 8,9 In cultured rat hepatocytes the proinflammatory cytokines interleukin 6 (IL-6), tumor necrosis factor (TNF- ), and IL-1 inhibited the glucagon-dependent induction of PCK gene transcription and accelerated PCK messenger RNA (mrna) degradation. 10,11 IL-6 attenuated the increase in camp levels stimulated by glucagon and prevented the transcriptional activation of the PCK gene promotor. 12 In vivo mouse liver endotoxin challenge attenuated the induction by cortisone of PCK enzyme activity. 13,14 Later it was shown that IL-6 and TNF- decreased PCK gene transcription in rat and mouse liver. 15,16 Yet, the principal mechanism by which the proinflammatory cytokines inhibited PCK gene expression remained unclear. 461 Also, in the septic rat, 17,18 in 12-hour TNF- treated mice, 19 and in IL-6 secreting tumor transplanted mice 20 the expression of the gluconeogenic enzyme, glucose-6-phosphatase, was inhibited, which together with the inhibition of PCK gene expression indicates a general impairment of gluconeogenesis by proinflammatory cytokines. In cultured rat hepatocytes 24-hour chronic treatment of hepatocytes with a mixture of IL-1, TNF-, interferon, and bacterial endotoxin inhibited gluconeogenesis. 21,22 Twenty-four hour treatment with either TNF- or IL-1 alone did not affect gluconeogenesis but IL-6 alone stimulated gluconeogenesis. 23 However, short-term 4-hour treatment of cultured rat hepatocytes with IL-1 or TNF- inhibited gluconeogenesis. 10 IL-6 is the main mediator of the hepatic acute phase response. Signal transduction is accomplished by the activation of the JAK/STAT pathway (reviewed in Heinrich et al. 24 ). However, recent reports showed that many other biological effects of IL-6 may also involve PI3-kinase, MAP-kinase, or protein kinase C Therefore, because IL-6 shares multiple signal transduction components with insulin it is feasible that the inhibition by IL-6 of glucagon-stimulated PCK gene expression also participates in insulin signal transduction

2 462 CHRIST, YAZICI, AND NATH HEPATOLOGY February 2000 components in cultured rat hepatocytes. It was the aim of this study to investigate by the use of inhibitors of PI3-kinase, of the MAP-kinase pathway, and of protein kinase C whether these kinases were involved in the inhibition by IL-6 or insulin of the stimulation by glucagon of the increase in camp and in PCK gene expression. Although the inhibition by IL-6 of PCK gene expression could be prevented by the combination of inhibitors of protein kinase C and PI3-kinase, the inhibition by insulin was attenuated by the inhibitor of PI3-kinase alone. Inhibitors of the MAP-kinase pathway were without effect. None of the inhibitors had any effect on the stimulation of camp increase by glucagon. MATERIALS AND METHODS Animals and Chemicals. Rat hepatocytes were prepared from male Wistar rats ( g) (Winkelmann, Borchen, Germany) fed a standard diet (Altromin). Chemicals were of pro analysis (p.a.) quality and purchased from local suppliers. Collagenase A, newborn calf serum, tools for the detection of digoxygenin-labeled nucleic acids, and chlorophenylthio (CPT)-cAMP were from Roche Biochemicals (Mannheim, Germany). Insulin, glucagon, and wortmannin were supplied by Sigma (Heidelberg, Germany). Culture medium M199 was from Life Technologies (Eggenstein, Germany). Tissue culture dishes, 60 mm and 35 mm, were from Greiner (Nürtingen, Germany). Percoll was purchased from Pharmacia (Freiburg, Germany) and recombinant human IL-6 was from Strathmann Biotech (Hannover, Germany). GF109203x and PD98059 were supplied by Calbiochem (Bad Soden, Germany). Experimental Setup. To isolate hepatic cells the rat liver was perfused with collagenase and parenchymal cells were separated from nonparenchymal cells and detriments by a Percoll density gradient. Hepatocytes were cultured at a density of /cm 2 either in 2.5-mL culture medium M199 on 60-mm plastic dishes for the preparation of total RNA for Northern blot analyses or in 1 ml M199 on 35-mm dishes for the determination of camp levels. The medium was supplemented with 100 nmol/l dexamethasone and 0.5 nmol/l insulin, which was added to improve culture maintenance. Cells were cultured for 48 hours under a gas atmosphere of 16% O 2,5%CO 2, and 79% N 2 with a medium change after 4 hours and 24 hours. For the first 4 hours, 4% newborn calf serum was added to the medium to support cell attachment. For the induction of PCK mrna, medium was sucked off after 48 hours and cells were washed twice with dexamethason-containing but insulin-free medium. Then fresh dexamethason-containing medium was supplied with either glucagon, at 0.1 nmol/l, or CPT-cAMP at 100 nmol/l, and IL-6 at 100 ng/ml, or insulin at 10 nmol/l where indicated. 30 Dexamethasone was included in the medium throughout the cell culture and the induction experiments, because it acted as a permissive hormone on the glucagon induction of PCK gene expression. 31 Inhibitors were applied together with the hormones where indicated at the following concentrations: 250 nmol/l wortmannin, 20 nmol/l GF109203x, 50 µmol/l PD After incubation for the times indicated in the figures, cells were washed twice in ice-cold phosphate-buffered saline (137 mmol/l NaCl, 2.68 mmol/l KCl, 3.2 mmol/l Na 2 HPO 4, 1.47 mmol/l KH 2 PO 4, ph 6.8), and the cells were scraped off for the preparation of total RNA. For the determination of camp levels the cells were washed twice with buffer A (20 mmol/l HEPES [ph 7.4], 120 mmol/l NaCl, 1.2 mmol/l MgSO 4, 1.2 mmol/l KH 2 PO 4, 2.5 mmol/l CaCl 2, 5 mmol/l glucose, and 2 mmol/l lactate) and further cultured for another 10-minute equilibration period. Then hormones and inhibitors were added simultaneously where indicated at the concentrations given above. After 3 minutes, the medium was sucked off, the cells frozen in liquid N 2 and processed further as described below. At this time-point camp levels were maximally increased by glucagon as was shown in previous studies. Thereafter camp decreased again and remained elevated at about half-maximal values for at least 30 minutes. IL-6 or insulin attenuated the maximal increase at 3 minutes. Thereafter levels declined again by one half and remained elevated at this level during the following 30 minutes. 12,32 Tools and Assays. Total RNA was prepared from the hepatocytes as described in detail elsewhere. 30,33 Fifteen micrograms of total RNA was separated on denaturing formaldehyde agarose gels. Equal lane-to-lane loading of RNA was determined by ethidium bromide staining of the gels before blotting onto nylon membranes according to standard protocols. 34 PCK mrna was hybridized to digoxygeninlabeled complementary RNA probes derived from the HindIIIlinearized plasmid pbs-pck by the use of T3-RNA polymerase. The plasmid contained a 1.2-kb rat PCK PstI complementary DNA fragment. 35 Digoxygenin-labeled hybrids were detected according to the manual of Roche-Diagnostics and quantitated by the use of a videodensitometer. For the determination of camp the frozen cells were disrupted in the cold in 1 ml of 10 mmol/l HCl containing in addition 100 µmol/l isobutylmethylxanthine to minimize camp degradation by phosphodiesterases. Denatured protein was pelleted by centrifugation and the supernatant was taken for the determination of camp by the use of a 125 I-cAMP radioimmunoassay according to the supplier s instructions (Amersham/Buchler). The results were calculated from the number of different cell cultures as indicated in the figure legends. They are expressed as means SEM. In case of camp determinations each culture was run in duplicate. Significance of differences between the means with IL-6 or insulin in the absence of inhibitors and in the presence of inhibitors was calculated by the use of Student s t test applying unpaired values. Significant inhibitor-caused attenuation of the inhibition by IL-6 or insulin was defined by the P values as indicated in the figure legends. RESULTS Attenuation of the Inhibition by IL-6 and Insulin of the Glucagon- Stimulated Increase in PCK mrna by the PI3-Kinase Inhibitor Wortmannin and the Protein Kinase C Inhibitor GF109203x But Not by the MAP-Kinase Inhibitor PD To investigate, whether PI3-kinase or protein kinase C contributed to the inhibition by IL-6 or insulin of glucagon-stimulated PCK gene expression, cultured rat hepatocytes were treated with either wortmannin (250 nmol/l) or GF109203x (20 nmol/l) along with 0.1 nmol/l glucagon and 100 ng/ml IL-6 or 10 nmol/l insulin. Glucagon transiently increased PCK mrna levels maximally 4.5-fold after 2 hours. IL-6 inhibited the increase by 70%. This inhibition was only 40% in the presence of wortmannin or GF109203x indicating that PI3-kinase and protein kinase C contributed to the inhibition of the increase in PCK mrna stimulated by glucagon (Fig. 1A). Insulin inhibited the stimulation by glucagon of the PCK mrna increase by 65%, which was prevented in the presence of wortmannin. GF109203x did not prevent the inhibition indicating that PI3-kinase but not protein kinase C contributed to the inhibition by insulin of the glucagon-stimulated increase in PCK mrna (Fig 1B). The 250 nmol/l concentration of wortmannin used in this study has been shown both in hepatocytes and in hepatoma cells to be saturating for the inhibition of the activation by insulin of PI3-kinase. 3,36-38 To underline this in the present study, a concentration dependence was performed. Cells were treated with 0.1 nmol/l glucagon for 2 hours in the absence or presence of 100 ng/ml IL-6 or 10 nmol/l insulin with or without increasing concentrations of wortmannin. A total of 50 nmol/l, 100 nmol/l, and 250 nmol/l wortmannin attenu-

3 HEPATOLOGY Vol. 31, No. 2, 2000 CHRIST, YAZICI, AND NATH 463 FIG. 1. Induction of PCK mrna levels by glucagon in cultured rat hepatocytes: inhibition by IL-6 (A) or by insulin (B) and attenuation of the inhibition by wortmannin and GF109203x. Cells were incubated for the times indicated with 0.1 nmol/l glucagon to induce PCK mrna. IL-6 or insulin (Ins) were added where indicated at a concentration of 100 ng/ml and 10 nmol/l, respectively. Wortmannin (Wom) at 250 nmol/l and GF109203x (GF) at 20 nmol/l were included where indicated. Total RNA was isolated from two pooled culture dishes and 15 µg subjected to electrophoretic separation for Northern blot analysis. Phosphoenolpyruvate carboxykinase mrna was detected with a digoxygenin-labeled crna hybridization probe and colored hybrids quantified by means of a videodensitometer. Lane-to-lane loading of total RNA was controlled by ethidium bromide staining before blotting. In each single experiment the highest value of PCK mrna was set equal to 100%, to which the other values are relative. Results are means SEM from 8-20 independent cell cultures in the (A) series and from 5-20 independent cell cultures in the (B) series. Statistics: Significant differences were calculated by the use of Student s t tests for unpaired values. In the presence of IL-6, values in the presence of wortmannin were significantly higher at the P.05 (*) level as compared with values in the absence of wortmannin. In the presence of IL-6, values in the presence of GF109203x were significantly higher at the P.05 (*) level as compared to values in the absence of GF109203x (A). In the presence of insulin, values in the presence of wortmannin were significantly higher at the P.05 (*) level and the P.01 (**) level as compared with values in the absence of wortmannin (B). ated the inhibition by IL-6 from 54% inhibition in the absence of wortmannin to 32%, 21%, and 33% inhibition, respectively (Fig. 2B), and the inhibition by insulin from 52% inhibition to 46%, 33%, and 7% inhibition, respectively (Fig. 2C). A total of 250 nmol/l concentrations of wortmannin were saturating. Wortmannin did not affect the glucagonstimulated increase in PCK mrna in the absence of IL-6 or insulin (Fig. 2A). A concentration dependence of GF109203x was performed with hepatocytes treated with 0.1 nmol/l glucagon for 2 hours in the absence or presence of 100 ng/ml IL-6 or 10 nmol/l insulin. Ten to 40 nmol/l GF109203x prohibited the inhibition by IL-6 of the PCK mrna increase from 60% inhibition in the absence of the inhibitor to only about 35% inhibition (Fig. 3B), whereas the inhibition by insulin was not affected (Fig. 3C). GF109203x at 20 nmol/l was saturat- FIG. 2. Concentration dependence of the attenuation by wortmannin of the inhibition by IL-6 and insulin of the glucagon-stimulated PCK mrna increase in cultured rat hepatocytes. Cells were incubated for 2 hours with 0.1 nmol/l glucagon alone (A) or in the presence of 100 ng/ml IL-6 (B) or 10 nmol/l insulin (C) and with the wortmannin concentrations as indicated. For Northern blot analysis cells were then processed as described in the legend to Fig. 1. In each single experiment the highest value of PCK mrna was set equal to 100%, to which the other values are relative. Data are expressed as means SEM from 6 independent cell cultures.

4 464 CHRIST, YAZICI, AND NATH HEPATOLOGY February 2000 FIG. 3. Concentration dependence of the attenuation by GF109203x of the inhibition by IL-6 and insulin of the glucagon-stimulated PCK mrna increase in cultured rat hepatocytes. Cells were incubated for 2 hours with 0.1 nmol/l glucagon alone (A) or in the presence of 100 ng/ml IL-6 (B) or 10 nmol/l insulin (C) and with the GF109203x concentrations as indicated. For Northern blot analysis cells were processed as described in the legend to Fig. 1. In each single experiment the highest value of PCK mrna was set equal to 100%, to which the other values are relative. Data are expressed as means SEM from 10 independent cell cultures. ing. In the absence of IL-6 or insulin the glucagon-stimulated increase in PCK mrna was lowered by 20% at 40 nmol/l GF109203x (Fig. 3A). To investigate whether the inhibition by IL-6 or insulin of the glucagon-stimulated increase in PCK mrna involved the MAP-kinase pathway, hepatocytes were treated with 50 µmol/l of the MAP/extracellular signal regulated kinase inhibitor PD98059, which prevents the activation of MAPkinase by the MAP/extracellular signal regulated kinase, for up to 6 hours in combination with 0.1 nmol/l glucagon and 100 ng/ml IL-6 or 10 nmol/l of insulin. Either in the absence or presence of PD98059, IL-6 or insulin inhibited the stimulation by glucagon of PCK mrna increase effectively, indicating that this inhibition did not involve the activation of MAP-kinase (data not shown). Prevention of the Inhibition by IL-6 of the Glucagon-Stimulated Increase in PCK mrna by the Combination of the PI3-Kinase Inhibitor Wortmannin and of the Protein Kinase C Inhibitor GF109203x. To show a potential additive effect of wortmannin and GF109203x in IL-6 inhibition of PCK mrna increase, cultured rat hepatocytes were stimulated with 0.1 nmol/l glucagon, and IL-6 at 100 ng/ml was added together with 250 nmol/l wortmannin and 20 nmol/l GF109203x. Although IL-6 inhibited the stimulation by glucagon of the increase in PCK mrna levels by 70% in the absence of the inhibitors, the stimulation by glucagon was totally restored in the presence of both inhibitors. Thus, PI3-kinase and protein kinase C contributed synergistically to the inhibition by IL-6 of the stimulation by glucagon of PCK mrna increase (Fig. 4). Missing Effect of Kinase Inhibitors on the Inhibition by IL-6 and by Insulin of Glucagon-Stimulated Increase in camp. To show whether or not the kinases investigated in this study were involved in the inhibition by IL-6 and insulin of glucagon-stimulated increase in camp, hepatocytes were treated for 3 minutes with 0.1 nmol/l glucagon and 100 ng/ml IL-6 or 10 nmol/l insulin in the absence or presence of wortmannin (250 mmol/l), GF109203x (20 nmol/l), and PD98059 (50 µmol/ L), respectively, and camp levels were determined by means of a radioimmunoassay. Glucagon increased camp levels by 2.7-fold from pmol/dish unstimulated values to FIG. 4. Induction of PCK mrna levels by glucagon in cultured rat hepatocytes; inhibition by IL-6 and prevention of the inhibition by the combination of wortmannin and GF109203x. Cells were incubated for the times indicated with 0.1 nmol/l glucagon to induce PCK mrna. IL-6 was added where indicated at a concentration of 100 ng/ml. Wortmannin (Wom) at 250 nmol/l and GF109203x (GF) at 20 nmol/l were included where indicated. For Northern blot analysis cells were then processed as described in the legend to Fig. 1. In each single experiment the highest value of PCK mrna was set equal to 100%, to which the other values are relative. Results are means SEM from 5-20 independent cell cultures. Statistics: Significant differences were calculated by the use of Student s t tests for unpaired values. In the presence of IL-6, values in the presence of wortmannin and GF109203x were significantly higher at the P.01 (*) level as compared with values in the absence of wortmannin and GF109203x.

5 HEPATOLOGY Vol. 31, No. 2, 2000 CHRIST, YAZICI, AND NATH 465 FIG. 5. Stimulation of camp increase by glucagon in cultured rat hepatocytes: inhibition by IL-6 (A) or insulin (B). Cells were incubated for 3 minutes with 0.1 nmol/l glucagon in the absence or presence of 100 ng/ml IL-6 (A) or 10 nmol/l insulin (B). Where indicated inhibitors were included at the following concentrations: 250 nmol/l wortmannin, 20 nmol/l GF109203x, 50 µmol/l PD Cells were quickly frozen and camp determined radioimmunologically in the homogenates after separation from cell debris. camp levels are expressed as increase over nonstimulated controls, which were in the range of pmol/dish. Results are expressed as means SEM from 2-11 independent cell cultures each performed in duplicate pmol/dish. IL-6 or insulin inhibited this increase by 58% and 45%, respectively. Neither of the kinase inhibitors prevented this inhibition indicating that PI3-kinase, protein kinase C, and MAP-kinase were not involved in the inhibition by IL-6 or insulin of the stimulation of camp increase. Also the combination of wortmannin and GF109203x was ineffective (Fig. 5). Attenuation of the Inhibition by IL-6 and by Insulin of the CPT-cAMP Stimulated Increase in PCK mrna by the PI3-Kinase Inhibitor Wortmannin and the Protein Kinase C Inhibitor GF109203x. Because the kinase inhibitors used in the present study did not prevent the inhibition by IL-6 or insulin of the increase in camp (Fig. 5), it should be investigated whether the restoration by wortmannin and GF109203x of the stimulation of FIG. 6. Induction of PCK mrna levels by CPT-cAMP in cultured rat hepatocytes: inhibition by IL-6 (A) or by insulin (B) and attenuation of the inhibition by wortmannin and GF109203x. Cells were incubated for the times indicated with 100 nmol/l CPT-cAMP to induce PCK mrna. IL-6 or insulin (Ins) were added where indicated at a concentration of 100 ng/ml and 10 nmol/l, respectively. Wortmannin (Wom) at 250 nmol/l and GF109203x (GF) at 20 nmol/l were included where indicated. For Northern blot analysis, cells were processed as described in the legend to Fig. 1. In each single experiment the highest value of PCK mrna was set equal to 100%, to which the other values are relative. Results are means SEM from 5-17 independent cell cultures in the (A) series and from 5-16 independent cell cultures in the (B) series. Statistics: Significant differences have been calculated by the use of Student s t tests for unpaired values. In the presence of IL-6, values in the presence of wortmannin were significantly higher at the P.05 (*) level as compared with values in the absence of wortmannin. In the presence of IL-6, values in the presence of GF109203x were significantly higher at the P.05 (*) level as compared with values in the absence of GF109203x (A). In the presence of insulin, values in the presence of wortmannin were significantly higher at the P.01 (**) level as compared with values in the absence of wortmannin. In the presence of insulin, values in the presence of GF109203x were significantly higher at the P.01 (**) level as compared with values in the absence of GF109203x (B).

6 466 CHRIST, YAZICI, AND NATH HEPATOLOGY February 2000 PCK mrna increase was independent of changes in camp levels. Cultured rat hepatocytes were treated with 100 nmol/l of CPT-cAMP to stimulate the increase in PCK mrna levels. A total of 100 ng/ml IL-6 or 10 nmol/l insulin were added along with CPT-cAMP in the absence or presence of 250 nmol/l wortmannin or 20 nmol/l GF109203x. IL-6 inhibited the increase in PCK mrna levels stimulated with CPT-cAMP by 75%. This inhibition was only about 45% to 55% in the presence of wortmannin and GF109203x (Fig. 6A). Insulin inhibited the increase in PCK mrna stimulated by CPTcAMP by 80%. The inhibition was prohibited in the presence of wortmannin and attenuated by 60% in the presence of GF109203x (Fig. 6B). PD98059 did not affect the inhibition of PCK mrna increase by IL-6 or insulin (data not shown). The data indicated that the inhibition by IL-6 of the stimulation by CPT-cAMP of PCK mrna increase in part involved PI3-kinase and protein kinase C. The inhibition by insulin seemed to be efficiently mediated by PI3-kinase alone. Thus, the PI3-kinase and protein kinase C mediated inhibition by IL-6 or insulin of PCK gene activation was independent of the inhibition of camp increase. Prevention of the Inhibition by IL-6 of the CPT-cAMP Stimulated Increase in PCK mrna by the Combination of the PI3-Kinase Inhibitor Wortmannin and the Protein Kinase C Inhibitor GF109203x. Wortmannin and GF109203x synergistically prohibited the inhibition by IL-6 of the stimulation by glucagon of PCK mrna increase (Fig. 4). Therefore, it is feasible that the inhibition by IL-6 of the stimulation by CPT-cAMP of PCK mrna increase could also be overcome by the combined application of the two inhibitors. Cultured rat hepatocytes were treated with 100 nmol/l CPT-cAMP to induce PCK mrna. Although IL-6 inhibited the stimulation by CPTcAMP of the increase in PCK mrna levels by 75% in the absence of the inhibitors, the stimulation by CPT-cAMP was totally restored in the presence of both inhibitors (Fig. 7). This indicated that the inhibition by IL-6 of CPT-cAMP stimulated increase in PCK mrna levels required the synergistic action of PI3-kinase and protein kinase C independent of changes in camp levels. DISCUSSION Involvement of PI3-Kinase and Protein Kinase C in IL-6 Inhibition of Glucagon-Stimulated PCK Gene Expression in Cultured Rat Hepatocytes. Either the PI3-kinase inhibitor, wortmannin, or the protein kinase C inhibitor, GF109203x, attenuated the inhibition of glucagon-stimulated PCK gene expression by IL-6 only partially (Fig. 1A). In combination the inhibitors completely prohibited the inhibition (Fig. 4) indicating that the inhibition of PCK gene expression by IL-6 required the synergistic action of PI3-kinase and protein kinase C. Because the inhibitors did not affect the stimulation by glucagon of the increase in camp levels (Fig. 5), it is supposed that PI3-kinase and/or protein kinase C acted downstream from the formation of camp. This is underlined by the fact that wortmannin and GF109203x also prevented the inhibition by IL-6 of the stimulation of PCK gene expression by the nonhydrolyzable camp analogue CPT-cAMP (Figs. 6A and 7). PI3-kinase plays an important role in mediating the effects of cytokines and growth factors in a number of systems. IL-6 and IL-3 promoted association of gp130 to the tyrosine kinase Tec and PI3-kinase through JAK 25 and tyrosine phosphorylation of the adapter protein Gab1 in response to IL-6, IL-3, or interferon alfa/beta stimulated ERK2 activity through a PI3- FIG. 7. Induction of PCK mrna levels by CPT-cAMP in cultured rat hepatocytes: inhibition by IL-6 and prevention of the inhibition by the combination of wortmannin and GF109203x. Cells were incubated for the times indicated with 100 nmol/l CPT-cAMP to induce PCK mrna. IL-6 was added at a concentration of 100 ng/ml. Wortmannin (Wom) at 250 nmol/l and GF109203x (GF) at 20 nmol/l were included where indicated. For Northern blot analysis cells were then processed as described in the legend to Fig. 1. In each single experiment the highest value of PCK mrna was set equal to 100%, to which the other values are relative. Results are means SEM from 5-17 independent cell cultures. Statistics: Significant differences were calculated by the use of Student s t tests for unpaired values. In the presence of IL-6, values in the presence of wortmannin and GF109203x were significantly higher at the P.01 (*) level as compared with values in the absence of wortmannin and GF109203x. kinase dependent mechanism. 27 The IFNAR1 chain of the type I interferon receptor bound the p85 regulatory subunit of PI3-kinase through the receptor-associated transcription factor STAT3. 39 The cytokine-dependent activation of STAT transcription factors requires in addition to the phosphorylation on tyrosine residues the phosphorylation by an as yet unknown serine/threonine protein kinase In the present study the specific protein kinase C inhibitor, GF109203x, has been used. Therefore, it can be argued that the inhibition by IL-6 of glucagon-stimulated PCK gene expression required in part protein kinase C. Yet the mechanism by which IL-6 through PI3-kinase and protein kinase C inhibited the glucagonstimulated expression of the PCK gene remains to be investigated. Involvement of PI3-Kinase in the Insulin Inhibition of Glucagon- Stimulated PCK Gene Expression in Cultured Rat Hepatocytes. The PI3-kinase inhibitor, wortmannin, totally prevented the inhibition by insulin of glucagon-stimulated PCK gene expression indicating the involvement of PI3-kinase (Fig. 1B). Because wortmannin did not reverse the inhibition of the increase in camp (Fig. 5), PI3-kinase mediated insulin inhibition of PCK gene expression at a site downstream. This

7 HEPATOLOGY Vol. 31, No. 2, 2000 CHRIST, YAZICI, AND NATH 467 was corroborated by the finding that also the inhibition by insulin of the stimulation of PCK gene expression by CPTcAMP was sensitive to wortmannin (Fig. 6B). The expression of the PCK gene was inhibited by insulin both in primary cultured rat hepatocytes and in rat hepatoma cells on the transcriptional level. 2,30 From studies in rat hepatoma cells it is evident that the expression of the PCK gene stimulated by CPT-cAMP was inhibited by insulin through the PI3-kinase pathway. 3-5 The present data show that this is also operative in primary cultured rat hepatocytes. However, in either case it remains unclear which is the downstream target of insulin finally mediating the transcriptional inactivation of the PCK gene promoter. Pathophysiological Significance. Patients suffering from trauma or sepsis after surgery exhibit increased catecholamine as well as glucagon serum levels concomitantly with insulin resistance, which favors the use of body glucose stores One major complication in the pathogenesis of the septic shock is the emergence of hypoglycemia during prolonged sepsis. 47 This has been attributed to the lowered capacity of the liver to supply glucose because of the inhibition of gluconeogenesis as a consequence of the inhibition of the expression of control enzymes of gluconeogenesis and because of the exhaustion of hepatic glycogen pools by proinflammatory cytokines From the presented results it is concluded that IL-6 and insulin share in part common signal transduction components. This may impair insulin signaling ultimately leading to insulin resistance. 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