Fasting induces nuclear factor E2-related factor 2 and ATP-binding Cassette transporters via protein kinase A and Sirtuin-1 in mouse and human

Abstract

Aims: The purpose of this study was to determine whether 3'-5'-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2-4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed.

Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1-dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter.

Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging.

Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway.

FIG. 1.

Fasting induces Abcc2–4 expression in vitro and in vivo. (A) Increased biliary disulfobromophthalein (DBSP) excretion upon fasting. DBSP (150 μmol/kg/5 ml, intraperitoneal injection) was injected into C57BL/6 mice (n=5 per group) either fed ad libitum or fasted for 18 h. 45 min after injection, gallbladders were collected and biliary DBSP content was determined spectrophotometrically after appropriate dilution and alkalinization of bile with 0.1 N NaOH. The data are represented as average±SEM μmol of DBSP/μl bile/kg body weight. (B) Induction of Abcc 2–4 expression in mouse liver after a 24 h fast-Total RNA was isolated from livers of C57BL/6 mice either fed ad libitum or fasted for 24 h. Relative Abcc 2–4 mRNA expression was quantified by real-time quantitative PCR. The data are represented as average±SEM fold change over the fed controls. All data have been normalized to 18S rRNA. (C) Induction of Abcc2–4 protein expression in mouse liver after 24 h fasting-Crude membrane proteins were isolated from livers of C57BL/6 mice either fed ad libitum or fasted for 24 h. Abcc2–4 protein expression was quantified by western blotting. (D) Induction of Abcc 2–4 mRNA expression in primary mouse hepatocytes by 8-Bromoadenosine-3′,5′-Cyclic Adenosine Monophosphate (8-Br-cAMP)-Primary mouse hepatocytes were treated with DMSO or 8-Br-cAMP (1 mM) for 24 h. Total RNA was isolated 24 h after treatment and Abcc2–4 mRNA expression was quantified using real time quantitative PCR. The data are represented as average±SEM (n=3) fold change over the fed controls. All data are normalized to 18S rRNA. Groups without a common letter are significantly different. (E) Time dependent expression of ABCC 2–4 mRNA in Huh-7 cells by 8-Br-cAMP- Huh-7 cells were treated with 1.0 mM 8-Br-cAMP for 0, 3, 6, 12, 24, and 48 h with media as the control (n=3). Total RNA was isolated and ABCC 2–4 mRNA expression was quantified using the Branched DNA Signal Amplification assay. The data are represented as average Relative Light Units (RLU) per 10 μg total RNA±SEM (n=3). (F) Induction of ABCC 2–4 mRNA expression in primary human hepatocytes by 8-Brc-AMP-Primary human hepatocytes were treated with media, DMSO, 8-Br-cAMP (0.1 and 1 mM), epinephrine (10 and 100 μM) or glucagon (0.1 and 1 μM) for 24 h. At the end of 24 h cells were lysed using Lysis Mixture^™. mRNA levels of ABCC 2–4 were quantified using Branched DNA signal amplification assay. The data are represented as relative mRNA expression per 25 μl lysate normalized to beta actin from three human hepatocyte donors.

FIG. 2.

Fasting induces nuclear factor-E2 related factor 2 (NRF2) target gene expression in vitro and in vivo. (A) Induction of Nadph:quinone oxidoreductase (Nqo1) and Nrf2 mRNA expression in mouse liver after 24 h fast- Total RNA was isolated from livers of C57BL/6 mice either fed ad libitum or fasted for 24 h. Gene expression of Nqo1 was quantified by real-time quantitative PCR. The data are represented as average±SEM fold change over the fed controls. All data have been normalized to 18S rRNA. (B) Induction of Nqo1 and NRF2 mRNA expression in primary mouse hepatocytes by 8-Br-cAMP-Primary mouse hepatocytes were treated with DMSO or 8-Br-cAMP (1 mM) for 24 h. Total RNA was isolated 24 h after treatment and Nqo1 and NRF2 mRNA expression was quantified using real time quantitative PCR. The data are represented as average±SEM (n=3) fold change over the fed controls. All data are normalized to 18S rRNA. Groups without a common letter are significantly different. (C) Fasting increases binding of NRF2 to its consensus binding sequence-Liver nuclear extracts were prepared from control and fasted C57BL/6 mice using Procarta TF nuclear extraction kit and analysis for transcription factor binding was determined using a Procarta TF binding assay. The results obtained are represented as data normalized to Transcription Factor II D. Data are expressed as Fluorescence Intensity/10 μg total liver nuclear extract normalized to C57BL/6 fed controls. p<0.05 was considered statistically significant. (D) Time dependent increase in NQO1 and NRF2 protein expression in Huh-7 cells by 8-Br-cAMP- Huh-7 cells were treated with1.0 mM 8-Br-cAMP for 0, 3, 6, 12, 24 h with media as the control (n=3). mRNA levels of NQO1 was quantified using Branched DNA signal amplification assay. The data are represented as fold change in NQO1 mRNA expression (n=3). NQO1 protein levels were determined by western blotting with Gapdh as the loading control. Nuclear NRF2 protein levels were measured by western blotting with laminB1 as loading control. (E) Induction of NQO1 mRNA expression in primary human hepatocytes by 8-Brc-AMP- Primary human hepatocytes were treated with media, DMSO, 8-Br-cAMP (0.1 and 1 mM), epinephrine (10 and 100 μM) or glucagon (0.1 and 1 μM) for 24 h. At the end of 24 h cells were lysed using Lysis Mixture. mRNA levels of NQO1 was quantified using Branched DNA signal amplification assay. The data are represented as relative mRNA expression per 25 μl lysate normalized to beta actin from three human hepatocyte donors.

FIG. 3.

Fasting increases Abcc2–4, Nqo1 expression in Nrf2 dependent manner. (A) Fasting for 24 h induces the expression of Nrf2 target genes in wild-type C57BL/6 mice livers but not in Nrf2 null mice livers-Total RNA was isolated from livers of C57BL/6 (n=6) and Nrf2-null (n=6) mice either fed ad libitum or fasted for 24 h. Nqo1and Abcc2–4 gene expression was quantified by real time quantitative PCR. The data are represented as average±SEM (n=6) fold change over the fed controls. (B) Immunohistochemical staining of Abcc2-Cryosections were incubated with anti-Abcc2 antibody followed by incubation with AlexaFluor-conjugated secondary antibodies (green). (C) Induction of Abcc2-3 protein expression in Nrf2-WT but not in Nrf2-null mouse liver after 24 h fasting-Crude membrane proteins were isolated from livers of C57BL/6 (n=2) and Nrf2 null (n=2) mice either fed ad libitum or fasted for 24 h. Nqo1, Abcc2, Abcc3 and beta actin protein expression was quantified by western blotting. The data are representative of three individual protein quantifications. (D) Fasting for 24 h decreases liver cAMP, while increasing serum cAMP levels in Nrf2 WT but not in Nrf2 null mice. Serum bile acids and serum and liver cAMP levels were determined from serum and livers obtained from C57BL/6 (n=4) and Nrf2 null (n=4) mice fasted for 24 h. Data are represented as average±SEM (n=4) fold change relative to control. (E) Fasting increases hPAP and Nqo1 mRNA in livers of ARE-hPAP mice-Total RNA was isolated from livers of C57BL/6 (n=3) and ARE-hPAP (n=3) mice either fed ad libitum or fasted for 24 h. Nqo1 and human alkaline phosphatase (hPAP) mRNA expression was quantified by real time quantitative PCR. The data are represented as average±SEM (n=3) fold change over the fed controls. All data have been normalized to 18S rRNA. (F) 8-Br-cAMP induces hPAP mRNA expression in primary mouse hepatocytes-ARE-hPAP mouse hepatocytes were treated with 1 mM 8-Br-cAMP for 24 h. Total RNA was isolated and ARE-hPAP mRNA expression was quantified using RT^2-PCR. Target gene expression was normalized to 18s rRNA. Groups without a common letter are significantly different. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Protein kinase A (PKA) pathway acts as upstream regulator of NRF2-ARE pathway. (A) 8-Br-cAMP induces antioxidant response element (ARE) in Huh-7 cells-Huh-7 cells were cultured to 85% conflucence and transiently transfected with an ARE–luciferase reporter construct (100 ng) for 24 h. The cells were then treated with 0.1 and 1.0 mM 8-Br-cAMP for 24 h. Luciferase activity was measured by dual luciferase assay with renilla luciferase as a control. The data are expressed as average fold induction in activity of the ARE-luciferase construct±SEM (n=4). (B) H89 inhibits 8Br-cAMP induced mRNA expression of hPAP-ARE-hPAP mouse hepatocytes were treated with 1 mM 8-Br-cAMP with or without H89 (10 μM) for 24 h. Total RNA was isolated and hPAP, expression was quantified using RT^2-PCR. Target gene expression was normalized to 18s rRNA. 8-Br-cAMP induced (C) NRF2, Nqo1 and (D) Abcc2 and 4 mRNA expression is muted by H89 cotreatment in NRF2 WT, but not NRF2-null hepatocytes- Primary mouse hepatocytes obtained from WT or Nrf2-null mice were treated with DMSO or 8-Br-cAMP (1 mM) with or without H89 (20 μM) for 24 h. Total RNA was isolated 24 h after treatment and Abcc2 mRNA expression was measured using real time quantitative PCR. The data are represented as average±SEM (n=3) fold change over the fed controls. All data are normalized to 18S rRNA. p<0.05 was considered statistically significant. Groups without a common letter are significantly different. (E) Fasting induced upregulation of hepatic NAD+/NADH levels occurs to the greater extent in NRF2 null mice-NAD+/NADH ratio was quantified in livers obtained from C57BL/6 mice fasted for 24 h according to manufacturer's protocol. The data are represented as average±SEM (n=4) fold change over the fed controls. (F) AMP-activated protein kinase (AMPK) activators do not induce expression of Nrf2 target genes-Primary mouse hepatocytes were treated with control, AICAR (0.5 mM), NAD+ (5 mM) or metformin (1 mM) for 24 h. Total RNA was isolated 6 h after treatment and mRNA expression of Nqo1 and Nrf2 was quantified using real time quantitative PCR. The data are represented as average±SEM (n=3) fold change over the fed controls. All data are normalized to 18S rRNA. Groups without a common letter are significantly different. (G) Total AMPKα and p-AMPKα is attenuated in Nrf2 WT and Nrf2 null mouse livers upon fasting-Proteins lysates were obtained from livers of C57BL/6 (n=2) and Nrf2 null (n=2) mice either fed ad libitum or fasted for 24 h. Total AMPK and p-AMPKα and Gapdh protein expression was quantified by western blotting.

FIG. 5.

Changes in Nrf2-ARE pathway during fasting are Sirtuin-1 (SIRT1) dependent. (A) SIRT1LKO mice demonstrate resistance to effects of fasting-Total RNA was isolated from livers of Sirt WT (n=5) and SIRT1LKO (n=3) mice either fed ad libitum or fasted for 24 h. Gene expression of NRF2, Abcc 2–4 was quantified by real time quantitative PCR. The data are represented as average±SEM fold change over the fed controls. All data have been normalized to 18S rRNA. p<0.05 was considered statistically significant. Groups without a common letter are significantly different. (B) SIRT1OE mice demonstrate enhanced induction of Abcc2–4 and Nrf2 target genes upon fasting-Total RNA was isolated from livers of SIRT1OE (n=8) mice either fed ad libitum or fasted for 19 h. Gene expression of NRF2, Abcc 2–4 was quantified by real time quantitative PCR. The data are represented as average±SEM fold change over the fed controls. Groups without a common letter are significantly different. All data are normalized to 18S rRNA. p<0.05 was considered statistically significant. Groups without a common letter are significantly different.

FIG. 6.

NRF2 expression increases coordinately with PPAR gamma coactivator alpha (Pgc-1α) and SIRT1 expression. (A) NRF2 mRNA coordinately increases with increasing activation of SIRT1-Huh-7 cells were cultured to 85% confluence and transiently transfected (50–100 ng) Myc-SIRT1, 75 ng Flag-NRF2 and 0.5 μg Flag-Pgc-1α expression plasmids using Lipofectamine LTX-plus reagent (Invitrogen) for 24 h. The cells were then treated with 1.0 mM 8-Br-cAMP for 24 h. Endogenous NRF2 and Pgc-1α mRNA expression was quantified by real time quantitative PCR. The data are represented as average±SEM (n=3) over control All data have been normalized to 18S rRNA. (B) NRF2 mRNA expression coordinately increases with increasing Pgc-1α expression-Huh-7 cells were cultured to 85% confluence and transiently transfected with (0.5–1.0 μg) Flag-Pgc-1α, 50 ng Myc-SIRT1, 75 ng Flag-NRF2 and 0.5 μg Flag-Pgc-1α expression plasmids for 24 h. The cells were then treated with 1.0 mM 8-Br-cAMP for 24 h. NRF2 and Pgc-1α mRNA expression was quantified by real time quantitative PCR. The data are represented as average±SEM (n=3) over control All data have been normalized to 18S rRNA. (C) Pgc-1α coimmunoprecipitates with NRF2 in a SIRT1 dependent manner-Huh-7 cells were cultured to 85% confluence in 6 cm dishes and transiently transfected with 3.0 μg Flag-tagged NRF2, with or without 0.75 μg cMyc-tagged SIRT1 plasmid for 24 h. At the end of 24 h, cells were treated with 1.0 mM 8-Br-cAMP for 45 min, and then lysed using lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40). 1 mg of the protein lysate was incubated with Anti-FlagM2 magnetic beads overnight in lysis buffer and immunoprecipitates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresie (SDS-PAGE) and immunoblotted for Flag-M2 and endogenous Pgc-1α. Gapdh was used as protein loading control. (D) Moderate SIRT1 overexpression (OE) increased Nrf2 mRNA expression in mouse liver. Total RNA was isolated from SIRT1 WT (n=5) and SIRT1 OE (n=5) mouse livers and NRF2 mRNA expression quantified using real-time quantitative PCR. The data are presented as average±SEM (n=2–3) fold change in RNA expression over SIRT1 WT controls. (E) Fasting slightly decreases SIRT1 protein in mouse liver. Liver lysates in RIPA buffer were subjected to SDS-PAGE followed by western blot for SIRT1 and Gapdh.

FIG. 7.

Activation of cAMP/PKA pathway increases NRF2 enrichment at ARE like elements in human ABCC2 promoter. (A) 8-Br-cAMP increases NRF2 enrichment at ARE-like sites in ABCC2 promoter. Huh-7 cells were treated with 8-Br-cAMP (0.1 mM) for 45 min and were washed and cross-linked for 10 min with 1% formaldehyde and crosslinking terminated by 125 mM glycine. The chromatin were sheared and prepared and incubated overnight with Protein G magnetic beads with Anti-NRF2 antibody or preimmune IgG. The antibody-bound chromatins and input DNA (1/10 dilution) were analyzed by PCR for the presence of DNA-fragments containing NRF2 bound-ARE-like element (MARE_ARE) with primers as indicated in Table 1. (B) 8-Br-cAMP increases NRF2 enrichment at other known ABCC2 AREs. Huh-7 cells were treated in a manner similar to (A) and the NRF2-antibody bound chromatin and input DNA were analyzed by RT²-PCR for the presence of DNA fragments containing MARE_ARE as well as ARE-like elements described by Stockel et al. (47) in ABCC2 promoter as well as negative primers directed at a non ARE-containing promoter region (Table 1). (C) 8-Br-cAMP increases SIRT1 enrichment at ABCC2 ARE-like elements in Huh-7 cells. Huh-7 cells were treated in a manner similar to (A), and the sheared chromatin fragments were incubated with anti-SIRT1 antibody and preimmune IgG overnight and the antibody-bound chromatins were analyzed by RT²-PCR for DNA fragments containing MARE_ARE (Table 1). (D) 8-Br-cAMP increases SIRT1 enrichment at the NQO1 ARE. Huh-7 cells treated in a manner similar to (A) and incubated with anti-SIRT1 antibody and preimmune IgG overnight and the antibody-bound chromatins were analyzed by PCR for DNA fragments containing ARE-like elements as described by Dhakshinamoorthy et al. (13) using primers indicated in Table 1.

FIG. 8.

Proposed model for fasting induced activation of NRF2-ARE pathway and expression of downstream target genes. Fasting increases circulating glucagon and epinephrine levels activating cAMP/PKA cascade, which eventually will increase Pgc-1α expression and also activate SIRT1. cAMP/PKA induces NRF2 binding to various AREs in a SIRT1-dependent manner leading to an increase in Nqo1, Abcc2–4 expression. Specifically, induction of Pgc-1α and SIRT1 can induce Nrf2 mRNA expression, which can lead to subsequent induction of Nrf2-regulated target genes, such as Nqo1 and Abcc2–4. In addition, activation of the PKA pathway increases NRF2 and SIRT1 recruitment to MARE_ARE elements in the ABCC2 promoter. It is hypothesized fasting will result in the production or accumulation of metabolic intermediates, which could be eliminated from hepatocytes via Abcc2–4. Fasting increases NAD+/NADH ratios, cAMP, bile acids, lipid peroxidation, glucuronide and glutathione (GSH) conjugation, and decreases GSH levels within the hepatocyte. GSH and glucuronide conjugates are substrates for Abcc2–4, some lipid peroxidation products are substrates for Abcc3. Abcc4 transports cyclic nucleotides, such as cAMP and cGMP. Overall, fasting could increase the hepatic efflux of metabolite conjugates and intermediates resulting from induction of the fasting response to maintain cellular homeostasis.