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), or N 6 -mercaptopurine riboside. Autophagic activity was inhibited by all three nucleosides, AICAR and N 6 -mercaptopurine riboside being more potent (IC 50 ‫؍‬ 0.3 mM) than adenosine (IC 50 ‫؍‬ 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.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"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 (2). 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...

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK alpha (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKalpha phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK alpha-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK beta-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the beta-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that beta-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR.

To identify phosphoproteins that might play a role in naringin-sensitive hepatocellular cytoskeletal disruption and apoptosis induced by algal toxins, hepatocyte extracts were separated by gel electrophoresis and immunostained with a phosphothreonine-directed antibody. Use of dilute (5%) polyacrylamide gels containing 6 M urea allowed the resolution of one very large (ϳ500-kDa) okadaic acid-and naringin-sensitive phosphoprotein, identified by tryptic fingerprinting, matrixassisted laser desorption/ionization time-of-flight mass spectrometry, and immunostaining as the cytolinker protein, plectin. The naringin-sensitive phosphorylation induced by okadaic acid and microcystin-LR probably reflected inhibition of a type 2A protein phosphatase, whereas the naringin-resistant phosphorylation induced by calyculin A, tautomycin, and cantharidin probably involved a type 1 phosphatase. Okadaic acid caused a collapse of the plectin-immunostaining bile canalicular sheaths and the general cytoskeletal plectin network into numerous medium-sized plectin aggregates. Inhibitors of protein kinase C, cAMP-dependent protein kinase, or Ca 2؉ /calmodulin-dependent kinase II had moderate or no protective effects on plectin network disruption, whereas naringin offered 86% protection. Okadaic acid induced a naringin-sensitive phosphorylation of AMP-activated protein kinase (AMPK), the stress-activated protein kinases SEK1 and JNK, and S6 kinase. The AMPK-activating kinase (AMPKK) is likely to be the target of inhibition by naringin, the other kinases serving as downstream components of an AMPKK-initiated signaling pathway.

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S -adenosylmethionine (AdoMet), into S -adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of (32)P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and (32)P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet-AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.