Biochemical and Biophysical Research Communications
Fenofibrate activates AMPK and increases eNOS phosphorylation in HUVEC☆
Section snippets
Materials and methods
Materials. Cell culture media and supplements were purchased from Kurabo Industries (Osaka, Japan). Fenofibrate was a kind gift from Kaken Pharmaceutical (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Gibco, Invitrogen (Carlsbad, CA). Wortmannin was purchased from EMD Biosciences (La Jolla, CA). Anti-phosphorylated AMPKα (Thr-172), AMPKα, phosphorylated acetyl-CoA carboxylase (ACC) (Ser-79), phosphorylated eNOS (Ser-1177), phosphorylated Akt (Thr-308), and Akt antibodies were from
Fenofibrate increased eNOS phosphorylation and NO production in HUVEC
First, we investigated the effects of fenofibrate on eNOS phosphorylation and expression in HUVEC. Fenofibrate treatment induced a rapid and significant increase in eNOS phosphorylation at Ser-1177. This increase was observed from 2.5 to 10 min after treatment (Figs. 1A and B). Although fenofibrate is known to increase eNOS expression, the total eNOS protein level was not altered over the 10 min.
We next examined the phosphorylation of AMPK and Akt, which are upstream kinases for eNOS [9]. In the
Discussion
In the present study, we demonstrated for the first time that fenofibrate can activate AMPK by increasing AMPK phosphorylation in HUVEC. Although we did not perform a strict kinase assay of AMPK, we are certain that fenofibrate indeed activates this kinase for following reasons. (1) The extent of AMPK phosphorylation at Thr-172 strongly reflects its activity [12]. (2) Fenofibrate indeed increased phosphorylation of its consensus substrate, ACC, at Ser-79. The ability of fenofibrate to activate
Acknowledgments
We thank Dr. Keiji Naruse (Department of Cardiovascular Physiology, Okayama University) and Mikie Takahashi (Department of Physiology II, Nagoya University) for their excellent technical assistance.
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Abbreviations: AMPK, 5′-AMP-activated protein kinase; eNOS, endothelial nitric oxide synthase; HUVEC, human umbilical vein endothelial cells.