Hepatic PGC-1α is not essential for fasting-induced cytochrome p450 regulation in mouse liver

Journal Pre-proofs Hepatic PGC-1α is not essential for fasting-induced cytochrome p450 regulation in mouse liver Rebekka Thøgersen, Caroline Maag Kristensen, Mette Algot Olsen, Hanne Christine Bertram, Henriette Pilegaard, Martin Krøyer Rasmussen PII: DOI: Reference:

S0006-2952(19)30435-6 https://doi.org/10.1016/j.bcp.2019.113736 BCP 113736

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

30 October 2019 20 November 2019

Please cite this article as: R. Thøgersen, C. Maag Kristensen, M. Algot Olsen, H. Christine Bertram, H. Pilegaard, M. Krøyer Rasmussen, Hepatic PGC-1α is not essential for fasting-induced cytochrome p450 regulation in mouse liver, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.113736

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Hepatic PGC-1α is not essential for fasting-induced cytochrome p450 regulation in mouse

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liver

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Rebekka Thøgersen1, Caroline Maag Kristensen2, Mette Algot Olsen2, Hanne Christine

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Bertram1, Henriette Pilegaard2 and Martin Krøyer Rasmussen1

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Department of Food Science, Aarhus University, Denmark

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2

Department of Biology, Copenhagen University, Denmark

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Corresponding author

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Martin Krøyer Rasmussen

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Department of Food Science, Aarhus University, Denmark

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Agro Food Park 48

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DK-8200 Aarhus N

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Tel.: + 45 22 16 93 90

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Fax: 87 15 48 91

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Email: [email protected]

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Abstract

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Fasting has been shown to regulate the expression of the cytochrome p450 (CYP) enzyme

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system in the liver. However, the exact mechanism behind the fasting-induced regulation of

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the CYP’s remains unknown. In the present study we tested the hypothesis that the peroxisome

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proliferator-activated receptor gamma coactivator 1α (PGC-1α), which is a key-regulator of

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energy metabolism, is responsible for the fasting-induced regulation of the CYP’s. Lox/lox and

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liver specific PGC-1α (LKO) mice of both sexes, fasted for 18 hours and the content of the

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CYP’s as well as the hepatic metabolome was assessed. Fasting increased the mRNA content

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of Cyp2a4, Cyp2e1, Cyp3a11 and Cyp4a10. The fasting-induced response in Cyp4a10 mRNA

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content was different between lox/lox and LKO mice, while the absence of PGC-1α had no

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effect on the fasting-induced response for the other Cyp’s. Moreover, the fasting-induced

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response in mRNA content of Sirtinus 1 and Perilipin 2 was different between lox/lox and LKO

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mice. Only the CYP1A isoform showed a fasting-induced response at the protein level.

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Absence of hepatic PGC-1α had no effect on the apparent metabolome, where fasting vs fed

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was the only discriminate in the following multivariate analysis. In conclusion, hepatic PGC-

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1α is not essential for the fasting-induced regulation of hepatic CYP’s.

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Keywords: Detoxification; Energy metabolism; Nuclear receptors; Liver; Metabolomics,

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PGC-1α

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1. Introduction

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The cytochrome p450 (CYP) enzyme system is in humans encoded by 57 genes [1], which are

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ordered into subfamilies. Of these, subfamily 1 to 3 contains the CYPs responsible for the bio-

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activation and elimination of a large number of toxins, drugs and endogenous compounds. The

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major expression site for CYP1-3 is the liver [2]. The CYPs display great adaptability in

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response to various physiological changes and their expression is dictated by age, medical

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history and nutritional status. In agreement, several studies have shown that both high-fat diet

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[3-6] and fasting [7-10] regulates the mRNA and protein content of the CYPs in the liver of

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both humans and animals. Although several molecules (e.g. cAMP) have been suggested to

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mediate the fasting-induced regulation of the CYPs [8], no clear description of the mechanisms

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underlying the transcriptional regulation of this response is provided in the literature.

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It is generally acknowledged that CYP1, 2 and 3 are under transcriptional regulation of the

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Aryl hydrocarbon receptor (AhR), Constitutive androstane receptor (CAR) and Pregnane X

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receptor (PXR), respectively. However, several other transcription factors and co-factors have

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also been reported to be involved in the transcriptional regulation of the CYPs [11, 12]. A

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number of these also contributes in regulating energy metabolism e.g. during fasting. The

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peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) is known as a

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master regulator of mitochondrial biogenesis, but has also been reported to regulate the

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antioxidant as well as key factors in hepatic gluconeogenesis [13, 14]. Interestingly, in a study

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using mouse primary hepatocytes, it was shown that PGC-1α mediated cAMP-induced

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expression of Cyp2a5 by co-activating HNF-4α. [15]. Likewise in mouse primary hepatocytes,

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PGC-1α overexpression upregulated the expression of PXR and potentiated pregnenolone-16a-

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carbonitrile dependent induction of Cyp3a11 [16]. Together, these studies suggests that PGC-

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1α might be responsible for the fasting induced response in Cyp expression. However, the

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importance of PGC-1α for the fasting-induced regulation of the CYP’s has not been 3

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investigated in in vivo settings before. Therefore, the aim of the study was to test the hypothesis

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that fasting-induced CYP regulation is mediated by hepatic PGC-1α. The fasting-induced CYP

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regulation, can be either through a direct interactions between PGC-1α and Cyp-regulating

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receptors or it can be indirect through PGC-1α dependent regulation of energy homeostasis,

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generating metabolites that might act as activators/deactivators of the transcription factors

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regulating CYP expression.

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To investigate the hypothesis, lox/lox (control) and liver-specific PGC-1α knock-out mice

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(LKO) were subjected to fasting for 18 hours and the response in gene and protein content of

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selected CYP’s and transcription factors were determined using real-time PCR and western

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blotting. To explore the possibility that PGC-1α might has an indirect effect on CYP

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transcription, we performed nuclear magnetic resonance spectroscopy (NMR) based

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metabolomics to elucidate if there were differences in the hepatic metabolome of lox/lox and

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LKO mice.

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2. Materials and methods

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2.1 Animals and experimental setup

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A total of 32 liver specific PGC-1α knock-out (LKO) mice (C57BL/6N) and corresponding

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wild type (lox/lox) mice were randomly divided into either a fasting or a fed group (n = 3 males

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and n =5 female in each group). The generation of the knock-out mice and post-genotyping is

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described elsewhere [17]. The mice were kept at a 12:12 light-dark cycle and had ad libitum

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access to water and standard chow (Altromin 1234; Brogaarden, Lynge, Denmark) until

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initiation of the study.

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At 14 weeks of age, food was removed from the fasting group at 2 a.m. and both fed and fasted

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mice were euthanized by cervical dislocation at 8–9 p.m. resulting in 18 h of fasting for the

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fasting group. The livers were quickly removed, frozen in liquid nitrogen and stored at - 80ºC

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until further analyses. The Danish Animal Experimental Inspectorate approved all

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experimental procedures.

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2.2 Gene expression

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Total RNA was extracted using TRI-reagent according to the manufactures protocol (Sigma-

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Aldrich, MO, USA) and equal amounts of RNA were subjected to reverse transcriptase using

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the iScript kit (Bio-Rad, California, USA).

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To determine the specific content of mRNA of selected genes, real-time PCR using TaqMan

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probes were performed as described elsewhere [18].

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Specific primer and probe pairs for the selected genes were designed according to Rasmussen

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et al., [10] and custom-made (LGC Biosearch Technologies, Risskov, Denmark). The used

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primers and TaqMan probe sequences are given in Table 1.

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Relative mRNA content was calculated from the obtained Ct-values using a standard curve and

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normalized to the mRNA content of β-actin. There were no significant difference in β-actin

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mRNA content between groups.

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2.3 Protein content

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Fifty mg liver tissue were homogenized in RIPA buffer (Sigma Aldrich, MO, USA)

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supplemented with 2 mM PSMF and incubated at 4 ºC for 60 minutes. Following centrifugation

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for 20 min at 20.000 x g, protein concentration in the supernatant was determined using Pierce

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BCA kit (Thermo Scientific, Roskilde, Denmark) with bovine serum albumin as standard.

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Equal amounts of protein were mixed 1:1 with Laemmli-buffer and separated by gel-

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electrophoresis using AnyKd gels (Bio-Rad, California, USA). Following transfer onto PVDF

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membranes using the TurboBlotter system (Bio-Rad, California, USA), membranes were

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blocked in 2% dry-milk powder dissolved in TBS-T (50 mM Tris, 500 mM NaCl, 0.1% Tween

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20; pH 7.4), before incubation overnight with primary antibodies (Cyp1a: Sc-53241 (Santa

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Cruz Biotechnology, Heidelberg, Germany); Cyp2b: Sc-53242 (Santa Cruz Biotechnology,

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Heidelberg, Germany); Cyp2e1: Ab28146 (Abcam, Cambridge, UK); Cyp3a: Sc-25845 (Santa

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Cruz Biotechnology, Heidelberg, Germany); Cyp4a: Sc-98988 (Santa Cruz Biotechnology,

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Heidelberg, Germany); α-tubulin: DM1A (Chalbiochem, Darmstadt, Germany)) at 4ºC. After

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several washes in TBS-T, the membranes were incubated with horseradish peroxidase

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conjugated secondary antibodies (Invitrogen, Paisley, UK) for 2 hours at room temperature.

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Specific protein bands were detected using ECL substrate and the ChemiDoc XRS+

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workstation (Bio-Rad, California, USA). Relative quantification was performed using the

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Image Lab software (Bio-Rad, California, USA) and normalized to the protein content of α-

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tubulin.

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2.4 1H NMR spectroscopy

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Chloroform-methanol extraction of liver samples was conducted according to a previously

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described method [19]. 1H NMR spectra of liver samples were obtained using a Bruker Avance

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III 600 MHz spectrometer operating at a frequency of 600.13 MHz and equipped with a 1H

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TXI probe (Bruker BioSpin, Rheinstetten, Germany). For analysis of the aqueous phase, water

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extract re-dissolved in 550 µL D2O, 25 µL ddH2O and 25 µL D2O with 0.05 wt. % TSP 3-

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(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP; Sigma-Aldrich, St. Louis, MO, USA) were

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transferred to a 5-mm NMR tube. 1H NMR spectra for the aqueous phase samples were

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obtained using a one-dimensional (1D) nuclear overhauser enhancement spectroscopy

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(NOESY)-presat pulse sequence (noesypr1d) at 298 K with the following acquisition

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parameters: 128 scans, a spectral width (SW) of 7288.63 Hz, 32768 data points (TD), an

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acquisition time (AQ) of 2.25 sec and a relaxation delay (D1) of 5 sec. For analysis of the

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chloroform phase, lipid extracts re-dissolved in 575 µL CDCl3 and 25 µL CDCl3 with 0.05 %

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v/v TMS (Cambridge Isotope Laboratories, Inc., MA, USA) were transferred to a 5-mm NMR

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tube. Spectra were obtained using a zg30 pulse sequence and the following acquisition

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parameters: 128 scans, a spectral width of 12019.23 Hz, 65536 data points, an acquisition time

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of 2.73 sec and a relaxation delay (D1) of 1 sec.

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Prior to Fourier transformation, free induction decays (FIDs) were multiplied by a line-

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broadening function of 0.3 Hz. In Topspin 3.0 (Bruker Biospin), the obtained spectra were

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baseline and phase corrected. Using MATLAB R2018b (Mathworks Inc., Natick, USA),

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spectra were referenced to TSP or TMS (0.0 ppm) for aqueous and chloroform phase samples,

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respectively, and corrected for chemical shifting using interval-correlation-shifting algorithm

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(icoshift) [20]. For aqueous phase spectra, spectral regions above 9.50 ppm and below 0.05

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ppm were removed in addition to region between 4.76 and 4.82 ppm containing the residual

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water signal. For 1H spectra obtained for the chloroform phase, spectral regions above 9.90

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ppm and below 0.05 ppm, the spectral region containing the chloroform signal between 6.90 7

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and 7.50 ppm and residual methanol signal between 3.32 and 3.46 ppm were removed. The

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spectra were normalized to the TSP signal or total area of the spectrum for aqueous phase and

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chloroform phase spectra, respectively. Subsequently, aqueous phase spectra were subdivided

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into regions of 0.005 ppm and chloroform phase spectra divided into regions of 0.01 ppm.

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Multivariate data analysis was conducted using SIMCA 15.0 (Sartorius Stedim Data Analytics

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AB, Umeå, Sweden). The data were pareto scaled and principal component analysis (PCA)

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was conducted. Orthogonal projections to latent structure discriminant analysis (OPLS-DA),

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with cross validation using seven segments, was used to investigate differences in the liver

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metabolome between the treatment groups. For the aqueous phase samples, one outlier was

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identified and removed based on PCA scores plot and Hotelling’s T2 plot revealing a T2 range

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value larger than the 95 % confidence interval limit. S-line plots were used to reveal metabolites

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important for the separation between treatment groups. Metabolites were identified using the

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software Chenomx NMR Suite 8.13 (Chenomx Inc, Edmonton, Canada) and literature [21].

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2.5 Statistics

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Data are presented as mean ± standard error of the mean. Three-way ANOVA was used to

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evaluate the effect of fasting, genotype and sex. If an overall effect was observed, Tukey’s post

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hoc test was used to identify difference between experimental groups. If equally variance test

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failed, data were log10 transformed before executing the ANOVA. For all tests, p < 0.05 was

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regarded as significant. Statistical tests were performed in SigmaPlot 11.0 (Systat Software,

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USA).

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3. Results

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3.1 Effect of PGC-1α on FIAF, Plin2 and SIRT1 mRNA content during fasting

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To evaluate the metabolic conditions of the mice, we examined the response of marker genes

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know to be induced by fasting. As expected, fasting for 18 hours induced a significant increase

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in the hepatic mRNA content of fasting induced adiponectin factor (FIAF) (6.6 – 17.6 fold),

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Perilipin 2 (Plin2) (5.0 – 8.2 fold) and sirtuin 1 (SIRT1) (1.4 – 3.0 fold) (Table 2). For Plin2,

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there was a difference between genotypes with lower Plin2 mRNA content in LKO than lox/lox

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mice in the fasted state. The induction of SIRT1 was significantly affected by genotype, and

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LKO had lower fasting-induced increases than lox/lox.

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3.2 Cytochrome P450 content

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There was no effect of fasting, genotype or sex on the mRNA content of Cyp1a1 and Cyp1a2

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in the liver (Table 2). On the other hand, mice subjected to fasting had significantly lower

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CYP1A protein content than fed mice (Figure 1). Moreover, male mice had higher CYP1A

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protein content than female mice.

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The mRNA content of Cyp2a4 in the liver was significantly higher in fasted mice than fed,

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which was not different between genotypes and sex (Table 2). Hepatic Cyp2b10 mRNA

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content was not affected by fasting and was not different between genotypes or sex. However,

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CYP2B protein content in the liver tended to be lower (P<0.1) in LKO than in lox/lox mice

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(Figure 1). Moreover, male mice had significantly higher hepatic CYP2B protein content than

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female mice, while fasting had no effect on CYP2B protein content. Cyp2e1 mRNA content in

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the liver was higher in fasted than fed mice, and also higher in male than female mice (Table

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2). At the protein level, no difference was observed in CYP2E1 content with fasting, genotype

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or sex (Figure 1).

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Hepatic mRNA content of Cyp3a11 was higher in fasting mice than in fed (Table 2). This effect

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was predominantly present in male mice. Moreover, male mice had higher content of Cyp3a11

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mRNA, than female mice. Fasting tended (p < 0.1) to increase hepatic CYP3A protein content

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compared to fed (Figure 1), while genotype and sex had no effect on the protein content of

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CYP3A.

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Fasting increased hepatic Cyp4a10 mRNA content relative to fed in both male and female mice,

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(Table 2). Moreover, LKO mice had lower fasting-induced increase in Cyp4a10 mRNA content

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in the liver than lox/lox mice. Fasting increased the protein content of CYP4A in the liver

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compared to fed (Figure 1), while genotype and sex had no effect on CYP4A protein content.

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3.3 AhR, CAR, PXR, PPARα and HNF4α mRNA

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Fasting, genotype and sex had no effect on the mRNA content of AhR in the liver (Table 2).

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Fasting increased the hepatic mRNA content of CAR, PXR, peroxisome proliferator-activated

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receptor α (PPARα) and hepatic nuclear factor 4α (HNF4α) compared to fed (Table 2). There

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were no differences with genotype and sex.

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3.4 Liver metabolome

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In order to investigate the effect of PGC-1α deficiency on liver metabolome, 1H NMR

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spectroscopy was used to identify water and lipid-soluble metabolites. Multivariate data

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analysis including PCA and OPLS-DA revealed that fed vs. fasting was the only factor by

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which the mice could be grouped. For the water-soluble metabolites, PCA revealed a clear

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separation between fed and fasted mice (data not shown). An OPLS-DA model for pairwise

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comparison of fasted vs. fed mice (Q2 = 0.51, Figure 2a) and associated s-line plot (Figure 2b)

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revealed that this separation was mainly due to higher glucose and maltose contents in the liver

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of fed mice and a higher 3-hydroxybutyrate content in the fasted mice. In addition, hepatic

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levels of lactate, alanine and glutathione were higher in fed mice than fasted mice. From the 10

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PCA scores plot for the lipid phase spectra, a grouping between fasted and fed mice was found

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(data not shown). However, an OPLS-DA model for pairwise comparison of fasted vs. fed mice

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showed only modest model predictability (Q2 = 0.3, Figure 2c). The associated S-line plot

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indicated higher content of lipids and glycerol in livers of fasting mice than fed mice (Figure

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2d).

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4. Discussion

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The main findings of the present study are that the observed fasting-induced increases in

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mRNA content of Cyp4a10 were dependent on the presence of PGC-1α, while the changes in

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the content of the other investigated CYP’s were independent of PGC-1α. Apart from CYP1A,

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the fasting-induced effects were not manifested at the protein level. Moreover, independent of

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PGC-1α, fasting also increased the mRNA content of selected transcription factors involved in

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the transcriptional regulation of the CYP’s. Likewise, hepatic PGC-1α was not required for the

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observed fasting-induced changes in the hepatic metabolome.

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To determine the significance of PGC-1α for the fasting-induced regulation of the CYP’s, we

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subjected both lox/lox and liver-specific PGC-1α knock-out mice to 18 h fasting. The used

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fasting protocol has in the same mice, been reported to result in > 90 % lower liver glycogen

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and > 35 %, lower plasma glucose as well as 2-3.5 fold higher plasma free fatty acid

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concentration and hepatic triglyceride content than the fed mice [17]. Notably, genotype had

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no effect on these metabolic parameters. Furthermore, in order to evaluate the changes induced

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to the metabolic pathways in these mice, the mRNA content of FIAF, also known as

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angiopoietin-like 4, and previously shown to regulate triglyceride metabolism [22] as well as

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Plin2, which is associated with lipid droplet formation [23] were determined. The observed

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increase in FIAF and Plin2 mRNA content in fasted mice, is in accordance with the previously

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observed upregulation of FIAF and Plin2 mRNA with fasting [24, 25] and confirms the

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metabolic state of the mice. Interestingly, we observed a genotype effect on the fasting-induced

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increase in Plin2. It is known that PPAR is involved in the transcriptional regulation of Plin2

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[25], and as PGC-1α is a co-factors for PPAR, this might explain the observed genotype-effect.

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Moreover, the observed fasting-induced increase in the mRNA content of SIRT1 is in

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accordance with previous studies [10, 26]. Taken together, these results confirm that the mice

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have been in a profound fasting state, and confirms that the model is valid for investigating the

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importance of hepatic PGC-1α for the fasting-induced response in CYP expression.

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The present finding that Cyp2a4, Cyp2e1, Cyp3a11 and Cyp4a10 mRNA content increased

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with fasting is in agreement with previous studies in mice [10, 27]. Surprisingly, we did not

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observe a fasting-induced increase in Cyp2b10 mRNA content. Several other studies have

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shown that this isoform is responsive to fasting [8, 10, 28]. It is not obvious what has caused

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the differences in the Cyp2b10 response, given that the present study used the same mouse

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strain and fasting time as the previous mentioned studies. Apart from AhR, all other

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investigated transcription factors were induced by fasting. This might partly explain the

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observed fasting-induced increases in CYP content. PGC-1α has been suggested to be a

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transcriptional regulator of the PXR gene in primary hepatocytes [16]. Moreover, PPARα has

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also been shown to regulate PXR content [29] and as we observed that the absence of PGC-1α

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affected other PPARα regulated genes (e.g. Cyp4a10), a genotype-effect on the fasting-induced

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increase in PXR could have be expected in the present study. However, this was not the case

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and suggests that more factor that the PGC-1α – PPARα axis is responsible for the

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transcriptional regulation of PXR.

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Acting as a coactivator, PGC-1α has been shown to regulate Cyp2a5 transcription in mouse

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primary hepatocytes together with HNF 4α [15]. Moreover, PGC-1α has also been suggested

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to mediate the fasting-induced increase in PXR and PXR-regulated genes, like Cyp3a11, in

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mice primary hepatocytes [16]. Contrary to the in vitro results, the present study show that

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PGC-1α is not essential for the fasting-induced regulation of the investigated CYP’s and the

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transcription factors; AhR, CAR and PXR. This finding suggests that PGC-1α is not the major

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convergence point between pathways regulating energy metabolism and detoxification or that

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alternative pathways can regulate CYP expression during fasting when PGC-1α is absent.

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Moreover, it should also be noted that the knockout of PGC-1α was restricted to the liver. Thus, 13

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systemic events mediated by PGC-1α could still be present and account for the observed

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fasting-induced modifications in CYP expression.

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PGC-1α is involved in the regulation of pathways for energy metabolism. Hence, PGC-1α

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deficiency may change the hepatic metabolome which eventually could be manifested in

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differentiated CYP regulation. Therefore, we determined the hepatic metabolome using NMR.

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Based on the hepatic metabolomes, the mice could only be differentiated according to whether

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they were in a fasting or a fed state. Thus, the hepatic knock-out of PGC-1α did not manifest

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into the metabolite profile of the liver. In Sprague-Dawley rats, Robertson et al [30] showed

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that 16 hours of fasting induced changes to the metabolome of the liver, demonstrating

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increased gluconeogenesis, which eventually leads to the generation of ketone bodies like 3-

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hydroxybutyrate. In accordance, the hepatic level of 3-hydroxybutyrate appeared to be one of

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the major discriminants for grouping the mice into the fasting and the fed state. Moreover, the

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observation that glucose, maltose and lactate decreased in fasting mice is in agreement with

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increased gluconeogenesis and the higher hepatic amounts of glycerol originates from an

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increased lipolysis. The finding that the lack of PGC-1α had no effect on the hepatic

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metabolome of the mice suggests, that the fasting-induced metabolome is not dictated by the

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presence of PGC-1α.

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It is well documented that mice display confound sex-differences in the abundance of a number

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of CYP’s and related transcription factors [31-33]. The present observation that the Cyp2e1

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and Cyp3a11 mRNA content was higher in male mice than in female mice and that Cyp2a4

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mRNA was higher in female mice than male are in accordance with previous studies [34-36].

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Similarly, the observation that CYP1A and CYP2B protein content was higher in male than

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female mice, is in agreement with previous observations for CYP1A [35], but not CYP2B [36].

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What causes this discrepancy in the observation of CYP2B protein content is not obvious, as

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the same mouse breed was used in both studies. 14

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Interestingly, the observation that the fasting-induced increase in Cyp3a11 mRNA content was

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greater in male mice than females corresponds with higher fasting-induced increases in PXR

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and HNF4α mRNA content in male than female mice, although no overall sex differences were

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shown for the two transcription factors. PXR and HNF4α are two important transcription

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factors in the regulation of the Cyp3a genes [37]. Hence, the sex-differentiated response in

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PXR and HNF-4α might explain the observed sex-dependent difference in the fasting-induced

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response of Cyp3a11.

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In conclusion, fasting increased the mRNA content of Cyp2a4, Cyp2e1, Cyp3a11 and

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Cyp4a10, as well as CAR, PXR and PPARα. Apart from Cyp4a10, none of the fasting-induced

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increases were affected by deficiency of hepatic PGC-1α. Hence, hepatic PGC-1α is not of

302

major importance for the fasting-induced increase in hepatic CYP 1, 2 and 3. Moreover, PGC-

303

1α is not required for the fasting-induced response in the hepatic metabolome. However,

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hepatic PGC-1α is of importance for the fasting-induced response in the PPARα regulated

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genes Cyp4a10 and Plin2.

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4.1 Study limitations

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It should be noticed that there are some methodological limitations in the present study. The

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deletion of a fragment of the PGC-1α gene was restricted to the liver, which enabled the

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investigation of the importance of PGC-1α in the liver for hepatic CYP expression. However,

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this also means that systemic factors regulated by extra-hepatic PGC-1α dependent pathways

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are not affected and could still be responsible for the fasting induced response in CYP

312

expression. Sex differences in the constitutive levels of CYP ‘s in mice are well known and

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also observed in the present study. However, the results should be taken with caution as the

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number of mice in the female group are relatively low.

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Acknowledgment 15

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This study was funded by a grant from the Danish Council for Independent Research - Medical

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Sciences to Henriette Pilegaard. In addition, Caroline Maag Kristensen was in part funded by

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a PhD stipend from the Danish Diabetes Academy.

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[34] M.J. Down, S. Arkle, J.J. Mills, Regulation and induction of CYP3A11, CYP3A13 and CYP3A25 in C57BL/6J mouse liver, Arch. Biochem. Biophys. 457(1) (2007) 105-10. [35] J.M. Chen, Q.S. Zhang, X.Y. Li, X. Gong, Y.J. Ruan, S.J. Zeng, L.L. Lu, X.X. Qi, Y. Wang, M. Hu, L.J. Zhu, Z.Q. Liu, Tissue Distribution and Gender-Specific Protein Expression of Cytochrome P450 in five Mouse Genotypes with a Background of FVB, Pharm. Res. 35(6) (2018) 114. [36] H.J. Renaud, J.Y. Cui, M. Khan, C.D. Klaassen, Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice, Toxicol. Sci. 124(2) (2011) 261-77. [37] R.G. Tirona, W. Lee, B.F. Leake, L.B. Lan, C.B. Cline, V. Lamba, F. Parviz, S.A. Duncan, Y. Inoue, F.J. Gonzalez, E.G. Schuetz, R.B. Kim, The orphan nuclear receptor HNF4alpha determines PXR- and CARmediated xenobiotic induction of CYP3A4, Nat. Med. 9(2) (2003) 220-4.

420 421

Table

422

Table 1. Primer and TaqMan probes for RT-PCR

423 424

Table 2. Relative mRNA content in fasted (Fast) and fed (Fed) male and female lox/lox or liver

425

specific PGC-1α knock-out mice (LKO).

426 427

18

428

Figure legends

429

Figure 1.

430

PGC-1α is not essential for the fasting induced response in CYP protein content in male and

431

female mice. Values are mean ± standard error of the mean. Lox, Lox/lox; LKO, liver specific

432

PGC-1α knockout mice, FED, fed mice; FAST, fasted mice. Representative protein blots; lane

433

1, female lox/lox fed; lane 2, female LKO fed; lane 3, female lox/lox fasted; lane 4, female

434

LKO fasted; lane 5, male lox/lox fed; lane 6, male LKO fed; lane 7, male lox/lox fasted; lane

435

8, male LKO fasted.

436

Figure 2.

437

PGC-1α is not essential for the fasting hepatic metabolome in mice following 18 hours of

438

fasting. OPLS-DA plot of metabolites in the water (A) (Q2 = 0.51) and lipid (C) (Q2 = 0.30)

439

phase of lox/lox and LKO mice. S-line plots of fasting vs. fed mice of metabolites in the water

440

(B) and lipid (D) phase.

441

Author contribution statement

442 443 444 445 446

M.K.R. and H.P. conceived the study. R.T, C.M.K., M.A.O. and M.K.R. did the experimental work. M.K.R., R.T. and H.C.B. did the data analysis. M.K.R. wrote the first draft of the manuscript supported by H.P. All authors contributed to writing of the final manuscript.

447 448

Highlights

449 450 451 452 453

   

Fasting-induced CYP regulation investigated in PGC-1α KO mice Fasting induced increase mRNA of Cyp2, Cyp3 and Cyp4 PGC-1α is not of major importance for fasting-induced CYP regulation PGC-1α absence did not effected the hepatic metabolome 19

454

Nam e

Forward (5´-3´)

Reverse (5´-3´)

TaqMan Probe (5´-3´)

Cyp1 A1

GACCTTCCGGCATTCAT CCT

GCCATTCAGACTTGTA TCTCTTGTG

CGTCCCCTTCACCATCCC CCA

Cyp1 A2

TGGAGCTGGCTTTGAC ACAG

CGTTAGGCCATGTCAC AAGTAGC

CACCACAGCCATCACCTG GAGCATTT

Cyp2 A4

TCGAGGAGCGCATCCA A

AATGAAAGCACCGTT CGTCTTC

AGGCGGGCTTTCTCATCG ATTCATTTC

Cyp2 B10

CCAGCCAGATGTTTGA GCTCTT

GGAGTTCCTGCAGGTT TTTGG

TTCCTGAAGTACTTTCCT GGTGCCCACA

Cyp2 E1

TTTCCCTAAGTATCCTC CGTGACT

GCTGGCCTTTGGTCTT TTTG

CCCGCATCCAAAGAGAG GCACACT

Cyp3 A11

AACTGCAGGATGAGAT CGATGAG

TTCATTAAGCACCATA TCCAGGTATT

CAACAAGGCACCTCCCA CGTATGATACTG

Cyp4 A10

TCCAGGTTTGCACCAG ACTCT

AGTTCCTGGCTCCTCC TGAGA

CGACACAGCCACTCATTC CTGCCC

AhR

GCGGCGCCAACATCA

GTCGCTTAGAAGGATT TGACTTAATTC

CAGAAAACAGTAAAGCC CATCCCCGC

CAR

TCAACACGTTTATGGTG CAGCCGCTCCCTTGAG CAACA AAG

ATCAAGTTCACCAAGGA TCTGCCGCTC

PXR

CACCTGGCCGATGTGT CA

AATAGGCAGGTCCCT AAAGTAGGATAT

CAAGGGCGTCATCAACTT CGCCAA

PPA Rα

CGCTGCCGCCAAGTTG

GAACTTGACCAGCCA CAAACG

AGGCCCTGCCTTCCCTGT GAACTG

HNF 4α

CCTGCAGGTTTAGCCG ACAA

AGCCCGGAAGCACTT CTTAAG

CCAGTGTCGTTACTGCAG

SIRT 1

AACGTCACACGCCAGC TCTA

CGAGGATCGGTGCCA ATC

CGCGGATAGGTCCATAT ACTTTTGTTCAGCA

FIAF

ATCAAAGCAGAATCTG AGAATACAGAAT

CTTTCCCCTCGAAGTC TTGTCTAC

CCAGATAGACCTCTTGGC CCCCACG

PLIN 2

CTGCACATCGAGTCAC GTACTCT

GGCGTTGACCAGGAC AGTCT

CTATCGCCCGCAACCTGA CCCA

20

βactin 455

GCTTCTTTGCAGCTCCT TCGT

GCGCAGCGATATCGTC ATC

CCGGTCCACACCCGCCAC C

Table 1. Primer and TaqMan probes for RT-PCR

456 457 458 459

AhR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor; PXR, pregnane x receptor; PPARα, peroxisome proliferator-activated receptor alpha; HNF4α, hepatic nuclear factor 4 alpha; SIRT1, sirtuin1; FIAF, fasting induced adiponectin factor; PLIN2, perilipin 2.

460 461

Table 2.

462 Female

Male

LOX

LKO

LOX

LKO

Fed

Fast

Fed

Fast

Fed

Fast

Fed

Fast

Cyp1a1

1.0 ± 0.7

0.1 ± 0.0

1.0 ± 0.8

0.4 ± 0.2

0.7 ± 0.3

1.5 ± 1.1

0.3 ± 0.2

0.9 ± 0.6

Cyp1a2

1.0 ± 0.2

1.0 ± 0.2

1.3 ± 0.4

0.9 ± 0.1

2.2 ± 0.4

2.4 ± 0.4

1.5 ± 0.1

1.9 ± 0.4

Cyp2a4

1.0 ± 0.0

1.8 ± 0.2

1.1 ± 0.1

1.7 ± 0.2

0.5 ± 0.1

1.4 ± 0.2

0.8 ± 0.2

1.4 ± 0.2

Cyp2b10

1.0 ± 0.4

2.1 ± 0.8

2.0 ± 0.9

1.8 ± 0.3

1.5 ± 0.2

1.4 ± 0.3

0.7 ± 0.2

0.7 ± 0.2

Cyp2e1

1.0 ± 0.2

2.3 ± 0.4

1.5 ± 0.2

1.8 ± 0.2

1.8 ± 0.4

4.2 ± 0.4

1.4 ± 0.2

3.8 ± 0.8

Cyp3a11

1.0 ± 0.1

1.2 ± 0.2

1.2 ± 0.2

0.9 ± 0.1

1.9 ± 0.7

4.6 ± 0.7

1.1 ± 0.2

2.6 ± 0.5

Cyp4a10

1.0 ± 0.3

7.7 ± 1.0

1.6 ± 0.2

4.2 ± 0.7

1.7 ± 0.5

13.6 ± 2.0

0.5 ± 0.0

6.6 ± 1.0

AhR

1.0 ± 0.2

0.8 ± 0.2

1.5 ± 0.3

0.6 ± 0.0

1.3 ± 0.5

2.9 ± 0.6

0.7 ± 0.1

1.7 ± 0.5

CAR

1.0 ± 0.2

2.5 ± 0.6

1.2 ± 0.2

1.7 ± 0.3

1.4 ± 0.4

2.8 ± 0.3

0.8 ± 0.2

2.3 ± 0.4

PXR

1.0 ± 0.0

2.8 ± 0.3

1.3 ± 0.1

2.3 ± 0.1

1.0 ± 0.2

3.8 ± 0.4

0.9 ± 0.1

3.0 ± 0.3

21

PPAR alpha

1.0 ± 0.1

3.0 ± 0.6

1.8 ± 0.4

2.0 ± 0.1

1.6 ± 0.5

5.1 ± 0.7

1.1 ± 0.2

3.5 ± 0.6

HNF4 α

1.0 ± 0.1

1.4 ± 0.1

1.0 ± 0.1

1.3 ± 0.1

0.8 ± 0.1

2.0 ± 0.2

0.6 ± 0.1

1.6 ± 0.2

SIRT1

1.0 ± 0.1

1.6 ± 0.2

0.9 ± 0.1

1.4 ± 0.1

1.2 ± 0.3

3.0 ± 0.5

0.7 ± 0.1

2.1 ± 0.3

FIAF

1.0 ± 0.1

9.0 ± 2.0

1.4 ± 0.2

6.6 ± 1.0

1.9 ± 0.7

17.6 ± 5.0

0.8 ± 0.2

10.6 ± 2.0

PLIN2

1.0 ± 0.1

7.3 ± 1.1

1.4 ± 0.1

5.0 ± 0.7

1.3 ± 0.2

8.2 ± 0.5

0.8 ± 0.1

5.4 ± 0.8

463 464 465 466

AhR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor; PXR, pregnane x receptor; PPARα, peroxisome proliferator-activated receptor alpha; HNF4α, hepatic nuclear factor 4 alpha; SIRT1, sirtuin1; FIAF, fasting induced adiponectin factor; PLIN2, perilipin 2.

467 468

469 470 471

22