Down-regulation of AMP-activated protein kinase by calorie restriction in rat liver Kazuo To, Haruyoshi Yamaza, Toshimitsu Komatsu, Takahiro Hayashida, Hiroko Hayashi, Hiroaki Toyama, Takuya Chiba, Yoshikazu Higami, Isao Shimokawa * Department of Investigative Pathology, Unit of Basic Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, 12-4 Sakamoto 1-chome, Nagasaki City 852-8523, Japan Received 26 December 2006; received in revised form 21 April 2007; accepted 5 July 2007 Available online 18 July 2007 Abstract AMP-activated protein kinase (AMPK) may act as a key enzyme for metabolic adaptation to calorie restriction (CR) or reduced growth hormone (GH)-insulin-like growth factor (IGF)-1 signaling, an experimental intervention for lifespan extension in animals. We investigated the protein levels of AMPKa and a downstream enzyme, acetyl-CoA carboxylase (ACC), by immunoblotting of liver and quadriceps femo- ris muscle (QFM) extracts from 6-month-old wild-type (W) and GH-suppressed transgenic (Tg) Wistar rats fed ad libitum (AL) or 30% CR diets from 6 weeks of age. A modified alternate-day feeding regimen for CR yielded a fed-fasted cycle in CR rats, and therefore the effects of overnight fasting in W-AL rats were also evaluated. CR decreased threonine-172-phosphorylated AMPKa (p-AMPKa; an activated form) levels in the liver, whereas the CR-fed-fasted cycle or overnight fasting did not significantly affect the p-AMPKa level. In the QFM, the p- AMPKa level was slightly elevated in the CR-fasted phase, but greatly increased in the AL-fasted phase. Suppression of GH did not affect the p-AMPKa level. The phosphorylated-ACC levels did not alter in parallel with the p-AMPKa level, particularly in the liver. The present results suggest that CR down-regulates the AMPK activity in the liver on a long-term basis. Ó 2007 Published by Elsevier Inc. Keywords: AMP-activated protein kinase; Acetyl-CoA carboxylase; Calorie restriction; Aging; Growth hormone; IGF-1; Metabolic adaptation 1. Introduction Moderate restriction of food intake while supplying essential nutrients, referred to as calorie restriction (CR), retards physiological aging and disease processes and pro- longs lifespan in laboratory animals, compared to control animals that are allowed to free access to food (Masoro, 2003; Weindruch and Walford, 1988). The effect of CR has been confirmed in many species, including laboratory rodents (Longo and Finch, 2003), thereby supporting the proposed hypothesis of an evolutionarily conserved mech- anism that responds to reduced food intake and regulates aging and longevity in animals (Holliday, 1989; Masoro and Austad, 1996). Recent studies on long-lived mutants of lower organisms and rodents have also indicated that reduced insulin-like signaling or growth hormone (GH)–IGF-1 signaling extends the lifespan of these animals (Longo and Finch, 2003). Since CR diminishes GH–IGF-1 signaling as well as serum insulin levels, the GH–IGF-1 axis could comprise one of the putative mechanisms by which CR affects aging and lifespan in animals (Shimokawa, 2006a). Analyses of the respiratory quotient in CR rats have indi- cated that a metabolic shift occurs during a feeding cycle, depending on food availability (Duffy et al., 1989; McCarter and Palmer, 1992). CR rats predominantly metabolize pro- teins and lipids in the period before feeding when the carbo- hydrate reserves are depleted (fasted phase), but metabolize carbohydrates immediately after feeding (fed phase). These findings also imply that anabolic processes, including lipo- genesis, proceed efficiently in CR rats when food is available, 0531-5565/$ - see front matter Ó 2007 Published by Elsevier Inc. doi:10.1016/j.exger.2007.07.003 * Corresponding author. Tel.: +81 95 849 7051; fax: +81 849 7052. E-mail address: shimo@nagasaki-u.ac.jp (I. Shimokawa). www.elsevier.com/locate/expgero Available online at www.sciencedirect.com Experimental Gerontology 42 (2007) 1063–1071