Title: Inhibition of Hepatocytic Autophagy by Adenosine, Aminoimidazole-4-carboxamide Riboside, and N 6-Mercaptopurine Riboside
Abstract: To examine the role of AMP-activated protein kinase (AMPK; EC 2.7.1.109) in the regulation of autophagy, rat hepatocytes were incubated with the AMPK proactivators, adenosine, 5-amino-4-imidazole carboxamide riboside (AICAR), orN 6-mercaptopurine riboside. Autophagic activity was inhibited by all three nucleosides, AICAR and N 6-mercaptopurine riboside being more potent (IC50 = 0.3 mm) than adenosine (IC50 = 1 mm). 2′-Deoxycoformycin, an adenosine deaminase (EC 3.5.4.4) inhibitor, increased the potency of adenosine 5-fold, suggesting that the effectiveness of adenosine as an autophagy inhibitor was curtailed by its intracellular deamination. 5-Iodotubercidin, an adenosine kinase (EC 2.7.1.20) inhibitor, abolished the effects of all three nucleosides, indicating that they needed to be phosphorylated to inhibit autophagy. A 5-iodotubercidin-suppressible phosphorylation of AICAR to 5-aminoimidazole-4-carboxamide riboside monophosphate was confirmed by chromatographic analysis. AICAR, up to 0.4 mm, had no significant effect on intracellular ATP concentrations. Because activated AMPK phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (EC 1.1.1.88), the rate-limiting enzyme in cholesterol synthesis, the strong inhibition of hepatocytic cholesterol synthesis by all three nucleosides confirmed their ability to activate AMPK under the conditions used. Lovastatin and simvastatin, inhibitors of HMG-CoA reductase, strongly suppressed cholesterol synthesis while having no effect on autophagic activity, suggesting that AMPK inhibits autophagy independently of its effects on HMG-CoA reductase and cholesterol metabolism. To examine the role of AMP-activated protein kinase (AMPK; EC 2.7.1.109) in the regulation of autophagy, rat hepatocytes were incubated with the AMPK proactivators, adenosine, 5-amino-4-imidazole carboxamide riboside (AICAR), orN 6-mercaptopurine riboside. Autophagic activity was inhibited by all three nucleosides, AICAR and N 6-mercaptopurine riboside being more potent (IC50 = 0.3 mm) than adenosine (IC50 = 1 mm). 2′-Deoxycoformycin, an adenosine deaminase (EC 3.5.4.4) inhibitor, increased the potency of adenosine 5-fold, suggesting that the effectiveness of adenosine as an autophagy inhibitor was curtailed by its intracellular deamination. 5-Iodotubercidin, an adenosine kinase (EC 2.7.1.20) inhibitor, abolished the effects of all three nucleosides, indicating that they needed to be phosphorylated to inhibit autophagy. A 5-iodotubercidin-suppressible phosphorylation of AICAR to 5-aminoimidazole-4-carboxamide riboside monophosphate was confirmed by chromatographic analysis. AICAR, up to 0.4 mm, had no significant effect on intracellular ATP concentrations. Because activated AMPK phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (EC 1.1.1.88), the rate-limiting enzyme in cholesterol synthesis, the strong inhibition of hepatocytic cholesterol synthesis by all three nucleosides confirmed their ability to activate AMPK under the conditions used. Lovastatin and simvastatin, inhibitors of HMG-CoA reductase, strongly suppressed cholesterol synthesis while having no effect on autophagic activity, suggesting that AMPK inhibits autophagy independently of its effects on HMG-CoA reductase and cholesterol metabolism. AMP-activated protein kinase 5-aminoimidazole-4-carboxamide riboside 2′-deoxycoformycin/pentostatinR 3-hydroxy-3-methylglutaryl-CoA 5-iodotubercidin lactate dehydrogenase N 6-mercaptopurine riboside 5-aminoimidazole-4-carboxamide riboside monophosphate 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino} ethanesulfonic acid high performance liquid chromatography. Numerous biochemical processes are involved in the maintenance and support of cell function and cellular growth. These processes need to be coordinated not only relative to each other but also in relation to the metabolic energy available to the cell. The coordination is generally thought to be effectuated in a diffuse fashion by the adenine nucleotides (AMP, ADP, and ATP), which are both carriers of energy (ATP and ADP) and capable of serving as direct regulators of the activity of many enzymes. Although the cellular energy metabolism is too complex to be described by a manageable algorithm, the activities of most metabolic pathways have been found to correlate reasonably well, positively or negatively, with the overall cellular energy state as embodied e.g. in the concept of “energy charge” ([ATP + ½ADP]/[ATP + ADP + AMP]) (1Atkinson, D. E. (1971) in Metabolic Pathways: Metabolic Regulation (Vogel, H. J., ed) Vol. 5, Chapter 1, pp. 1–21, Academic Press, New YorkGoogle Scholar). In recent years, the enzyme AMP-activated protein kinase (AMPK)1 (EC 2.7.1.109) has been emerging as a “master switch” that mediates at least part of the coordination between energy state and metabolism (2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar). The activity of this enzyme is regulated in a complex fashion by adenine nucleotides as well as by an upstream kinase (itself activated by AMP) and becomes active when the AMP level is high relative to ATP. Activated AMPK in turn shuts down energy-requiring pathways like fatty acid and cholesterol synthesis through phosphorylation and inactivation of the pertinent key enzymes while indirectly activating an energy-producing pathway like fatty acid oxidation (2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar). However, so far the documented regulatory effects of AMPK have been largely confined to lipid metabolism. It would obviously be of interest to study other metabolic pathways to assess the generality of this enzyme's role in metabolic regulation. Autophagy occupies a central position in cellular metabolism, supplying small molecules both for anabolic and catabolic purposes through the bulk degradation of proteins and other cellular macromolecules. By a poorly understood sequestration process, pieces of cytoplasm become encapsulated by cellular membranes, forming autophagic vacuoles that eventually fuse with lysosomes to have their contents degraded. The autophagic-lysosomal pathway is subject to complex regulation at the initial sequestration step, by hormones, growth factors, metabolites, and various signaling molecules (3Seglen P.O. Bohley P. Experientia. 1992; 48: 158-172Crossref PubMed Scopus (370) Google Scholar). Two previous sets of observations are particularly pertinent to the energy-metabolic control of autophagy: (i) ATP depletion has been shown to reduce the autophagic activity (4Plomp P.J.A.M. Wolvetang E.J. Groen A.K. Meijer A.J. Gordon P.B. Seglen P.O. Eur. J. Biochem. 1987; 164: 197-203Crossref PubMed Scopus (78) Google Scholar), the overall autophagic-lysosomal protein degradation (5Seglen P.O. Grinde B. Solheim A.E. Eur. J. Biochem. 1979; 95: 215-225Crossref PubMed Scopus (355) Google Scholar), and the fractional volume of autophagosomes (6Schellens J.P.M. Vreeling-Sindelárová H. Plomp P.J.A.M. Meijer A.J. Exp. Cell Res. 1988; 177: 103-108Crossref PubMed Scopus (37) Google Scholar) in isolated hepatocytes, apparently by inhibiting several steps in the autophagic-lysosomal pathway (7Plomp P.J.A.M. Gordon P.B. Meijer A.J. Høyvik H. Seglen P.O. J. Biol. Chem. 1989; 264: 6699-6704Abstract Full Text PDF PubMed Google Scholar). (ii) Adenosine and, more potently, N-substituted adenosine analogues are capable of suppressing hepatocytic autophagy (8Seglen P.O. Gordon P.B. Holen I. Høyvik H. Kovács A.L. Strømhaug P.E. Berg T.O. Courtoy P.J. Endocytosis. Springer-Verlag, Berlin1992: 247-254Crossref Google Scholar) and protein degradation (9Gordon P.B. Seglen P.O. Arch. Biochem. Biophys. 1982; 217: 282-294Crossref PubMed Scopus (24) Google Scholar). It is noteworthy that a large increase in hepatocytic AMP levels is a characteristic feature of both ATP depletion (10Hue L. Biochem. J. 1982; 206: 359-365Crossref PubMed Scopus (54) Google Scholar, 11Aw T.Y. Jones D.P. Am. J. Physiol. 1989; 257: C435-C441Crossref PubMed Google Scholar, 12Bontemps F. Vincent M.F. Van den Berghe G. Biochem. J. 1993; 290: 671-677Crossref PubMed Scopus (58) Google Scholar) and adenosine addition (13Carabaza A. Ricart M.D. Mor A. Guinovart J.J. Ciudad C.J. J. Biol. Chem. 1990; 265: 2724-2732Abstract Full Text PDF PubMed Google Scholar). The possibility should, therefore, be considered that AMP, perhaps through activation of AMPK, might be a mediator of autophagy suppression under both conditions. Because autophagy apparently requires energy (4Plomp P.J.A.M. Wolvetang E.J. Groen A.K. Meijer A.J. Gordon P.B. Seglen P.O. Eur. J. Biochem. 1987; 164: 197-203Crossref PubMed Scopus (78) Google Scholar, 7Plomp P.J.A.M. Gordon P.B. Meijer A.J. Høyvik H. Seglen P.O. J. Biol. Chem. 1989; 264: 6699-6704Abstract Full Text PDF PubMed Google Scholar), it would seem logical that it be included it among the processes shut down by AMPK under conditions of energy depletion (2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar). In the present work we have therefore investigated whether the autophagy-inhibitory effect of adenosine might be mediated by its phosphorylation to AMP. Furthermore, we tested the effect of 5-amino-4-imidazolecarboxamide riboside (AICAR), shown to be a potent activator of AMPK after its intracellular phosphorylation to AICA-ribotide or ZMP (14Sullivan J.E. Brocklehurst K.J. Marley A.E. Carey F. Carling D. Beri R.K. FEBS Lett. 1994; 353: 33-36Crossref PubMed Scopus (417) Google Scholar, 15Henin N. Vincent M.F. Van den Berghe G. Biochim. Biophys. Acta. 1996; 1290: 197-203Crossref PubMed Scopus (94) Google Scholar). Both adenosine and AICAR were found to inhibit autophagy strongly. An inhibitor of adenosine kinase (EC2.7.1.20), 5-iodotubercidin (ITu) (16Henderson J.F. Paterson A.R.P. Caldwell I.C. Paul B. Chan M.C. Lau K.F. Cancer Chemother. Rep. 1972; 3: 71-85Google Scholar), suppressed the autophagy-inhibitory effects of both treatments, indicating that formation of AMP and ZMP, respectively, was necessary to inhibit autophagic activity. The results thus support the contention that AMPK may be involved in the regulation of hepatocytic autophagy. [2-14C]acetic acid, (50–62 mCi/mmol, 200 μCi/ml) and [32P]phosphoric acid (∼200 mCi/mmol) were purchased from Amersham International Inc. (Little Chalfont, Buckinghamshire, UK). TLC plates (silica Gel F 1500) were purchased from Schleicher & Schuell (Dassel, Germany). Analytical reagents for lactate dehydrogenase (LDH) (EC 1.1.1.27) measurements were from Boehringer Mannheim (Mannheim, Germany). ATP-monitoring reagents were from Pharmacia (Uppsala, Sweden). Leupeptin was purchased from Peptide Institute Inc. (Osaka, Japan). 5-Iodotubercidin (ITu) was purchased from Research Biochemicals Inc. (Natick, MA). 2′-Deoxycoformycin (dCF) was generously donated by Parke-Davis (Ann Arbor, MI). The luciferin/luciferase assay kit was purchased from LKB-Wallac Oy (Oulu, Finland). Metrizamide was from Nycomed A/S (Oslo, Norway). All disposable accessories for reverse phase HPLC, including 0.45- and 0.22-μm HA filters, were from Millipore (Milford, MA). Collagenase (type IV, from Clostridium histolyticum) and other biochemicals, including AICAR, 5-amino-4-imidazolecarboxamide ribotide (ZMP), N 6-mercaptopurine riboside (N 6-MPR), and adenosine, were purchased from Sigma (St. Louis, MO) or from other suppliers of analytical grade chemicals. Hepatocytes were isolated by two-step collagenase perfusion (17Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5225) Google Scholar) from male albino Wistar rats (250–300 g) fasted for 18 h. After isolation and washing with wash buffer (148 mm Na+, 6.7 mm K+, 1.2 mm Ca2+, 151 mm Cl−, pH 7.4) (17Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5225) Google Scholar), the hepatocytes were suspended in fortified suspension buffer (122 mmNa+, 6.5 mm K+, 1.2 mmCa2+, 2.0 mm Mg2+, 77 mm Cl−, 1.1 mmPO43−, 0.7 mmSO42−, 30 mm Hepes, 30 mm Tes, 30 mm Tricine, 20 mmpyruvate, pH 7.6, 37 °C) (17Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5225) Google Scholar). By this method we obtained about 4 g of wet mass of >90% intact hepatocytes (cells able to exclude trypan blue). The wet mass was measured by weighing sedimented cell pellets, which have a composition corresponding exactly to intact liver tissue (17Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5225) Google Scholar). The cells were incubated as 0.4-ml aliquots (70–80 mg cellular wet mass/ml) at 37 °C for 2 h in rapidly shaking glass tubes; the shaking ensures adequate oxygenation as indicated by the maintenance of high physiological ATP levels (4Plomp P.J.A.M. Wolvetang E.J. Groen A.K. Meijer A.J. Gordon P.B. Seglen P.O. Eur. J. Biochem. 1987; 164: 197-203Crossref PubMed Scopus (78) Google Scholar, 7Plomp P.J.A.M. Gordon P.B. Meijer A.J. Høyvik H. Seglen P.O. J. Biol. Chem. 1989; 264: 6699-6704Abstract Full Text PDF PubMed Google Scholar, 9Gordon P.B. Seglen P.O. Arch. Biochem. Biophys. 1982; 217: 282-294Crossref PubMed Scopus (24) Google Scholar). Sequestration of a long-lived cytosolic enzyme, LDH, was used to measure autophagy (18Kopitz J. Kisen G.Ø. Gordon P.B. Bohley P. Seglen P.O. J. Cell Biol. 1990; 111: 941-953Crossref PubMed Scopus (191) Google Scholar). Leupeptin (0.3 mm), a cysteine proteinase inhibitor (5Seglen P.O. Grinde B. Solheim A.E. Eur. J. Biochem. 1979; 95: 215-225Crossref PubMed Scopus (355) Google Scholar), was added to the cell suspensions during incubation to prevent the lysosomal degradation of LDH (18Kopitz J. Kisen G.Ø. Gordon P.B. Bohley P. Seglen P.O. J. Cell Biol. 1990; 111: 941-953Crossref PubMed Scopus (191) Google Scholar). After the incubation, the cells were washed twice with 4.0 ml of isotonic sucrose solution (10% w/v) followed by centrifugation (1600 × g, 4 min). The cells were then resuspended in 500 μl of 10% sucrose, briefly warmed to 37 °C, electrodisrupted by a single high voltage pulse (2 kV/cm), and transferred to a new tube containing 0.5 ml of ice-cold post-disruption buffer (100 mm potassium phosphate, 2 mm EDTA, 2 mm dithiothreitol, and 0.05 mm sucrose, pH 7.5). To separate autophagic vacuoles from the rest of the cellular material, 0.6 ml of the cell suspension was carefully layered on top of a 4-ml density cushion (2.2% sucrose, 8% metrizamide, 50 mm potassium phosphate, 1 mm dithiothreitol, and 1 mm EDTA, pH 7.5) and centrifuged at 3700 ×g for 30 min (i.e. approximately 1.1·105 g·min−1). After centrifugation and removal of the supernatant (containing the cytosol), the pellet (containing the autophagic vacuoles) was washed in 10% sucrose and resuspended in resuspension buffer (50 mmpotassium phosphate, 1 mm EDTA, and 1 mmdithiothreitol, pH 7.5) and frozen at −70 °C. The remaining 0.4 ml of the cell suspension was also frozen at −70 °C, to be used for measuring the total cellular LDH. LDH activity in resuspended pellets (autophagocytosed LDH) and in the disrupted cells (total LDH) was measured spectrophotometrically with a Technicon RA 1000 autoanalyzer. The amount of sequestered LDH relative to total cellular LDH was taken to indicate the autophagic sequestration rate and was expressed as percent/h. The intracellular ATP content was measured with a luminometer from LKB-Wallac Oy (Oulu, Finland) using a luciferin/luciferase assay procedure. Immediately after incubation the cells were acid-precipitated by ice-cold perchloric acid (final concentration, 2% w/v), followed by a 10-min incubation on ice and a 15-min centrifugation at 3700 ×g. A fixed volume of supernatant was transferred to a new tube, neutralized with freshly made 1 n KOH, and frozen at −20 °C. On the measurement day, the samples were diluted 40 times with Tris acetate buffer (0.1 mm Tris acetate, 2 mm EDTA, pH 7.75), before measuring the intensity of the produced light in each sample relative to an ATP standard. Intracellular nucleosides and nucleotides were separated and quantified by reverse phase HPLC as described by Shevchuket al. (19Shevchuk I. Chekulayev V. Moan J. Berg K. Int. J. Cancer. 1996; 67: 791-799Crossref PubMed Scopus (25) Google Scholar) using a reverse phase Supelcosil LC-18-T column (25 cm × 4.6 mm) from SUPELCO (Park Bellefonte, PA). The HPLC instrument system was from Waters (chromatographic division of Millipore Co., Milford, MA), consisting of a Waters 486 tunable absorbance detector adjusted to 254 nm, a WISP 710B automated injector and pump, and a Waters multisolvent delivery system. Data were analyzed by a Waters Maxima 820 chromatography work station. After incubation, the samples were immediately treated with 2% perchloric acid and incubated for 15 min at 0 °C. The samples were then centrifuged for 15 min at 3700 × g, the supernatants were transferred to a new set of tubes, neutralized with freshly made 1N NaOH, and stored at −20 °C until the measurement day. All samples and solutions used in the HPLC system were filtered through a 0.22-μm Millipore filter. The mobile phase consisted of two buffers: buffer A (100 mmKH2PO4, pH 5.0, 1.5% acetonitrile, 0.08% tetrabutyl ammonium bromide) and buffer B (150 mmKH2PO4, pH 5.0, 10% acetonitrile, 0.08% tetrabutyl ammonium bromide). The measurement time for each sample was 45 min at a constant flow rate of 1 ml/min with detection at 254 nm. The chromatographic procedure was divided into three phases: (i) the gradient phase (in which the mobile phase gradually changed from 100% buffer A to 100% buffer B within 2 min), (ii) the constant phase (100% buffer B for the next 30 min), and (iii) the wash phase (100% buffer A for 15 min). Each peak was identified by coelution of the samples with added standards. Cholesterol synthesis in isolated hepatocytes was measured as the incorporation of [2-14C]acetate according to Rustan et al.(20Rustan A.C. Nossen J.O. Osmundsen H. Drevon C.A. J. Biol. Chem. 1988; 263: 8126-8132Abstract Full Text PDF PubMed Google Scholar). After incubation in the presence of [2-14C]acetate (100 μm; 1 μCi/ml), the cells were washed in 10% isotonic sucrose (4 min at 1600 × g) to remove dead cells and excess [2-14C]acetate. The cells were then dissolved in 1 ml of 50 mm potassium phosphate (pH 7.5) and frozen at −70 °C until the next day, when extraction was performed. The cell samples were thawed and homogenized by sonication (60 mA/s), and 400 μl of homogenate was transferred to a new tube. Cellular lipids were extracted by mixing with 20 times the volume of chloroform/methanol (2:1, v/v), incubation for 30 min, addition of four times the volume of 0.9% NaCl solution, and incubation for 15 min, which allowed the mixture to separate into two phases. After centrifugation (5000 × g for 5 min), the inorganic phase, and the protein layer was gently removed, and the organic phase was dried under nitrogen at 40 °C. The residual lipid extract was redissolved in 200 μl of hexane and separated by thin layer chromatography (acid-resistant silica gel TLC-foils, F 1500, from Schleicher & Schuell) using hexane/diethyl ether/acetic acid (80:20:1, v/v/v) as the developing system. Lipids were recognized by visualizing added standards in iodine vapor and cut out, and their radioactivity was quantified by liquid scintillation counting in an LKB Wallac beta counter (1261 multigamma). Previous studies have shown that adenosine and (more potently) some of itsN 6-substituted derivatives,e.g. N 6-dimethyladenosine and N 6-methylmercaptopurine riboside, inhibit protein degradation in isolated rat hepatocytes (9Gordon P.B. Seglen P.O. Arch. Biochem. Biophys. 1982; 217: 282-294Crossref PubMed Scopus (24) Google Scholar). As shown in Fig. 1 A, adenosine was able to suppress hepatocytic autophagy (measured as the sequestration of endogenous LDH), which would explain its degradation-inhibitory ability. The effect of adenosine was strongly potentiated by dCF, a specific adenosine deaminase (EC 3.5.4.4) inhibitor (21Henderson J.F. Brox L. Zombor G. Hunting D. Lomax C.A. Biochem. Pharmacol. 1977; 26: 1967-1972Crossref PubMed Scopus (103) Google Scholar) that lowered the IC50 of adenosine from 1 to 0.2 mm. The inhibition of autophagy by adenosine would thus normally seem to be restrained by rapid adenosine deamination. Incubation in the presence of 10 μm ITu, a potent and specific inhibitor of adenosine kinase (16Henderson J.F. Paterson A.R.P. Caldwell I.C. Paul B. Chan M.C. Lau K.F. Cancer Chemother. Rep. 1972; 3: 71-85Google Scholar), virtually completely abolished the inhibitory effect of adenosine + dCF on autophagy (Fig. 1 B). This absolute requirement for adenosine phosphorylation would suggest that its inhibitory effect on autophagy is mediated by AMP. Measurements of intracellular AMP levels (by HPLC) indeed revealed a 10-fold increase (transiently peaking at 3 μmol/g wet mass) after the addition of adenosine + dCF. 2A. L. Kovács, P. B. Gordon, E. M. Grotterød, and P. O. Seglen, unpublished results. One possible mechanism for the inhibition of autophagy by AMP might be through activation of the AMPK. To investigate this possibility, we incubated hepatocytes with the nucleoside analogue, AICAR, which in its monophosphorylated form (AICA-ribotide, or ZMP) is a potent activator of hepatocytic AMPK (22Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1036) Google Scholar). As shown in Fig. 2 A, AICAR inhibited hepatocytic autophagy in a dose-dependent manner (IC50 of about 0.3 mm). Its effect on autophagy was completely suppressed by 10 μm ITu, consistent with a requirement for phosphorylation to ZMP.N 6-MPR, an adenosine analogue thiolated at the N 6 position, also inhibited autophagic seqestration in an ITu-sensitive manner suggestive of a phosphorylation requirement (Fig. 2 B).N 6-MPR was more potent than adenosine, perhaps because the thiol group at theN 6-position prevented its deamination by adenosine deaminase, as previously suggested to explain the potent protein degradation-inhibitory effects of otherN 6-substituted adenosine derivates (9Gordon P.B. Seglen P.O. Arch. Biochem. Biophys. 1982; 217: 282-294Crossref PubMed Scopus (24) Google Scholar). The intracellular phosphorylation of AICAR to form ZMP and suppression of this process by ITu were confirmed by reverse phase HPLC analysis of intracellular metabolites in hepatocytes. As shown in Fig. 3 A, addition of 0.4 mm AICAR to the hepatocyte suspension resulted in its rapid intracellular accumulation. Accumulated AICAR was effectively phosphorylated to form ZMP, which at 2 h had almost completely replaced AICAR (Fig. 3 B). This phosphorylation was suppressed by the addition of 10 μm ITu (Fig. 3 C), indicating the involvement of adenosine kinase. Autophagic sequestration has previously been shown to correlate positively with intracellular ATP levels (4Plomp P.J.A.M. Wolvetang E.J. Groen A.K. Meijer A.J. Gordon P.B. Seglen P.O. Eur. J. Biochem. 1987; 164: 197-203Crossref PubMed Scopus (78) Google Scholar), suggesting a requirement for energy. To check whether AICAR might inhibit autophagy by altering the intracellular ATP concentration, its effect on hepatocellular ATP levels was measured. As shown in Fig. 4, AICAR concentrations below 0.6 mm had no effect on intracellular ATP levels, although autophagic sequestration was inhibited 80%. At higher AICAR concentrations, some reduction in ATP (about 30% at 1 mm) was observed. Apparently, AICAR does not exert its inhibitory effect on autophagy by altering intracellular ATP. One of the established biological roles of activated AMPK its to phosphorylate and inactivate HMG-CoA reductase (EC 1.1.1.88), the rate-limiting enzyme in cholesterol synthesis (23Corton J.M. Gillespie J.G. Hardie D.G. Curr. Biol. 1994; 4: 315-324Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 24Henin N. Vincent M.-F. Gruber H.E. Van den Berghe G. FASEB J. 1995; 9: 541-546Crossref PubMed Scopus (226) Google Scholar). If adenosine, AICAR, and N 6-MPR really activate AMPK, they would therefore be expected to inhibit hepatocellular cholesterol synthesis. As shown in Fig. 5, all these compounds inhibited cholesterol synthesis in isolated hepatocytes, with about the same potencies as in their inhibition of autophagy. ITu (10 μm) supressed these effects on cholesterol synthesis completely, demonstrating the need for phosphorylated intermediates (AMP, etc.). The results in Fig. 5 thus support the notion that adenosine, AICAR, and N 6-MPR are able to activate hepatocellular AMPK under the experimental conditions employed. The parallel suppression of autophagy and HMG-CoA reductase activity by adenosine and its analogues would be consistent with a role for HMG-CoA reductase in the regulation of autophagy. To examine this possibility, we incubated hepatocytes with lovastatin or simvastatin, two potent and specific drugs that lower cholesterol synthesis by inhibiting HMG-CoA reductase (25Alberts A.W. Cardiology. 1990; 77: 14-21Crossref PubMed Scopus (103) Google Scholar). As shown in Fig. 6, neither lovastatin (panel A) nor simvastatin (panel B) exerted any effect on autophagy, although they potently inhibited hepatocellular cholesterol synthesis (IC50 < 5 μm). HMG-CoA reductase would thus not seem to be involved in regulation of autophagy. Autophagy, or macroautophagy, is a cellular process that plays a major role in the bulk sequestration and subsequent lysosomal degradation of long-lived cytosolic proteins, ribosomal RNA, and cytoplasmic organelles (3Seglen P.O. Bohley P. Experientia. 1992; 48: 158-172Crossref PubMed Scopus (370) Google Scholar, 26Holtzman E. Lysosomes. Plenum Press, New York1989Crossref Google Scholar, 27Mortimore G.E. Khurana K.K. Int. J. Biochem. 1990; 22: 1075-1080Crossref PubMed Scopus (6) Google Scholar, 28Dunn W.A. Trends Cell Biol. 1994; 4: 139-143Abstract Full Text PDF PubMed Scopus (444) Google Scholar). The initial autophagic sequestration step is highly regulated by growth factors, hormones, and metabolites such as amino acids and adenosine (which can be regarded as negative feedback inhibitors of autophagy) and by protein phosphorylation (8Seglen P.O. Gordon P.B. Holen I. Høyvik H. Kovács A.L. Strømhaug P.E. Berg T.O. Courtoy P.J. Endocytosis. 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Biochem. 1996; 236: 163-170Crossref PubMed Scopus (51) Google Scholar), and protein tyrosine kinase activity (34Holen I. Strømhaug P.E. Gordon P.B. Fengsrud M. Berg T.O. Seglen P.O. J. Biol. Chem. 1995; 270: 12823-12831Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) have been implicated in the regulation of autophagy in isolated rat hepatocytes. Depletion of ATP inhibits autophagic sequestration, suggesting that autophagy is an energy-dependent process (4Plomp P.J.A.M. Wolvetang E.J. Groen A.K. Meijer A.J. Gordon P.B. Seglen P.O. Eur. J. Biochem. 1987; 164: 197-203Crossref PubMed Scopus (78) Google Scholar, 7Plomp P.J.A.M. Gordon P.B. Meijer A.J. Høyvik H. Seglen P.O. J. Biol. Chem. 1989; 264: 6699-6704Abstract Full Text PDF PubMed Google Scholar). However, ATP depletion also leads to increases in intracellular AMP and adenosine (10Hue L. Biochem. J. 1982; 206: 359-365Crossref PubMed Scopus (54) Google Scholar, 12Bontemps F. Vincent M.F. Van den Berghe G. Biochem. J. 1993; 290: 671-677Crossref PubMed Scopus (58) Google Scholar). Because adenine, adenosine, and some of their thiolated or methylated derivates also inhibit autophagic protein degradation (8Seglen P.O. Gordon P.B. Holen I. Høyvik H. Kovács A.L. Strømhaug P.E. Berg T.O. Courtoy P.J. Endocytosis. Springer-Verlag, Berlin1992: 247-254Crossref Google Scholar, 9Gordon P.B. Seglen P.O. Arch. Biochem. Biophys. 1982; 217: 282-294Crossref PubMed Scopus (24) Google Scholar, 35Seglen P.O. Gordon P.B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1889-1892Crossref PubMed Scopus (1181) Google Scholar, 36Caro L.H.P. Plomp P.J.A.M. Wolvetang E.J. Kerkhof C. Meijer A.J. Eur. J. Biochem. 1988; 175: 325-329Crossref PubMed Scopus (114) Google Scholar), the posibility should be considered that at least part of the autophagy-inhibitory effect of ATP depletion might be mediated by adenosine or AMP, perhaps through AMPK. AMPK is the central component in a multisubstrate protein kinase cascade involved in the regulation of lipid metabolism (37Hardie D.G. Carling D. Sim A.T.R. Trends Biochem. Sci. 1989; 14: 20-23Abstract Full Text PDF Scopus (151) Google Scholar). AMPK is activated in response to elevations of the AMP/ATP ratio, causede.g. by hypoxia, environmental stress, starvation, cell damage, heat, etc. (23Corton J.M. Gillespie J.G. Hardie D.G. Curr. Biol. 1994; 4: 315-324Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 38Vincent M.F. Bontemps F. Van den Berghe G. Biochem. J. 1992; 281: 267-272Crossref PubMed Scopus (68) Google Scholar). Upon its activation, AMPK shuts down major ATP-consuming biosynthetic processes like cholesterol and fatty acid synthesis, thereby preserving ATP for more essential and immediate cellular needs such as the maintenance of ion gradients. It has been suggested than AMPK may serve as a general integrator of metabolic responses to changes in energy availability likely to have regulatory effects extending beyond lipid metabolism (2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar). In the present study, the ability of the adenosine kinase inhibitor ITu (16Henderson J.F. Paterson A.R.P. Caldwell I.C. Paul B. Chan M.C. Lau K.F. Cancer Chemother. Rep. 1972; 3: 71-85Google Scholar) to abolish the autophagy-suppressive effect of adenosine clearly shows that the suppression requires AMP formation and most likely is mediated by AMP. This effect of ITu would effectively rule out the possibility that adenosine might mediate its effect through purinergic (adenosine) receptors, known to be present at the surface of rat hepatocytes (39Okajima F. Tokumitsu Y. Kondo Y. Ui M. J. Biol. Chem. 1987; 262: 13483-13490Abstract Full Text PDF PubMed Google Scholar, 40Olah M.E. Stiles G.L. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 581-606Crossref PubMed Google Scholar). The administration of adenosine alone has previously been shown to raise the concentration of AMP in isolated rat hepatocytes (13Carabaza A. Ricart M.D. Mor A. Guinovart J.J. Ciudad C.J. J. Biol. Chem. 1990; 265: 2724-2732Abstract Full Text PDF PubMed Google Scholar). Under the conditions used in the present experiments, with the adenosine and adenylate deaminase inhibitor, dCF (21Henderson J.F. Brox L. Zombor G. Hunting D. Lomax C.A. Biochem. Pharmacol. 1977; 26: 1967-1972Crossref PubMed Scopus (103) Google Scholar), included to prevent the deamination of adenosine and AMP, a rapid increase in intracellular AMP to very high levels has been demonstrated.2 The marked synergism between adenosine and dCF in suppressing autophagy would thus be consistent with a mediation by AMP. Both adenosine and dCF are also capable of elevating the hepatocytic levels of other adenosine metabolites, likeS-adenosylhomocysteine and (secondarily)S-adenosylmethionine (41Hoffman D.R. Marion D.W. Cornatzer W.E. Duerre J.A. J. Biol. Chem. 1980; 255: 10822-10827Abstract Full Text PDF PubMed Google Scholar, 42Helland S. Ueland P.M. Cancer Res. 1982; 42: 1130-1136PubMed Google Scholar), but this pathway would be potentiated rather than antagonized by ITu (43Bontemps F. Van den Berghe G. Hers H.-G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2829-2833Crossref PubMed Scopus (89) Google Scholar). AMP can directly activate or inactivate a number of enzymes,e.g. in glucose/glycogen metabolism (2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar, 13Carabaza A. Ricart M.D. Mor A. Guinovart J.J. Ciudad C.J. J. Biol. 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AICAR was rapidly converted to ZMP under the present conditions, and the autophagy-suppressive effect of AICAR was abolished by ITu, strongly suggesting that ZMP rather than the nucleoside itself was the active autophagy suppressant. Furthermore, the ability of AICAR to virtually completely inhibit hepatocellular cholesterol synthesis, reflecting the inactivating phosphorylation of the rate-limiting enzyme HMG-CoA reductase by AMPK (24Henin N. Vincent M.-F. Gruber H.E. Van den Berghe G. FASEB J. 1995; 9: 541-546Crossref PubMed Scopus (226) Google Scholar, 37Hardie D.G. Carling D. Sim A.T.R. Trends Biochem. Sci. 1989; 14: 20-23Abstract Full Text PDF Scopus (151) Google Scholar, 45Sato R. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9261-9265Crossref PubMed Scopus (141) Google Scholar), clearly demonstrated that AMPK was activated by AICAR under our experimental conditions. AICAR, at concentrations that inhibited autophagy by 80%, had no significant effect on hepatocellular ATP levels, in accordance with previous observations (22Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1036) Google Scholar), nor did it detectably reduce cellular viability. Its effect on autophagy would therefore be likely to be mediated by AMPK activation rather than by energy depletion. N 6-MPR, an aminothiolated adenosine analogue, inhibited autophagy more potently than did adenosine in our experiments. Like in the case of adenosine and AICAR, its autophagy-suppressive effect was abolished by ITu, indicating mediation by a 5′-phosphorylated derivative, i.e. an AMP analogue, likely to activate AMPK. However, previous work (15Henin N. Vincent M.F. Van den Berghe G. Biochim. Biophys. Acta. 1996; 1290: 197-203Crossref PubMed Scopus (94) Google Scholar) indicated thatN 6-MPR 5′-monophosphate did not activate purified rat liver AMPK, in contrast to AMP and ZMP. To see if the situation might be different in intact cells, we measured the effect of N 6-MPR on hepatocellular cholesterol synthesis, as an indirect measure of in situ AMPK activity.N 6-MPR was found to strongly inhibit cholesterol synthesis, and in an ITu-sensitive manner, suggesting that its 5′-monophosphate was in fact able to activate AMPK in intact cells. The discrepancy between this observation and the inability of N 6-MPR 5′-monophosphate to activate purified AMPK can perhaps be explained by the complex manner in which AMPK is regulated. The direct allosteric activation of AMPK by 5′-nucleotides may increase its activity maximally 5-fold (46Carling D. Clarke P.R. Zammit V.A. Hardie D.G. Eur. J. Biochem. 1989; 186: 129-136Crossref PubMed Scopus (344) Google Scholar), whereas its phosphorylation by an upstream AMP-activated protein kinase kinase may cause a more than 20-fold activation (47Moore F. Weekes J. Hardie D.G. Eur. J. Biochem. 1991; 199: 691-697Crossref PubMed Scopus (194) Google Scholar, 48Weekes J. Hawley S.A. Corton J. Shugar D. Hardie D.G. Eur. J. Biochem. 1994; 219: 751-757Crossref PubMed Scopus (67) Google Scholar). These two effects can act synergistically to produce, potentially, a more than 50-fold activation of AMPK (48Weekes J. Hawley S.A. Corton J. Shugar D. Hardie D.G. Eur. J. Biochem. 1994; 219: 751-757Crossref PubMed Scopus (67) Google Scholar). Because the effects of 5′-nucleotides on the two enzymes are independent (49Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar), it is theoretically quite possible for a given nucleotide, like N 6-MPR, to cause a substantial activation of AMPK by binding to the upstream kinase rather than to AMPK itself. In the present work, hepatocellular cholesterol synthesis has been used as an indirect measure of AMPK activity. However, the data also demonstrate that all the investigated nucleosides cause a strong suppression of HMG-CoA reductase activity and of cholesterol formation, raising the possibility that the latter effects could be instrumental in mediating the inhibition of autophagy. The effects of lovastatin and simvastatin, two potent and specific inhibitors of HMG-CoA reductase (25Alberts A.W. Cardiology. 1990; 77: 14-21Crossref PubMed Scopus (103) Google Scholar), were therefore investigated. Both inhibitors completely blocked cholesterol synthesis while having no effect on autophagy. It would thus seem clear that neither HMG-CoA reductase activity nor de novo cholesterol formation is required for autophagic activity. The effects of adenosine, AICAR, and N 6-MPR on cholesterol metabolism and on autophagy should probably be regarded as two independent consequences of AMPK activation: HMG-CoA reductase phosphorylation/inhibition and modification of another as yet unknown protein. In conclusion, the present results suggest that hepatocytic autophagy can be suppressed by AMP through activation of AMPK. The adenosine-antagonistic effect of ITu would rule out a direct regulation by adenosine, making it unlikely that adenosine (an RNA degradation product) can function as a physiological feedback inhibitor of autophagic degradation. Although 0.5 mm added adenosine could elevate intracellular AMP levels dramatically in the presence of dCF, physiological adenosine concentrations would probably be too low to have much regulatory effect in the absence of inhibitors. On the other hand, high levels of AMP can be reached under hypoxia and other conditions of energy depletion (10Hue L. Biochem. J. 1982; 206: 359-365Crossref PubMed Scopus (54) Google Scholar, 11Aw T.Y. Jones D.P. Am. J. Physiol. 1989; 257: C435-C441Crossref PubMed Google Scholar, 12Bontemps F. Vincent M.F. Van den Berghe G. Biochem. J. 1993; 290: 671-677Crossref PubMed Scopus (58) Google Scholar, 13Carabaza A. Ricart M.D. Mor A. Guinovart J.J. Ciudad C.J. J. Biol. Chem. 1990; 265: 2724-2732Abstract Full Text PDF PubMed Google Scholar), which also cause a suppression of autophagy (4Plomp P.J.A.M. Wolvetang E.J. Groen A.K. Meijer A.J. Gordon P.B. Seglen P.O. Eur. J. Biochem. 1987; 164: 197-203Crossref PubMed Scopus (78) Google Scholar, 5Seglen P.O. Grinde B. Solheim A.E. Eur. J. Biochem. 1979; 95: 215-225Crossref PubMed Scopus (355) Google Scholar, 6Schellens J.P.M. Vreeling-Sindelárová H. Plomp P.J.A.M. Meijer A.J. Exp. 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