Tài liệu Báo cáo khoa học: The diacylglycerol and protein kinase C pathways are not involved in insulin signalling in primary rat hepatocytes doc

Eur J Biochem 270, 4635–4646 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03853.x The diacylglycerol and protein kinase C pathways are not involved in insulin signalling in primary rat hepatocytes Irmelin Probst1, Ulrich Beuers2, Birgit Drabent1, Kirsten Unthan-Fechner1 and Peter Butikofer3 ă Institut fuăr Biochemie und Molekulare Zellbiologie, Georg-August Universitaăt Goăttingen, Germany; 2Medizinische Klinik IIGroòhadern, Ludwig-Maximilians-Universitaăt Muănchen, Germany; 3Institut fuăr Biochemie und Molekularbiologie, Universitaăt Bern, Switzerland Diacylglycerol (DAG) and protein kinase C (PKC) isoforms have been implicated in insulin signalling in muscle and fat cells We evaluated the involvement of DAG and PKC in the action of insulin in adult rat hepatocytes cultured with dexamethasone, but in the absence of serum, for 48 h Our results show that although insulin stimulated glycolysis and glycogen synthesis, it had no effect on DAG mass or molecular species composition Epidermal growth factor showed the expected insulin-mimetic effect on glycolysis, whereas ATP and exogenous phospholipase C acted as antagonists and abolished the insulin signal Similarly to insulin, epidermal growth factor had no effect on DAG mass or molecular species composition In contrast, both ATP and phospholipase C induced a prominent increase in several DAG molecular species, including 18:0/20:4, 18:0/20:5, 18:0/22:5 and a decrease in 18:1/18:1 These changes were paralleled by an increase in phospholipase D activity, which was absent in insulin-treated cells By immunoblotting or by measuring PKC activity, we found that neither insulin nor ATP translocated the PKCa, -d, -e or -f isoforms from the cytosol to the membrane in cells cultured for six or 48 h Similarly, insulin had no effect on immunoprecipitable PKCf Suppression of the glycogenic insulin signal by phorbol 12-myristate 13-acetate, but not by ATP, could be completely alleviated by bisindolylmaleimide Finally, insulin showed no effect on DAG mass or translocation of PKC isoforms in the perfused liver, although it reduced the glucagon-stimulated glucose output by 75% Together these results indicate that phospholipases C and D or multiple PKC isoforms are not involved in the hepatic insulin signal chain Among the three major insulin-sensitive organs, i.e liver, muscle and fat tissue, the liver plays a key role in the regulation of blood glucose homeostasis by channelling excess glucose into glycogen after food uptake and by producing glucose through glycogenolysis and gluconeogenesis in the states of hunger and starvation Insulin, the dominant hormone of the absorptive phase, acts via receptor-mediated tyrosine phosphorylation of insulin receptor substrates (IRSs) Two well established signalling cascades are initiated when adaptor proteins are recruited to the IRSs through their src homology domains (a) the growth factor receptor binding protein activates the ras/ mitogen-activated protein kinase pathway and (b) phosphatidylinositol 3-kinase activates the protein kinase B/glycogen synthase kinase-3 cascade Recent data suggest that a third signalling pathway, downstream of phosphatidylinositol 3-kinase, may also be involved: phospholipase D (PLD)-dependent generation of phosphatidic acid (PA) and diacylglycerol (DAG), with subsequent activation of DAG-insensitive atypical protein kinase C (PKC) isozymes such as f and k, as well as activation of DAG-sensitive PKC isozymes [1–3] These studies, which were performed on muscle and fat cells, showed insulindependent increases in lipid mediator concentrations [4–7] and translocation and activation of various PKC isoforms [6–13], suggesting their probable involvement in insulin action [8,10,14,15] In contrast, the available data on hepatic systems are scarce, controversial and have been obtained using primary adult rat hepatocyte suspensions and cultures, and different hepatoma cell lines, as model systems In hepatocyte suspensions, insulin provoked increases in DAG mass [16,17], whereas activation of PLD was demonstrated by two groups [17,18], but not by another group [19] Similarly, activation of PKC was demonstrated, in two reports, in both cytosolic and membrane fractions of crude extracts [16,20], but not in a third [21] Furthermore, activation of atypical PKCf was demonstrated in hepatocytes cultured without glucocorticoid for days [22], whereas two other reports showed enhanced translocation of the d isoform in different hepatoma cell lines [23,24] Correspondence to I Probst, Institut fur Biochemie und Molekulare ă Zellbiologie, Humboldtallee 23, 37073 Gottingen, Germany ă Fax: + 49 551 395960, Tel.: + 49 551 395961, E-mail: iprobst@gwdg.de Abbreviations: DAG, diacylglycerol; EGF, epidermal growth factor; IRSs, insulin receptor substrates; ODN, oligodesoxynucleotides; PA, phosphatidic acid; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; TGF-a, transforming growth factor-a Enzymes: phospholipase C (EC 3.1.4.3); phospholipase D (EC 3.1.4.4); protein kinase C (EC 2.7.1.37) (Received 25 April 2003, revised 26 August 2003, accepted 25 September 2003) Keywords: hepatocytes; insulin; ATP; diacylglycerol molecular species; protein kinase C Ó FEBS 2003 4636 I Probst et al (Eur J Biochem 270) The aim of the present work was to study the possible involvement of lipid signalling and PKC during hepatic insulin action in a differentiated model for the adult organ, the primary adult rat hepatocyte cultured serum-free with dexamethasone This system shows high insulin sensitivity and responsiveness towards a multitude of insulin-dependent parameters [25–27] The effects of insulin were compared with those of epidermal growth factor (EGF), ATP and exogenous phospholipase C (PLC) lactate (2 mL per dish) For the determination of glycogen synthesis and glycolysis, the medium was supplemented with [14C]glucose (30 kBq per dish) After a 30-min preincubation, zero-time samples were taken and the experiment was started by the addition of agonists to the dishes Inhibitors were added 10 before the agonists The incubation was terminated by rapidly aspirating the medium and immersing the dishes in liquid N2 Glycolysis and glycogen synthesis Materials and methods Materials Enzymes, M199 medium, collagenase A and the transfection agent DOSPER were from Roche Molecular Biochemicals (Mannheim, Germany) Bovine insulin was from Serva (Heidelberg, Germany) Bisindolylmaleimide I and protein G–agarose were from Calbiochem (Bad Soden, Germany) Phorbol 12-myristate 13-acetate (PMA), rottlerin, PLC from Clostridium perfringens, IGEPAL and dexamethasone were from Sigma (Taufkirchen, Germany) A stock solution of PMA (10 mM) was made in dimethylsulfoxide; before use it was diluted : 100 in M199 medium containing 0.2% (w/v) bovine serum albumin D-[U-14C]Glucose, [32P]ATP[cP], [9,10-3H]myristic acid, [9,10-3H]palmitic acid and the Renaissance Western blot chemiluminescence reagent were from New England Nuclear (Dreieich, Germany) The DAG quantification test kit and the PKC enzyme assay system were purchased from Amersham (Braunschweig, Germany) The PKCf isoenzyme-specific pseudosubstrate [Ser159]PKC-e-[153–164]-NH2 was from Bachem (Heidelberg, Germany) Silica gel 60 TLC plates with concentration zones were from Merck (Hannover, Germany) Whatman P-81 paper was from Herolab (Wiesloch, Germany) Rabbit anti-PKC peptide Igs, anti-a, -b, -c, -d, -e, and -f for immunoblotting were obtained from Gibco (Grand Island, NY, USA) Rabbit anti-PKCf for immunoprecipitation and activity determination, and the antiPKCf blocking peptide, were from Santa Cruz (Heidelberg, Germany) PKCf antisense oligodesoxynucleotides were from Biognostik (Goettingen, Germany) and cytofectin from Eurogentec/Glen Research (Koln, Germany) ă Cell culture Hepatocytes from fed male Wistar rats (of weight 180–250 g) were isolated by recirculating collagenase perfusion in situ, purified by centrifugation through Percoll and cultured in M199 medium on 6-cm plastic dishes [28] For the first h, medium contained 4% newborn calf serum, nM insulin and 0.1 lM dexamethasone Serum was then omitted and the cells were cultured for the next or 43 h with nM insulin and 0.1 lM dexamethasone Medium was changed at 22 h The gas atmosphere contained CO2/O2/N2 (5 : 17 : 78) Cell experiments After or 46 h of continuous culture, dishes were washed twice and incubated in M199 (2.5 mL per dish) After h the medium was replaced with M199 containing mM Glycolysis was determined by the rate of lactate release into the culture supernatant Labelled glucose was separated from labelled lactate by chromatography of 100 lL of culture supernatant on Dowex · (formate form), as outlined previously [25] The rate of glycogen synthesis was determined by extracting and quantifying the 14 C-labelled glycogen from one cm dish, as described previously [27] Liver perfusion Rat livers were perfused in situ, via the portal vein, with Krebs-Henseleit bicarbonate buffer, pH 7.4 (5 mM glucose, mM lactate, 0.2 mM pyruvate; 95% O2/5% CO2; 37 °C; constant flow without recirculation, 5.5–6 mLỈmin)1Ỉg)1 of liver) Experiments were performed between 09.00 h and 11.00 h; preperfusion lasted for 20 before the onset of sampling from the inferior vena cava Liver samples were taken from the front lobe at 35 Lipid extraction and separation by TLC Hepatocytes from one 6-cm dish were scraped into mL of methanol and transferred into a glass tube Chloroform (1 mL) was added and lipids were extracted for 10 at °C Subsequently, mL of chloroform and 1.7 mL of M NaCl were added under vigorous mixing After min, samples were centrifuged (400 g for min), the aqueous phase was discarded and the organic phase dried under N2 at room temperature Lipids were concentrated in the V-shaped tip of the tube by repetitive solvent evaporation and resuspension using diminishing volumes of chloroform Dried lipids were stored under N2 at )20 °C For separation of lipids by TLC, extracts were redissolved in 50 lL of chloroform/methanol (2 : 1, v/v) and applied in a 1-cm zone on a 20 · 20 cm2 silica gel plate with concentration zone The solvent system used for separation of DAG was heptan/diisopropylether/acetic acid (60 : 40 : 8, v/v/v) Alkylacyl and alk-1-enylacyl subclasses co-migrate on this TLC system with the DAG species For the determination of PLD activity in hepatocytes, 0.3% (v/v) butanol was added to cells incubated in the presence or absence of insulin or ATP After lipid extraction, phosphatidylbutanol (formed by PLD-mediated phosphatidyltransfer onto butanol) was separated by TLC using ethyl acetate/isooctane/acetic acid/H2O (130 : 20 : 30 : 100, v/v/ v/v) as the solvent system [17] Phosphatidylbutanol was quantified by a procedure that chars saturated and unsaturated lipids equally [29] followed by densitometry using authentic phosphatidylbutanol, prepared as described previously [30], as a standard Ó FEBS 2003 Insulin signalling in rat hepatocytes (Eur J Biochem 270) 4637 Determination of DAG mass DAG content was quantified radioenzymatically by incubating aliquots of the lipid extract with DAG kinase and [32P]ATP[cP], as described by Preiss et al [31] The manufacturer’s instructions for the commercially available DAG test kit were followed 32P-labelled PA was purified using chloroform/methanol/acetic acid (65 : 15 : 5, v/v/v) as a solvent system and quantified with a Storm 860 phosphoimager (Pharmacia, Freiburg, Germany) Analysis of DAG molecular species Hepatocytes from one 10-cm dish were extracted as outlined above After drying the lipid extract under nitrogen, DAGs were extracted with ether and immediately benzoylated, as described by Blank et al [32] Diradylglycerobenzoates were separated into their subclasses (diacyl, alkylacyl, and alk-1enylacyl types) by TLC using benzene/hexane/ether (50 : 45 : 4, v/v/v) as a solvent system, and the individual molecular species were separated by HPLC using an octadecyl reverse-phase column in acetonitrile/isopropanol (80 : 20, v/v) as the mobile phase Individual peaks were quantified by measuring absorbance at 230 nm To identify individual molecular species, representative samples were analysed by combined HPLC/MS [33] using the instrumentation described in Butikofer et al [34] Briey, after the UV ă detector, methanol/0.2 M aqueous ammonium acetate (10 : 90; v/v) was added via a T-connector, and the total flow was introduced through a thermospray interface into a Finnigan MAT model TSQ70 mass spectrometer The M + NH4+ ions of the diradylglycerobezoates were monitored by selected ion recording The positional distribution of the fatty acyl and fatty alcohol chains of individual molecular species was not determined The inclusion of the antioxidant, butylated hydroxytoluene, in the different solvents was found not to be necessary PKC activity in cytosol and membranes of crude extracts Hepatocytes from two 6-cm dishes were homogenized in 500 lL of lysis buffer (20 mM Hepes, pH 7.5, 250 mM sucrose, mM EGTA, mM sodium vanadate, mM sodium pyrophosphate, mM NaF, 20 lgỈmL)1 leupeptin, 20 lgỈmL)1 aprotinin, mM phenylmethanesulfonyl fluoride, 20 mM 2-mercaptoethanol) and centrifuged at 100 000 g for 30 Supernatant and membrane fraction were diluted with lysis buffer (without sucrose and EGTA) and 8–14 lg of protein from each fraction was assayed for the ability to phosphorylate a synthetic EGF-receptor peptide (RKRTLRRL) The Amersham assay contained 25 lL of sample, 25 mM Tris/HCl, pH 7.5, 34 lgỈmL)1 phosphatidylserine, 2.7 lgỈmL)1 PMA, 102 lM receptor peptide, 3.4 mM dithiothreitol, 1.36 mM calcium acetate, 109 lM ATP, and 6.5 mM MgCl2 in a total volume of 55 lL For the specific measurement of the atypical PKC isoenzyme-f, Ca2+ was omitted from the assay, 80 lgỈmL)1 phosphatidylserine was substituted for the kit lipid reagent and the kit peptide substrate was replaced by 50 lM [Ser159]PKC-e-[153–164]-NH2 Assays were conducted in the presence or absence of the substrate for 3–9 at 30 °C and stopped with 0.3 M phosphoric acid Aliquots were spotted on P-81 filter paper, washed three times with 75 mM phosphoric acid and counted Immunoblotting of PKC isoforms Hepatocyte preparation and immunoblotting were performed exactly as described previously [35] Samples from the perfused liver (200 mg) were homogenized in mL of lysis buffer by sonication (5 · 10 s) and centrifuged at 8000 g for min; the supernatant was processed as outlined previously [35] The bands of PKC isoforms were identified by (a) comparison with molecular mass markers run on each gel, (b) comparison with the bands of a rat brain cytosol sample rich in all relevant PKC isoforms run on each gel, (c) PMA-induced PKC translocation from the cytosol to the membrane fraction (except for the nonmobile f-isoform; samples of control and PMA-treated cells were run on each gel for comparison), and (d) comparison of bands after incubation of a membrane blot with buffer in the presence or absence of an antigen (PKC isoform) of the respective PKC antibody The bands on the immunoblots at about 80 000 molecular mass, representing PKC isoforms a, d and f, and at 90 000 molecular mass, representing PKC isoform e, were quantified by densitometry Immunoprecipitation and activity assay of PKCf Hepatocytes from one cm dish were homogenized in 500 lL of PKC lysis buffer (see above) supplemented with 0.5% IGEPAL (Nonidet P-40) and 1% Triton X-100 The lysate was sonicated for 10 s and centrifuged for 20 at 20 000 g after 30 of incubation at °C Supernatants (200 ll, mg of protein) were incubated under mild agitation for h at °C with lg of anti-PKCf, which had been coupled to protein G–agarose (30 lL of agarose in NaCl/Pi, h, °C) Immobilized immune complexes were recovered by centrifugation, washed three times with complete lysis buffer and twice in kinase buffer (50 mM Tris pH 7.5, 10 mM MgCl2, mM sodium vanadate, mM dithiothreitol, 10 lgỈmL)1 leupeptin, 10 lgỈmL)1 aprotinin, 0.2 mM phenylmethanesulfonyl fluoride) Kinase buffer (25 lL) was added to the beads and the enzyme was assayed in a total volume of 50 lL containing 80 lgỈmL)1 phosphatidylserine, 50 lM [32P]ATP[cP] (15 kBq per assay) and 50 lM PKC e-peptide Enzyme activity showed time linearity for at least 15 Assays were conducted for 10 and processed, as described for PKC, in crude extracts Immunoblot analysis showed that insulin treatment of the cells did not alter the amount of PKCf in the immunoprecipitate Results Hepatocytes used in the present study were routinely cultured serum-free in the presence of 0.1 lM dexamethasone for 46 h In each subsequent short-term experiment, measurement of lipid mediators or PKC was always paralleled by the determination of the physiological action of insulin on glucose metabolism ATP and exogenous PLC, which both stimulate DAG formation [36–38], were used as positive controls In addition, EGF, an insulin-mimetic as 4638 I Probst et al (Eur J Biochem 270) well as an insulin-antagonistic factor [39–41], was included in some experiments Metabolic effects We found that the addition of insulin to our primary rat hepatocyte cultures stimulated glycolysis 4.5-fold, with a 50% effective dose (ED50) of  0.3 nM, whereas EGF increased glycolysis twofold, with an ED50 of  0.5 ngỈmL)1 (Fig 1B) Similar results have been reported before for Ó FEBS 2003 other hepatocyte culture systems [27,41] Furthermore, transforming growth factor-a (TGF-a) completely mimicked EGF action in the lower concentration range (0.1–3 ngỈmL)1); however, it elicited an extrastimulatory response (+30%) at higher concentrations (Fig 1B) In contrast to its known inhibitory action on glycogen synthase [36], PLC was insulin-mimetic at low concentrations (0.1–3 mmL)1) and stimulated glycolysis by up to 3.5-fold (Fig 1A) However, at concentrations of > mmL)1, the effect of PLC diminished with increasing concentrations At these higher concentrations, PLC strongly antagonized the action of insulin As reported previously [42], ATP (> 10 lM) inhibited basal and insulinactivated glycolysis (results not shown) Furthermore, we observed that the addition of insulin stimulated glycogen synthesis ninefold, whereas ATP, PLC and EGF/TGF-a severely inhibited both basal and insulinactivated rates of glycogen synthesis (Fig 2) These findings are in good agreement with previous reports using other hepatocyte culture systems [28,37,40,43] Unexpectedly, however, EGF and TGF-a were found to be insulinmimetic at a low concentration (1 ngỈmL)1) (Fig 2) In some experiments, cells were cultured for only h; these cells were less insulin-responsive (as shown by a threefold increase of glycogenesis) Determination of DAG mass and DAG molecular species Fig Insulin-mimetic effects of epidermal growth factor (EGF), transforming growth factor a (TGF-a) and phospholipase C (PLC) on glycolysis Hepatocytes were cultured for 46 h in the presence of nM insulin and 0.1 lM dexamethasone Subsequently, they were washed free of hormones and incubated for 30 in M199 medium containing 0.1 lM dexamethasone and mM lactate before the agonists were added [14C]Lactate production from mM [14C]glucose was measured for h Data represent mean values ± SD from three different hepatocyte preparations Increases in DAG and PA, through insulin-dependent activation of PLC and PLD, have been reported previously for rat hepatocytes [16–18] In contrast to these studies, we found no increase in DAG mass when the cells were stimulated with 1–100 nM insulin, either in 6-h cultures (data not shown) or in 48 h cultures (Fig 3) Similarly, the addition of 10 ngỈmL)1 EGF or 10 ngỈmL)1 TGF-a also showed no effect As shown previously [36,37], ATP and PLC are capable of rapidly elevating the level of DAG In agreement with these reports, we found that the addition of 100 lM ATP doubled DAG mass within min; interestingly, the presence of PLC increased DAG mass at both insulin-mimetic (5 mmL)1) and insulin-antagonistic (100 mmL)1) concentrations (Fig 3) It has been previously shown that the addition of tritiumlabelled fatty acids to hepatocytes results in the incorporation of label into the phospholipid fraction [17,18]; subsequent addition of insulin led to an increase in the production of [3H]DAG and [3H]PA We investigated such a possible mechanism by labelling cells from 24 h to 46 h of culture with 110 kBqỈmL)1 of [3H]myristate or [3H]palmitate and determined the amount of radioactivity recovered in the DAG fraction Again, our results showed no differences between cells incubated in the presence or absence of insulin (results not shown) To study whether the observed increase in DAG mass after stimulation of rat hepatocytes with ATP or PLC was specific for certain molecular species, diacyl, alkylacyl and alk-1-enylacyl subclasses were separated and their molecular species composition was determined by combined HPLC/ MS The results in Table show a typical molecular species composition of the diacylglycerol subclass from untreated hepatocytes; the corresponding HPLC trace is shown in Fig 4A Control and agonist-stimulated hepatocytes Ó FEBS 2003 Insulin signalling in rat hepatocytes (Eur J Biochem 270) 4639 Fig Modulation of basal and insulin-stimulated glycogen synthesis by epidermal growth factor (EGF), transforming growth factor a (TGF-a), ATP and phospholipase C (PLC) Hepatocytes were cultured as described in the legend to Fig Incorporation of [14C]glucose into glycogen was measured for h Data represent mean values ± SD from four to seven different hepatocyte preparations Fig Total cellular diacylglycerol (DAG) mass of hepatocytes after treatment with ATP, phospholipase C (PLC), insulin and epidermal growth factor (EGF)/transforming growth factor a (TGF-a) Hepatocytes were cultured as described in the legend to Fig The DAG content was quantified in lipid extracts using the DAG kinase assay Data represent mean values ± SD from three to five different hepatocyte preparations contained almost exclusively diacyl-type molecular species (> 98% of total species) The HPLC profile, and thus the composition of DAG species, was not altered when cultures were treated with insulin (100 nM), EGF (10 ngỈmL)1) or TGF-a (10 ngỈmL)1), for various periods of time (0.5–60 min) at different cell densities (results not shown) These results are entirely consistent with our observation that insulin, EGF and TGF-a have no effect on DAG levels in primary rat hepatocytes In contrast, 100 lM ATP and 100 mmL)1 PLC showed a dramatic change in the HPLC profile (Fig 4B) Relative increases were seen for peak 11 (18:0/20:5), peak 17 (18:0/22:5) and peak 18 (18:0/20:4), whereas peak 20 (18:1/ 18:1) was reduced (Fig 5) The most prominent effect was a 3.9-fold enrichment of the species 18:0/20:4 (peak 18), which was observed for both agonists Our results clearly contrast those of Baldini and coworkers who showed an insulin-dependent increase in DAG and PA in hepatocytes [17,18] However, their studies were carried out either with hepatocyte suspensions or with cells cultured for 24 h in the absence of dexamethasone and insulin, but in the presence of 10% (v/v) fetal bovine serum We therefore investigated the effect of insulin on DAG molecular species composition using their culture conditions In agreement with their results, we found that in cells cultured for 24 h, insulin provoked the elevation of two molecular species of DAG (18:0/20:4 and 18:0/20:5), while one species was decreased (18:1/18:1) Thus, insulin indeed mimicked the effects of ATP and PLC although the changes were smaller, i.e 30–50% of the ATP responses (results not shown) However, when we studied the metabolic insulin responsiveness of the cells cultured under these steroid-free conditions, we found that the activation of glycogen Ó FEBS 2003 4640 I Probst et al (Eur J Biochem 270) Table Diacylglycerol molecular species composition of rat hepatocytes cultured for 48 h Diacylglycerols were analysed as diacylglycerobenzoate derivates by combined HPLC/MS Individual molecular species are listed in order of their elution from the HPLC column (Fig 4A) Values are mean ± SD of three determinations from a typical experiment Peak no 1+2 6+7 8+9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 a Molecular species 16:1, 20:4 18:2, 20:4 + 16:0, 20:5a 16:2, 18:2 18:2, 18:2 + 18:1, 18:3a + 16:1, 20:3a 16:1, 18:2 + 16:0, 22:6a + 14:0, 14:0a 16:1, 16:1 + 14:0, 18:2a + 14:0, 16:1a 16:1, 22:4 + 18:0, 20:5a 16:0, 20:4 18:1, 18:2 + 18:0, 22:6a + 16:1, 18:1a 16:0, 18:2 + 18:0, 18:3a 16:0, 16:1 18:0, 22:5 18:0, 20:4 17:0, 18:2 + 16:1, 17:0a 18:1, 18:1 16:0, 18:1 + 18:0, 18:2a 16:0, 16:0 + 18:0, 22:4a 18:0, 18:1 16:0, 18:0 18:0, 18:0 Composition (%) 3.2 ± 0.1 1.2 ± 0.1 4.4 ± 0.4 5.1 ± 0.2 1.7 ± 0.2 enhanced in the presence of ATP (positive control, data not shown) Translocation of PKC Rat hepatocytes in culture expressed PKC isoforms a, d, e and f The a-isoform was mainly associated with the cytosolic fraction, and the d-, e- and f-isoforms were approximately equally distributed between the cytosol and membrane fraction (Table 2) In control experiments, the conventional cPKCa and the novel nPKCs d and e, but not the atypical aPKCf, were translocated to the membrane fraction by the phorbol ester, PMA (Table 2) These results are in good agreement with a previous study [35] Neither insulin nor ATP were able to translocate any of the isoforms within 1–15 after agonist addition (Table 2) 2.6 ± 0.3 Measurement of PKC activity 4.4 ± 0.2 2.4 ± 0.2 3.8 ± 0.2 12.5 ± 0.3 8.8 ± 0.5 0.7 ± < 0.5 4.1 ± 4.3 ± 3.6 ± 0.1 0.6 0.9 0.7 13.6 ± 0.8 14.3 ± 0.4 1.4 ± 0.1 < 0.5 < 0.5 2.5 ± 0.01 3.8 ± 0.3 1.0 ± 0.4 These species co-elute from the HPLC column synthesis was severely reduced by 90% compared to cells cultured with dexamethasone (Fig 2) Measurement of PLD activity A possible involvement of PLD in insulin signalling was investigated in cells using our serum-free culture conditions, in the presence of dexamethasone, by determining transphosphatidylation activity with 0.3% butanol as the acceptor [17] We found that cell exposure to insulin in the presence of butanol did not increase the formation of phosphatidylbutanol As reported previously [19], transphosphatidylation was, however, five- to 10-fold In a first series of experiments, PKC activity was determined as overall activity in cytosol and membranes using the EGFreceptor peptide as a non isoform-specific substrate and the PKCe pseudosubstrate as a preferred substrate for PKCf Translocation of the PKC by PMA from the cytosol to the membrane was clearly demonstrated by the cytosolic decrease and membranous increase of enzyme activity (Table 3); in contrast, insulin showed no effect on PKC activity In a second series of experiments, PKCf was immunoprecipitated and its activity was determined in precipitates from cells treated with or without insulin for 1–15 We were unable to detect an insulin-dependent increase in the activity of the immunoprecipitated enzyme, which agrees with the inability of insulin to translocate PKCf Inhibitor studies Stimulation of glycogen synthesis by insulin could not be inhibited by the relatively selective PKC inhibitor bisindolylmaleimide I, which predominantly inhibits conventional and novel isoforms, i.e the a-, d- and e-isoforms (Fig 6) Owing to its isoform specificity, the inhibitor completely alleviated the insulin-antagonistic effect of PMA, which is mediated via DAG-dependent PKC isoforms In contrast, bisindolylmaleimide I was unable to revert the ATP-mediated blockade of the insulin signal (Fig 6) Selective inhibition of PKCd by the inhibitor rottlerin (5–10 lM) was also without effect on insulin signalling (data not shown) Finally, we tried to inhibit insulin signalling by transfecting hepatocytes with antisense oligodesoxynucleotides (ODN) targeted against PKCf We found that cells transfected with 2.5 lgỈmL)1 cytofectin and 0.125 nM fluorescent ODN, or with 2–10 lgỈmL)1 DOSPER and 0.5–2.5 lM fluorescent ODN, showed up to 80% fluorescent nuclei, and the amount of PKCf was reduced slightly (< 30%) after days of culture when PKCf antisense ODN was added It should be noted, however, that both cell vitality (measured by the release of lactate dehydrogenase) and insulin signalling (measured as glycogen synthesis) were significantly decreased by the transfection agents as well as by the control ODN alone (results not shown) Thus, Ó FEBS 2003 Insulin signalling in rat hepatocytes (Eur J Biochem 270) 4641 Fig HPLC profile of diacylglycerol (DAG) molecular species of control and ATP-stimulated hepatocytes Hepatocytes cultured for days were incubated for with M199 as vehicle (A) or 100 lM ATP (B), and the molecular species of DAG were analysed as described in the Materials and methods Data represent mean values ± SD of three determinations from a representative experiment of 10 although this method has been successfully applied to down-regulate specific PKC isoforms and to study PKC involvement in signal transduction in other cell systems previously [44], it seems to not (yet) be applicable to primary hepatocytes Insulin effects in the perfused liver The effects of insulin on DAG mass and PKC isoform translocation were examined in the intact organ to exclude the possibility that the data obtained with hepatocytes were restricted to the isolated cell system The anti-glucagon action of insulin was chosen to demonstrate the hormone’s metabolic activity The perfused liver received 50 pM glucagon for 5–10 min; this first bolus served as an internal metabolic vitality control From 30 to 35 min, the liver received no agonist (basal control), lM PMA (positive control for PKC translocation), a second bolus of 50 pM glucagon or a staggered infusion of 10 nM insulin (25–35 min) and 50 pM glucagon (Fig 7B,C) The antiglucagon effect of insulin was demonstrated by a 75% reduction of the glucagon-stimulated glucose output PMA alone stimulated glucose production (data not shown) [45] Of all agonists used, only PMA translocated PKC isoforms a, d and e (Fig 7A) Differences in DAG mass (lgỈmg)1 of protein) were not observed between control liver (8.3) and livers treated with glucagon (7.9), insulin/glucagon (8.2), or PMA (8.2, n ¼ for all treatments) Discussion In muscle and fat tissue, lipid messengers such as DAG and PA, as well as DAG-dependent and -independent PKC isoforms, have recently been proposed to play a role in the insulin signal leading to activation of glucose uptake [1–3] Ó FEBS 2003 4642 I Probst et al (Eur J Biochem 270) Fig Changes in hepatocyte diacylglycerol (DAG) molecular species composition in response to ATP and phospholipase C (PLC) stimulation Hepatocytes cultured for days were incubated with vehicle (control, s), 100 lM ATP (d), or (n) or 100 (m) mmL)1 PLC, and the molecular species of DAG were analysed as described in the Materials and methods The figure shows time-dependent changes of four DAG species expressed as percentages of total DAG Data represent the mean values ± SD from four to six different hepatocyte preparations Table Effect of 4b-phorbol 12-myristate 13-acetate (PMA), insulin and ATP on the distribution of protein kinase C (PKC) isoforms Hepatocytes cultured for h and 48 h were incubated with 0.1 lM PMA, 10 nM insulin or 0.1 lM ATP for min, and subsequently homogenized and separated into cytosol and particulate membrane fraction The membrane-bound fraction of the PKC isoforms is expressed as the percentage of the total (membrane + cytosol) signal from immunoblots Results are given as mean values ± SD from five to eight experiments using different hepatocyte preparations Percentage of membrane-bound PKC Agonist Culture PKCa Control 48 48 48 48 19.5 13.3 39.7 44.0 20.0 20.7 27.8 19.0 PMA Insulin ATP h h h h h h h h ± ± ± ± ± ± ± ± PKCd 7.7 11.4 11.0* 12.3* 8.0 15.3 4.9 7.3 51.5 37.7 77.3 67.9 52.8 39.2 41.2 37.1 ± ± ± ± ± ± ± ± PKCe 11.1 5.9 10.0* 9.7* 9.6 13.3 9.6 7.4 41.5 40.1 66.5 54.7 41.6 35.6 58.8 42.7 ± ± ± ± ± ± ± ± PKCf 5.8 4.0 12.2* 11.1* 12.1 7.4 8.7 10.4 41.2 41.7 43.5 37.5 38.8 36.7 39.0 36.7 ± ± ± ± ± ± ± ± 7.7 11.7 7.0 16.6 2.3 10.1 9.9 7.9 * P < 0.05 vs control In contrast, in liver preparations these novel insulin signalling pathways have been poorly studied and the available data are confusing and controversial [16–22] The results presented in this report were obtained using (a) the highly insulin-sensitive in vitro liver system of cultured hepatocytes and (b) the perfused liver, and speak clearly against an involvement of phospholipases and PKC isoforms in hepatic insulin signalling, for the following reasons First, we found that the addition of insulin to rat hepatocytes did not increase DAG mass or change the Ó FEBS 2003 Insulin signalling in rat hepatocytes (Eur J Biochem 270) 4643 Table Determination of protein kinase C (PKC) activity in crude extracts (pmolỈmin-1Ỉmg-1 of protein) and PKCf immunoprecipitates (pmolỈmin-1Ỉmg-1 of lysate protein) Hepatocytes cultured for h and 48 h were exposed to vehicle, 0.1 lM phorbol 12-myristate 13-acetate (PMA) or 10 nM insulin for 10 Data represent mean values ± SD from three different hepatocyte preparations Protein kinase C activity Crude extracts Treatment Culture Cytosol Control 48 48 48 48 48 121.3 243.2 58.0 141.5 123.9 220.0 48.7 165.5 55.1 146.0 PMAa Insulina Controlb Insulinb h h h h h h h h h h ± ± ± ± ± ± ± ± ± ± Membrane 16 8d 33c 16 35 38 a PKCf-immunoprecipitate 95.1 148 218.5 249.1 91.6 171.7 161.4 188.3 151.5 199.7 ND ND ND ND ND ND ND 2.26 ± 0.14 ND 2.13 ± 0.21 ± ± ± ± ± ± ± ± ± ± 10 34 19d 40c 15 29 18 40 14 29 Assay with Ca2+ and the epidermal growth factor-receptor peptide (Amersham test kit) as substrate peptide-e as substrate ND, not determined, c P < 0.05, d P < 0.005 Fig Sensitivity of insulin-, 4b-phorbol 12-myristate 13-acetate (PMA)- and ATP-modulated glycogen synthesis to bisindolylmaleimide Hepatocytes cultured for 48 h were incubated with the agonists and the inhibitor for h Data represent the mean values ± SD from three different hepatocyte preparations DAG molecular species composition Second, an involvement of PLD could not be demonstrated as insulin-stimulated hepatocytes showed no evidence for transphosphatidylation activity Third, we found no evidence of translocation of PKC isoforms from the cytosol to the membrane fraction after stimulation of hepatocytes with insulin Fourth, insulin-stimulated cells showed no b Assay without Ca2+ and with increase in membrane-bound PKC activity and did not increase the activity of immunoprecipitated PKCf Fifth, the action of insulin on glycogen synthesis was not abolished by the specific PKC inhibitor, bisindolylmaleimide, whereas it completely reversed the insulin-antagonistic effect of PMA Sixth, insulin did not alter DAG mass and PKC isozyme distribution in the perfused liver Our results are in good agreement with two previous reports showing a lack of PLD [19] and PKC [21] activation upon stimulation of rat hepatocytes with insulin In contrast, they clearly contradict several other recent studies showing insulin-mediated activation of PLD and PKC activities in hepatocyte suspensions and cultures [16–18,20,22] We suggest that this controversy may be a result of the use of different cell systems: hepatocytes in suspension often show reduced insulin responsiveness, whereas primary cultured cells can easily lose their insulin sensitivity when cultured without dexamethasone There is ample evidence that, for a number of insulin-sensitive metabolic parameters, hormone responsiveness is only retained when the cells are cultured long term in the presence of a glucocorticoid [26] Interestingly, the reports showing insulin-dependent increases in DAG, and activation of PLD and/or PKC, all used cell suspensions or glucocorticoid-deprived cultures [16–18,20,22], whereas the hepatocytes used in this report were cultured in the presence of dexamethasone A clear example of how dramatically the results may change, depending on the culture conditions, was obtained when we incubated hepatocytes in the absence of dexamethasone; this led to insulin-dependent increases in DAG molecular species rich in stearate and arachidonate, which agree with Baldini’s data for steroid-deprived cells [17,18] However, our parallel observation, that the cells cultured under these conditions showed a dramatic reduction of insulin-stimulated glycogen synthesis, casts serious doubts on the validity of these steroid-free cultures A similar controversy also exists concerning the mechanism of action of EGF in hepatocytes A review of the literature shows that EGF-dependent phospholipase activation, and Ó FEBS 2003 4644 I Probst et al (Eur J Biochem 270) steroid-treated and untreated cultures might well reflect the differences in cytoskeletal cell architecture and thus point to a major regulatory role of actin fibers in the propagation of hormone and growth factor signals The recent finding that focal adhesion kinase regulates protein kinase B, glycogen synthase kinase-3 and glycogen synthase, in an insulindependent manner [49], supports the hypothesis of crosstalk between insulin and integrin-signalling pathways The lack of an insulin-elicited increase in DAG, shown here for dexamethasone-treated hepatocytes and for the perfused liver, excludes the involvement of conventional and novel PKCs, but not that of atypical PKCf in signal transduction Our results indicate that PKCf is not involved in the activation of glycogen synthesis by insulin This finding is in good agreement with a previous report showing that a specific inhibitor of PKCf had no effect on the activation of glycogen synthase, although the authors observed the insulin-dependent activation of PKCf in their glucocorticoid-deprived hepatocyte cultures [22] Recent observations also showed insulin-mediated activation of PKCs in hepatoma cell lines [23,24,50] However, in our view, these data not support a role for PKCs in the adult hepatic insulin signalling cascade because hepatoma cells are in an abnormal proliferative state Recently, doubts have been raised regarding whether atypical PKCs are indeed involved in glucose transport in L6 myotubes [51] and in 3T3-L1 adipocytes transiently transfected with wild-type or mutant PKCk and f [52] Thus, activation of atypical PKCs by insulin might depend on cell differentiation status (via culture conditions), and PKC isoforms may indirectly modulate insulin action by interfering with enzyme compartmentalization and association with the cytoskeleton Acknowledgments Fig Insulin effects on glucose metabolism and protein kinase C (PKC) distribution in the perfused liver After the first glucagon bolus, livers were further perfused without agonist (C, control), with glucagon (Ggn) (B), with insulin/glucagon (Ins/Ggn) (C) or with lM phorbol 12-myristate 13-acetate (PMA; shown only for PKC distribution) (A) Liver samples of the front large lobe were sampled at 35 Data represent the mean values from three different perfusions for each agonist *P < 0.05 were indicated (A vs control) and for the insulin values from 34 to 41 (C vs glucagon) increases in DAG, PA, inositoltrisphosphate and cytosolic calcium, were detected to various degrees when hepatocyte suspensions or glucocorticoid-free hepatocyte cultures were used [36,39,41,46,47] In contrast, in our dexamethasonetreated cultures, EGF had no effect on DAG levels, which is in agreement with the results of Dajani et al [38] who used similar culture conditions Working with hepatocyte suspensions and cultured cells, Nojiri & Hoek [47] pointed out that EGF-induced inositoltrisphosphate formation was effectively reduced by actin rearrangement, which occurs during the transition of the cells from the suspended to the cultured state As dexamethasone is known to retain cuboidal hepatocyte morphology in cultures and to influence actin polymerization [48], the differences in insulin signalling (and also in EGF signalling) observed between We are very grateful to Frank Rhode for his expert help with liver perfusions and we thank Dr Ralf Wimmer for the measurements of PKC distribution in liver tissue samples This work was supported by grants from the Swiss National Science Foundation (to P.B.) and the Deutsche Forschungsgemeinschaft (to I.P and U.B.) 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