Skeletal Muscle Interleukin-6 Regulates Hepatic Cytochrome P450 Expression: Effects of 16-Week High-Fat Diet and Exercise

TOXICOLOGICAL SCIENCES, 162(1), 2018, 309–317 doi: 10.1093/toxsci/kfx258 Advance Access Publication Date: November 20, 2017 Research Article Jakob G. Knudsen,* Lærke Bertholdt,* Anders Gudiksen,* Sabine Gerbal-Chaloin,† and Martin Krøyer Rasmussen‡,1 *Department of Biology, Copenhagen University, DK-2200 Copenhagen, Denmark; †IRMB, INSERM, University of Montpellier, F-34290 Montpellier, France; and ‡Department of Food Science, Aarhus University, DK-8830 Tjele, Denmark 1 To whom correspondence should be addressed at Department of Food Science, Aarhus University, Blichers Alle 20, PO Box 50, DK-8830 Tjele, Denmark. Fax: 8715 4891; E-mail: . ABSTRACT High-fat diet (HFD) induces several changes to the pathways regulating energy homeostasis and changes the expression of the hepatic cytochrome p450 (Cyp) enzyme-system. Despite these pervious findings, it is still unclear how the effects of HFD and especially HFD in combination with treadmill running affect hepatic Cyp expression. In this study, we investigated the mRNA and protein expression of selected Cyp’s in mice subjected to 16 weeks of HFD and treadmill running. To understand the regulatory mechanisms behind the exercise-induced reversion of the HFDinduced changes in Cyp expression, we used a model in which the exercise-induced myokine and known regulator of hepatic Cyp’s, interleukin-6 (IL-6), were knocked out specifically in skeletal muscle. We found that HFD increased the mRNA expression of Cyp1a1 and Cyp4a10, and decreased the expression of Cyp2a4, Cyp2b10, Cyp2e1, and Cyp3a11. HFD in combination with treadmill running reversed the HFD increase in Cyp4a10 mRNA expression. In addition, we observed increased Cyp1a and Cyp3a protein expression as an effect of exercise, whereas Cyp2b expression was lowered as an effect of HFD. IL-6 effected the response in Cyp3a11 and Cyp1a expression. We observed no changes in the content of the aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, or peroxisome proliferation activator receptor alpha. In conclusion, we show that both HFD and exercise in HFD-fed animals can regulate hepatic Cyp expression and that changes in Cyp3a in response to HFD and exercise are dependent on skeletal muscular IL-6. Key words: detoxification; endurance training; IL-6; nuclear receptors; drug metabolism. The liver is the main organ for the biotransformation of xenobiotics, presenting high expression and activity of the cytochrome P450 (CYP) system. In general, biotransformation is conducted in two phases, where the CYPs belonging to family 1–3 are part of phase I, usually adding reactive groups to the parent compound. Accordingly, the regulation and expression of the CYP system is very important for the wellbeing of the individual and has gain a lot of attention during the last decades especially from the pharmaceutical industry. In humans, at least 57 genes coding for CYPs have been identified (Lewis, 2004). In the liver, CYP3A4 is the most predominant isoform taking care of >50% of all prescribed drugs (Guengerich, 2008). The transcriptional regulation of CYP is mediated by a number of transcription factors, including nuclear receptors (NR). It is generally accepted that the CYP subfamily 1A is regulated by aryl hydrocarbon receptor (AhR), 2A and 2B by constitutive androstane receptor (CAR), 3A by pregnane X receptor (PXR), and 4A by peroxisome proliferation activator C The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. V All rights reserved. For Permissions, please e-mail: 309 Skeletal Muscle Interleukin-6 Regulates Hepatic Cytochrome P450 Expression: Effects of 16-Week High-Fat Diet and Exercise 310 | TOXICOLOGICAL SCIENCES, 2018, Vol. 162, No. 1 MATERIALS AND METHODS Animals and intervention. Mice with skeletal muscle specific knockout of the IL-6 gene (IL-6 MKO) were generated by crossing C57Bl/6 mice with a LoxP site surrounding the second exon of the IL-6 gene with mice expressing CRE recombinase under the control of the myogenin promoter as previously described in Ferrer et al. (2014) and Knudsen et al. (2015). At the age of 12 weeks, IL-6 MKO and control (Floxed; control) mice were divided into 3 groups, with 10 in each, receiving either (1) standard rodent chow (Chow), (2) HFD, or (3) HFD combined with exercise for 16 weeks (HFD ExTr). The exercise regimen consisted of voluntary wheel running for the first 12 weeks of the intervention and voluntary wheel running combined with 3 h of weekly treadmill running for the last 4 weeks as previously described in Knudsen et al. (2015). The mice were given ad libitum access to food and water in a 12:12-h light:dark environment at a constant temperature of 22  C. At the end of the experiment, mice were euthanized by cervical dislocation and the liver removed and snap frozen in liquid nitrogen. Details about the mice have been published before in Knudsen et al. (2015, 2016). RNA isolation and reverse transcription. Total RNA were isolated using TriReagent, according to the manufactures protocol (Sigma-Aldrich, Schnelldorf, Germany). Equal amounts of RNA were converted into cDNA using iScript, according to the manufactures protocol (Bio-Rad, Solna, Sweden). Real-time PCR. For the analysis of the expression of the specific mRNA primers and TaqMan probes were designed using Primer Express (Version 2, Applied Biosystems, Californien, USA) and mouse-specific gene sequences obtained from Ensembl (http:// www.ensembl.org/Mus_musculus/Info/Index). All primers and probe pair were designed to expand exon-exon junctions and target specificity verified using BLAST searching. Primers and probe sequences are given in Table 1. The RT-PCR reaction were performed using the StepOne Plus thermocycler executing the following temperature profile: 50  C for 2 min, 95  C for 10 min and 40 cycles of 95  C for 15 s and 60  C for 1 min. All samples were analyzed in duplicates. The relative gene expression was normalized to the expression of GAPDH. There were no difference in the obtained Ct-values for GAPDH values between the different experimental groups. Western blotting. Protein lysate for western blotting were prepared as described elsewhere (Knudsen et al., 2015). Western blotting was done according to Rasmussen et al. (2016b). The used antibodies are given in Supplementary data 1. The relative protein expression of the analyzed Cyp’s were normalised to the protein expression of b-actin. Statistics. Data are presented as the mean 6 SEM. Two-way ANOVA was used to evaluate the effect of intervention (Chow, HFD, or HFD ExTr) and genotype (Floxed and IL-6 MKO). The data were log10 transformed if they failed an equal variance test. If an overall effect was observed, Tukey’s post hoc test was used to identify differences between groups. For all tests, p < .05 was regarded as significant. RESULTS Impact of HFD, Exercise Training and Skeletal Muscle IL-6 on Hepatic CYP The mRNA express (...truncated)