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), 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...

Inhibition of Hepatic Phosphatidylcholine Synthesis by 5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside Is Independent of AMP-activated Protein Kinase Activation

“…Our results cast doubt on this assertion, since, in our hands, ATP levels decreased dose-dependently above 50 M AICAr. This is consistent with previous reports that concentrations of AICAr above 0.5 mM deplete ATP levels in both hepatocytes and liver (27,37,40). Although the exact mechanism is unknown, it has been suggested that AICAr inhibits hepatic oxidative phosphorylation and glycolysis by an AMPK-independent mechanism (37,41).…”

Recycle or die: The role of autophagy in cardioprotection

“…Generation of ATP via oxidative phoshorylation is inhibited during ischemia and reduced ATP levels have been shown to induce autophagy [1,10]. AMP kinase (AMPK) is involved in activating energy-generating pathways to maintain or restore ATP levels [54] and has been reported to upregulate autophagy [55][56][57]. AMPK is activated during ischemia and was recently shown to play a role in upregulaing autophagy during ischemia [10].…”

Hsp90 regulates processing of NF-κB2 p100 involving protection of NF-κB-inducing kinase (NIK) from autophagy-mediated degradation

“…As shown in Figure 4A, AICAR, an inhibitor of autophagy [21,29], efficiently prevented NIK degradation induced by GA (top panel, lane 3). The role of AICAR is specific, because it failed to protect Akt, a well-known client of Hsp90, from GA-mediated degradation (middle panel, lane 3).…”