Eight-week-old C57BL/6N female mice were obtained from Beijing Vital River Laboratory Animal Technology Company (Beijing, China). Mice were kept in a 12:12 h light:dark cycle at 25 ± 0.5°C and 50–60% (vol./vol.) humidity and fed a standard diet with water ad libitum. The Ethics Committee of Shandong Provincial Hospital affiliated to Shandong University approved the procedures for animal experiments.
Eight-week-old female mice were randomly divided into four groups: (1) sham-operation (sham), (2) bilateral ovariectomy (OVX) with diet-supplemented oestradiol (E2) (OVX+E2), (3) OVX+E2 with low-dose FSH (L-FSH; 30 U/kg body weight recombinant human follitropin alfa solution, Merck, Kenilworth, New Jersey, USA) (OVX+E2+L-FSH) and (4) OVX+E2 with high-dose FSH (H-FSH; 60 U/kg body weight) (OVX+E2+H-FSH). Groups (1) and (2) were injected with vehicle and used as controls. To exclude the effect of E2, the mice were placed on a hormone replacement diet supplemented with 2.6 ppm desiccated E2 powder (Bayer, Leverkusen, Germany). After E2 was added for 5 days, FSH was given i.p. daily for 2 weeks, until mice were killed.
Additionally, OVX+E2 mice were randomly divided into four groups (n = 6/group): control (saline [154 mmol/l NaCl]), H-FSH, GRK2 inhibitor (200 μg/kg body weight) and GRK2 inhibitor + FSH (see Electronic supplementary material (ESM) Methods for further details).
Fshr and Crtc2 knockout mice, and Fshr siRNA
Fshr+/− mice (Wuhan Kangweida Gene Technology Company, Wuhan, China) were intercrossed to produce Fshr−/− mice. Female Fshr−/− mice were fed a 2.6 ppm E2-supplemented diet from 3 weeks of age. Female wild-type (Fshr+/+) littermates were used as the controls. At 8–10 weeks, mice were ovariectomised (Fshr+/+ mice were then also fed 2.6 ppm of E2) and injected with H-FSH (see ESM Methods for details).
Additionally, OVX+E2 mice were injected with Fshr siRNA adenovirus and, 7 days later, treated with FSH or vehicle. Mice were killed on day 22 (see ESM Methods for details).
Crtc2 knockout mice (Crtc2−/−) and their Crtc2+/+ littermates were also ovariectomised, fed 2.6 ppm of E2 and injected with H-FSH or vehicle daily for 2 weeks (see ESM Methods for details).
All the samples were randomised using the random number table method. All group assignments and outcome assessments carried out in this study were blinded. Illnesses including dermatitis, dehydration, inflammation and weight loss were exclusion criteria for animals; however, no animals were excluded. No experimental data was excluded using the Grubbs outlier test.
After treatment, mice underwent OGTTs, insulin tolerance tests (ITTs) and pyruvate tolerance tests (PTTs) (see ESM Methods for details).
Mice were fasted for 8 h or refed and then killed for blood and tissue collection. Blood was collected immediately before mice were killed, and glucose, FSH (Abnova ELISA Kit, #KA2330, Wuhan, China) and E2 (Demeditec Oestradiol-Sensitive ELISA Kit, #DE4399, Kiel, Germany) levels were assessed. Tissues were rapidly obtained after mice were killed and repetitive freeze–thaw cycles were avoided (see ESM Methods for details).
H&E and PAS staining
Paraffin-embedded liver tissues were sectioned and stained with haematoxylin and eosin (H&E). For hepatic glycogen staining, samples were stained with periodic acid–Schiff (PAS) according to the manufacturer’s instructions (Yili Company, Beijing, China).
Optimal cutting temperature compound (OCT)-embedded liver tissues were incubated with rabbit anti-GRK2 antibodies (1:200, Santa Cruz, Dallas, TX, USA) and then with fluorescein (tetramethylrhodamine [TRITC])-conjugated goat anti-rabbit IgG (1:300, Invitrogen, Beijing, China). Nuclei were stained with DAPI. Confocal fluorescence microscopy (LSM 780, Zeiss, Oberkochen, Germany) was used for imaging (see ESM Methods).
Hepatic cell lines, mouse primary hepatocyte cultures
HepG2 cells (Cell Library of the Chinese Academy of Sciences, Shanghai, China), mice hepatocyte NCTC 1469 cells (Xiehe Cell Library, Beijing, China) and mouse primary hepatocytes were used. HepG2 cells were treated with FSH (0, 10, 50 and 100 ng/ml) for 6 h or 24 h, or insulin (100 nmol/l) for 4 h or 6 h. Other regents including glucagon (100 nmol/l), protein kinase A (PKA) inhibitor (PKI; 10 μmol/l) and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 1 mmol/l) were added 2 h before FSH. HepG2 and NCTC 1469 cells were authenticated by the cell library from which they were sourced and were not contaminated with mycoplasma.
NCTC cells were infected with Grk2 shRNA or scrambled shRNA and collected for protein analysis.
HepG2 cells were transfected with AMPK siRNA (targeting human AMPKα1, AMPKα2, AMPKβ1 and AMPKγ1) (see ESM Methods for details).
Glucose production measurements
After treatment with FSH for 22 h or insulin for 2 h, HepG2 cells were incubated for 2 h in glucose-free and phenol red-free DMEM media (Gibco, Grand Island, NY, USA) supplemented with 10 mmol/l lactate and 1 mmol/l pyruvate (and FSH/insulin). Glucose was measured in media using a glucose detection kit (Applygen Technologies, Beijing, China). Data were analysed relative to the controls (see ESM Methods).
Plasmid construction and transfection and dual luciferase activity assays
The plasmid encoding wild-type GRK2 (a gift from J. Staňková ), Firefly luciferase reporter plasmids containing wild-type PEPCK and G6Pase promoters (provided by H.S. Choi ), plasmids encoding green fluorescent protein (GFP)-tagged wild-type AMPK and a nonphosphorylatable mutant of AMPK substituted with alanine (AMPK S485A; Shanghai Genechem, Shanghai, China), and the Firefly luciferase reporter plasmid containing the CRTC2-CREB complex-binding site (CRE)-mutant PEPCK promoter (Hanbio Biotechnology, Shanghai, China) were used for transfection. A Renilla luciferase plasmid (Promega, Madison, WI, USA) served as an internal control. Luciferase activity was measured using the dual luciferase reporter assay (Promega), according to the manufacturer’s protocol. Firefly luciferase activity was normalised to Renilla luciferase activity. Data were analysed relative to controls (see ESM Methods).
RNA isolation and real-time quantitative RT-PCR
Total RNA from cells and mice liver tissue was isolated and real-time quantitative RT-PCR was used to determine relative mRNA expression of mouse Pepck (also known as Pck1), G6pase (also known as G6pc), Crtc2, Pgc1a (also known as Ppargc1a), Gck, Pfkfb1, Pygl, Gys2 and β-actin (Actb), and human PEPCK (also known as PCK1), G6Pase, GLUT2 (also known as SLC2A2), GLUT4 (also known as SLC2A4), AMPK (also known as PRKAA1) and β-actin (ACTB) (see ESM Methods for further details and primers are listed in ESM Table 1).
Protein extraction and western blotting
Protein was extracted from hepatocytes and tissues, separated on 10% (wt/vol.) SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). Anti-p-Thr172 AMPKα (1:1000), p-Ser485 AMPKα (1:1000), AMPKα (1:1000), AMPKβ1 (1:1000), p-Ser428 liver kinase B1 (LKB1; 1:1000), p-Ser9 glycogen synthase kinase 3β (GSK3β; 1:1000), GSK3β (1:1000), p-Ser307 IRS1 (1:1000), p-Ser133 cyclic AMP response element-binding protein (CREB; 1:1000), CREB (1:1000), p-Ser473 Akt (1:1000) and Akt (1:1000) antibodies were obtained from Cell Signaling Technology (Boston, MA, USA). Other antibodies used in the study included anti-GLUT2 (1:1000) and anti-GLUT4 (1:1000) (Abcam, Cambridge, UK); anti-LKB1 (1:1000, Merck Millipore); anti-CRTC2 (1:2000, Calbiochem, Merck Millipore); anti-GRK2 (1:200, Santa Cruz); anti-AMPKγ1 (1:1000, Thermo Scientific, Waltham, MA, USA); anti-FSHR (1:500) and anti-glucose-6-phosphatase (G6Pase) (1:1000) (OriGene, Rockville, MD, USA); anti-PEPCK (1:2000), PGC1α (1:1000), GFP (1:5000), LMB1 (1:5000) and GAPDH (1:7500) (Proteintech, Wuhan, China), and anti-p-Ser670 GRK2 (1:200, Bioss, Beijing, China). All the antibodies used in the article were validated by companies from which they were bought respectively, using methods such as, siRNA knock-down, phosphatase and activator treatment, positive/negative cell line and lot-to-lot testing. The appropriate secondary antibodies conjugated to horseradish peroxidase (HRP; ZSGB-Bio, Beijing, China) were used at 1:5000. Immune complexes were detected using the chemiluminescence (see ESM Methods).
Co-immunoprecipitation was carried out to test the binding of GRK2 with AMPK. Protein was extracted from cells and incubated with rabbit anti-AMPKα or rabbit anti-AMPKβ1 antibodies. The pre-immune sample was generated by combining protein extracts with rabbit IgG (Cell Signaling Technology). The following day, Protein A/G plus agarose slurry beads (Santa Cruz) were added to samples for 2 h. The eluted proteins were separated on 10% (wt/vol.) SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with an anti-GRK2 antibody (Santa Cruz) and then with a secondary HRP-conjugated antibody (1:5000). Immune complexes were detected using chemiluminescence (see ESM Methods for details).
The data are expressed as the means ± SEMs. Statistical analyses were performed using either two-tailed unpaired Student’s t tests (two groups) or one-way ANOVA tests (more than two groups). A p < 0.05 is considered to be statistically significant.