Original Basic 1 Leyendecker M et al. Insulin Lowers Ceruloplasmin Expression … Horm Metab Res 2011; 43: 1–7 HMR/2010-11-0358/27.1.2011/Macmillan ■ Proof copy for correction only. All forms of publication, duplication or distribution prohibited under copyright law. ■ received 30.11.2010 accepted 13.01.2011 Bibliography DOI http://dx.doi.org/ 10.1055/s-0031-1271692 Horm Metab Res 2011; 43: 1–7 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Dr. L-O. Klotz University of Alberta Faculty of Pharmacy and Pharmaceutical Sciences 3126 Dentistry/Pharmacy Centre Edmonton, Alberta Canada T6G 2N8 Tel.: + 1/780/492 5783 Fax: + 1/780/492 1217 larsoliver.klotz@ualberta.ca Key words ● ▶ ceruloplasmin ● ▶ liver ● ▶ insulin ● ▶ FoxO ● ▶ oxidative stress ● ▶ copper ● ▶ diabetes mellitus Ceruloplasmin Expression in Rat Liver Cells is Attenuated by Insulin: Role of FoxO Transcription Factors On the other hand, CP may be considered a pro- oxidant. For example, it was demonstrated that CP is capable of oxidizing low-density lipopro- tein (LDL) in vitro [6], likely mediated by a single redox-active copper ion exposed on the protein surface [7]. Interestingly, this process appears to be stimulated by enhanced plasma glucose lev- els, suggesting a link between the pro-oxidant activity of CP and atherosclerotic processes in diabetes mellitus [8]. Furthermore, exposure of CP to reactive oxygen species (ROS) was demon- strated to induce a conformational change of the protein, resulting in the release of loosely bound copper ions, thus potentiating pro-oxidative and damaging ROS effects [9, 10]. CP plasma levels were reported to be increased in diabetic patients [11–13]. Similar to CP, levels of proteins involved in glucose metabolism are altered in diabetic conditions, such as glucose 6- phosphatase (G6Pase), which was found upregu- lated in diabetic rats [14], and the expression of Introduction ▼ Ceruloplasmin (CP) is a 132 kDa plasma protein with 6 to 7 copper ions per molecule synthesized by hepatocytes. More than 95 % of the plasmatic copper is found in CP [1]. Both anti- and pro-oxi- dant properties were described for CP [2]. On the one hand, CP harbors a ferroxidase activity essen- tial to iron homeostasis in that oxidation of fer- rous to ferric iron is a prerequisite for its incorporation into transferrin. Moreover, decreas- ing the availability of the ferrous iron form pre- vents its involvement in Fenton-type reactions causing the generation of peroxyl or hydroxyl radicals [3, 4]. Loss-of-function mutations in the CP gene result in aceruloplasminemia, which is characterized by a deficiency of serum CP, and subsequent pathological iron accumulation in brain and visceral organs due to impaired iron homeostasis [1, 5]. Authors M. Leyendecker 1,10 , P. Korsten 2,10 , R. Reinehr 3 , B. Speckmann 4 , D. Schmoll 5 , W. A. Scherbaum 6 , S. R. Bornstein 7 , A. Barthel 7,8,11 , L-O. Klotz 1,9,11 Affiliations Affiliation addresses are listed at the end of the article Abstract ▼ The phosphoinositide 3 ′-kinase (PI3 K)/Akt path- way controls the activity of a number of proteins important in the regulation of apoptosis and cell proliferation. FoxO (forkhead box, class O) tran- scription factors, substrates of the Ser/Thr kinase Akt, control the expression of several target genes that are crucial to the defense against oxidative stress, the regulation of cell cycle, and apoptosis in mammalian cells. Here, expression of ceru- loplasmin (CP), the major copper-containing protein in blood released by the liver, was inves- tigated. We observed a significant downregula- tion of CP mRNA levels after insulin treatment in H4IIE rat hepatoma cells. The PI3K inhibitor wortmannin counteracted this insulin effect on CP mRNA levels, indicating that the PI3K/Akt cas- cade is involved in the regulation of CP expres- sion. Stimulation of FoxO1 was induced in H4IIE rat hepatoma cells expressing a conditionally active FoxO1 construct, resulting in significant upregulation of CP mRNA levels. This upregula- tion was prevented in the presence of insulin. In parallel, mRNAs of established FoxO target genes were analyzed: like CP mRNA, selenopro- tein P and glucose 6-phosphatase mRNAs were upregulated by FoxO1, which was prevented by insulin. The same effects of insulin on CP mRNA levels were detected in primary rat hepatocytes. Furthermore, CP release into cell culture media was analyzed with primary hepatocytes and found to be attenuated by insulin. In line with its insulin-mimetic effects on cultured cells, Cu 2 + imitated the effect of insulin on CP expression and caused a downregulation of CP mRNA levels in rat hepatoma cells.