3‐Methyladenine, an inhibitor of autophagy, has multiple effects on metabolism

Abstract

3-Methyladenine is generally used as an inhibitor of autophagy [P. 0. Seglen & P. B. Gordon (1982) Proc.Natl Acad. Sci. U S A 79, . Using isolated hepatocytes, we observed that 3-methyladenine has other effects as well.1. 3-Methyladenine promoted glycogen breakdown and inhibited flux through phosphofructokinase and pyruvate kinase. These effects proved to be unrelated to inhibition of autophagic proteolysis and were caused by CAMP, which slightly increased in the presence of 3-methyladenine.2. Addition of 3-methyladenine to intact hepatocytes increased the intralysosomal pH and caused a lower density of the lysosomal population upon centrifugation in a Percoll density gradient. No increase in the intralysosomal pH was effected by 3-methyladenine in isolated lysosomes.3-Methyladenine is a powerful inhibitor of autophagy and primarily interferes with the formation of autophagosomes [1 -31. In the course of a study of factors involved in the control of proteolysis in isolated rat hepatocytes we observed that 3-methyladenine has other effects on cellular functions in addition to its action as an inhibitor of autophagy. Since 3-methyladenine is now widely used as a specific inhibitor of autophagy, the interpretation of results obtained with this compound should take such additional effects into account. MATERIALS AND METHODS HepatocytesHepatocytes were isolated from the livers of male Wistar rats (200-250 g) using the method of Berry and Friend [4] with the modifications described by Groen et al. [5].Hepatocytes (5 -10 mg dry mass/ml) were incubated at 37 "C in 25-ml plastic counting vials with Krebs-Ringer bicarbonate buffer, containing 1.3 mM Ca2+ and the additions mentioned in the legends to the figures and tables. The gas phase was 95% O2 plus 5% C 0 2 and the final volume was 2 -4 ml. Incubations were terminated by the addition of cold sulfosalicylic acid (final concentration 4%, mass/vol.) for the analysis of valine, or by the addition of HC104 (final concentration 3.5%, mass/vol.) for measurement of glucose and lactate. Samples for valine analysis were brought to pH 2.2 with 3 M LiOH. Perchloric acid extracts were neutralised to pH 7 with a mixture of 2 M KOH and 0.3 M Mops.For determination of the intracellular content of metabolites, the cells were centrifuged through a layer of silicone oil (Wacker, AR 200: AR 20 = 3: 2) into a layer of 14% HC104.Proteolysis was measured as the rate of appearance of valine in the incubation medium [6].

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

“…[174][175][176] Similarly, 3-methyladenine affects multiple facets of cellular metabolism. 177 Thus, while tests with pharmacological inhibitors constitute useful starting points for experimentation, they cannot be employed as surrogates of genetic studies based on gene knockout or RNAi.…”

Biogenesis of Multilamellar Bodies via Autophagy

“…3-MA treatment is associated with increased lysosomal pH and decreased lysosomal density and with inhibition of late endosome to lysosome transport (Caro et al, 1988;Punnonen et al, 1994), which may explain the slight enlargement of LAMP-2-positive structures in both GlcNAc-TV-transfected and untransfected Mv1Lu cells ( Figure 6, B and D). Nevertheless, 3-MA treatment of GlcNAc-TV transfectants results in the disappearance of large LAMP-2-labeled vacuoles corresponding to MLBs as well as the significant reduction in morphologically identifiable MLBs by electron microscopy.…”

Does Autophagy Contribute To Cell Death?

“…83 Secondly, 3-MA is not a pharmacologically potent compound, and is commonly utilized at millimolar concentrations; at these concentrations, additional effects have been reported, including the inhibition of Jun N-terminal kinase (JNK) and p38 kinase, both of which are involved in the regulation of stress-induced cell death. 84,85 Clearly, more specific loss-of-function approaches to interrogate the role of various atg genes in cell death have been long overdue. As discussed in the previous section, several studies have identified the www.landesbioscience.com Autophagyrequirement for atg orthologues in certain cell death processes.…”