Forskolin attenuates the action of insulin on the Akt–mTOR pathway in human skeletal muscle Scott R. Richmond, Chad D. Touchberry, and Philip M. Gallagher Abstract: Forskolin (FSK) is capable of both stimulating and inhibiting the intracellular signaling pathways of protein syn- thesis tissues other than skeletal muscle. The purpose of this investigation was to determine if FSK administration affects various elements of the protein kinase B (Akt)–mammalian target of rapamycin (mTOR) pathway in human skeletal muscle. Ten (n = 10) healthy, young (21.6 ± 1.3 years), nonobese (body mass index = 25.5 ± 3.5 kgÁm –2 ), recreationally active males were selected for participation. Following an 8 h fast, 2 muscle biopsies of the vastus lateralis were per- formed. The samples were sectioned and exposed to 4 in vitro treatment conditions: basal, FSK, insulin (INS), and FSK+INS. The samples were then analyzed for total and phosphorylated levels of Akt, mTOR, S6 kinase (S6K1), and 4E binding protein (4EBP1). Akt phosphorylation was significantly greater in the INS-treated samples compared with the basal and FSK conditions (p = 0.007). Furthermore, the ratio of phosphorylated Akt to total Akt (P/T) was higher in the INS samples compared with the basal and FSK samples (p = 0.001). There were no differences in mTOR phosphorylation among the 4 groups; however, total mTOR was significantly greater in the FSK+INS group (p = 0.006). There were also no differences in phosphorylated or total levels of S6K1 among the 4 groups. However, 4EBP1 phosphorylation was sig- nificantly greater in the INS-treated samples compared with the basal (p = 0.003) and FSK (p = 0.004) treatments. There were no differences in the ratio of phosphorylated 4EBP1 to total 4EBP1 (P/T) among the 4 groups. These results indicate that FSK does not activate the Akt–mTOR pathway in human skeletal muscle; however, these results suggest that FSK may inhibit the actions of INS on this pathway. Key words: insulin, protein kinase B (PKB), p70 ribosomal protein S6 kinase 1 (S6K1), 4EBP1, cAMP. Re ´sume ´: La forskoline (FSK) a la capacite ´ de stimuler et d’inhiber les voies intracellulaires de signalisation de la syn- the `se des prote ´ines tissulaires autres que dans le muscle squelettique. Cette e ´tude se propose d’analyser si l’administration de la FSK affecte plusieurs constituants de la voie de signalisation de l’Akt–mTOR dans le muscle squelettique humain. Dix jeunes hommes (21,6 ± 1,3 ans) en bonne sante ´, non obe `ses (IMC = 25,5 ± 3,5 kgÁm –2 ) et pratiquant des loisirs actifs sont choisis pour cette e ´tude. Apre `s une pe ´riode de jeu ˆne d’une dure ´e de 8 h, on pre ´le `ve deux e ´chantillons de tissu muscu- laire du vastus lateralis. Les e ´chantillons segmente ´s sont soumis a ` quatre conditions in vitro : contro ˆle, FSK, insuline (INS) et FSK+INS. Par la suite, on de ´termine la concentration totale et la concentration phosphoryle ´e d’Akt, de mTOR, de S6K1 et de 4EBP1. Comparativement aux conditions de contro ˆle et de FSK, la phosphorylation de l’Akt est significative- ment plus grande (p = 0,007) dans les e ´chantillons expose ´s a ` l’INS. De plus, comparativement aux conditions de contro ˆle et de FSK, on calcule un plus haut ratio d’Akt phosphoryle ´e / Akt totale (P/T) dans les e ´chantillons expose ´s a ` l’INS (p = 0,001). On n’observe pas de diffe ´rences de degre ´ de phosphorylation de la mTOR dans les quatre conditions, mais on note une plus grande concentration significative de mTOR totale dans le groupe FSK+INS (p = 0,006). On n’observe pas de diffe ´rences de concentration de S6K1 totale ou phosphoryle ´e d’une condition a ` l’autre. Ne ´anmoins, on observe une plus grande phosphorylation significative de 4EBP1 dans la condition INS que dans la condition de contro ˆle (p = 0,003) et FSK (p = 0,004). Dans les quatre conditions, on n’observe pas de diffe ´rence du ratio 4EBP1 phosphoryle ´e / 4EBP1 totale (P/T). D’apre `s ces observations, la FSK n’active pas la voie de l’Akt–mTOR dans le muscle squelettique humain; cepen- dant la FSK semble inhiber les actions de l’INS dans cette voie. Mots-cle ´s : insuline, prote ´ine kinase B, p70 S6 kinase ribosomique (S6K1), 4EBP1, cAMP. [Traduit par la Re ´daction] Introduction Forskolin (FSK) is capable of inducing cell differentiation through the direct activation of adenylate cyclase (AC) and the stimulation of cyclic AMP (cAMP) production (Adnot et al. 1982; Litosch et al. 1982; Gonza ´lez et al. 1983; Gesta et al. 2003). Although FSK has been shown to increase in- tracellular cAMP levels in various cell cultures and tissues, the end results of this change can be quite different, depend- ing on cell type (Adnot et al. 1982; Graves et al. 1995; Ii- jima et al. 2002; Mei et al. 2002; Sakamoto et al. 2002; Kwon et al. 2004; Machida et al. 2005). Most research has Received 23 February 2009. Accepted 14 May 2009. Published on the NRC Research Press Web site at apnm.nrc.ca on 10 October 2009. S.R. Richmond, C.D. Touchberry, and P.M. Gallagher. 1 Applied Physiology Laboratory, University of Kansas, Lawrence, KS 66045, USA. 1 Corresponding author (e-mail: philku@ku.edu). 916 Appl. Physiol. Nutr. Metab. 34: 916–925 (2009) doi:10.1139/H09-096 Published by NRC Research Press