Cell extracts were immunoblotted with an antibody against Ser79-phosphorylated ACC. Piperidolate hydrochloride 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 -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 -subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the -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 -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 JTK12 as by AICAR. for 4?min in the cold (4?C), resuspended in 4?ml of ice-cold unbuffered (electrolyte-free) 10% (w/v) sucrose and Piperidolate hydrochloride centrifuged again. The resuspension and centrifugation were repeated once more and, finally, the cell pellet was resuspended in 0.5?ml of 10% sucrose, briefly warmed to 37?C and electrodisrupted by a single high-voltage (2?kV/cm) pulse. The disrupted cells were centrifuged through a metrizamide/sucrose cushion, and the amount of LDH in the resulting cytosol-free sediment was measured and expressed as a percentage (autophagically sequestered per hour) of the total cellular LDH in the sample [27]. RESULTS Stimulation of AMPK phosphorylation by AICAR but not by amino acids Autophagic activity in isolated rat hepatocytes has been shown to be strongly suppressed by adenosine, AICAR and various adenosine analogues [7,28]. The suppression of autophagy can be eliminated by the adenosine kinase inhibitor, 5-iodotubercidin, suggesting mediation by AMP and AMP analogues and a possible involvement of AMPK [7,28]. Hepatocytic autophagy can also be inhibited by amino acid mixtures [11], as well as by OA and other algal toxins [22], but the mechanisms of action of these agents are not known. AMPK is activated allosterically by AMP; however, in addition, its activity is absolutely dependent on phosphorylation at Thr172 by an upstream protein kinase [29], recently identified as LKB1 [30,31]. The availability of a commercial, phosphospecific antibody that detects phosphorylation of AMPK at Thr172 has allowed a closer examination of the effects of autophagy suppressants on AMPK activation. As shown in Figure 1(A), treatment of freshly isolated rat hepatocytes with AICAR induced a dosedependent phosphorylation of AMPK at Thr172, indicating activation of the enzyme. Probably, the direct binding of AMP to AMPK alters its susceptibility towards phosphorylation by LKB1, although it has also been suggested that the AMPK-phosphorylating enzyme itself could be allosterically activated by AMP [29]. Some basic AMPK phosphorylation could usually be detected, but it varied, probably reflecting various degrees of hypoxia sustained during hepatocyte preparation. This variable background probably accounted for the variability (in the range Piperidolate hydrochloride 0.03C1?mM) in the AICAR concentrations needed to obtain a detectable effect. Open in a separate window Figure 1 Stimulation of AMPK phosphorylation by AICARFreshly isolated rat hepatocytes were incubated for 1?h at 37?C with (A) AICAR or (B) a physiological amino acid mixture, at the concentrations indicated. Cell extracts were immunoblotted with an antibody against the Thr172-phosphorylated AMPK -subunit. The position of a 66?kDa marker protein is indicated. In addition to increasing the pThr172 immunoreactivity of the single AMPK band seen in unstimulated cells, AICAR treatment induced immunoreactivity at two adjacent bands of lower mobility (Figure 1A, arrows). Since Thr172 phosphorylation could be detected in all three bands, additional structural or conformational changes associated with AMPK activation.