4-hydroxy-3-methoxycinnamic acid regulates orexigenic peptides and hepatic glucose homeostasis through phosphorylation of FoxO1


Animal experiments

All animal experiments were approved by the Institutional Animal Care and Use Committee of Yonsei University, Wonju College of Medicine. C57BL/6 male mice were housed in a controlled environment with a 12 h dark/light cycle at a room temperature of 22–24 °C. Mice were divided into three experimental groups. The first group (n=4) was maintained on normal chow diet purchased from Zeigler Bros., Inc. (Cat. No. NIH-31M Auto, Gardners, PA, USA). The second group (n=5) was fed a HFD obtained from Research Diets Inc. (Cat. No. D12429, New Brunswick, NJ, USA) for 12 weeks. The third group (n=6) was fed a HFD together with an intraperitoneal injection of 10 mg kg−1 FA28 every other day for 12 weeks. Food intake was monitored for 3 consecutive days at 8am and 5pm on 14-week-old mice. FA was purchased from Sigma (Cat. No 128708, St. Louis, MO, USA) and dissolved in 1% Tween 80, 1% DMSO and 98% saline. The control groups (normal chow and high fat diet) were injected with vehicle.

Glucose (GTT), pyruvate (PTT) and insulin (ITT) tolerance tests

For the GTT and PTT, mice were fasted overnight for 16–18 h and provided with water ad libitum. The following morning, mice were housed in individual cages and allowed to acclimatize for 2 h followed by intraperitoneal injection of 1.2 g kg−1 glucose (Sigma) for GTT and 2 gkg−1 sodium pyruvate (Sigma) for PTT. GTT was performed on 15-week-old mice; the NC (normal chow)-fed cohort (n=4) had a body weight of 27.6±0.3 g, the HFD-fed cohort (n=5) had a body weight of 41.7±1.2 g, and the HFD+FA cohort (n=6) had a body weight of 35.6±0.6 g. PTT was performed on 17-week-old mice; the NC-fed cohort (n=4) had a body weight of 29.7±0.5 g, the HFD cohort (n=4) had a body weight of 48.2±1.4 g and the HFD+FA (n=6) had a body weight of 42.4±0.8 g. For ITT, the mice were fasted and stabilized for 2 h in individual cages with free access to water. Insulin, 0.9 U kg−1, (Eli Lilly and Company, IN, USA) was administered intraperitoneally. ITT was performed on 16-week-old mice; the NC-fed cohort (n=4) had a body weight of 29.6±0.2 g, the HFD cohort (n=5) had a body weight of 48.9±0.6 g and the HFD+FA cohort (n=6) had a body weight of 40.9±0.7 g. Blood samples were obtained from a tail nick and the blood glucose levels were measured at 0, 20, 40, 60, 90, 120 and 150 min using a commercial glucometer (Bayer HealthCare, Mishawaka, IN, USA).

Hormone measurement

Blood serum was collected at 10:00 am from 18 week old mice and the insulin and leptin levels were measured using an ELISA kit obtained from the Morinaga Institute of Biological Science (Yokohama, Japan) following the manufacturer’s protocol.

Cell culture

The hypothalamic N1,29 hepatic HepG230 and human embryonic kidney (HEK293) cells10 were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were maintained at 37 °C and 5% CO2.

RNA isolation and real-time quantitative PCR

RNA was isolated from cells and tissue samples using Trizol (Life Technologies, Carlsbad, CA, USA), following the manufacturer’s protocol. One microgram of total RNA was used to synthesize cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Q-PCR was performed using the SYBR Green PCR master mix from Applied Biosystems. The mouse Q-PCR primer sequences are listed below: PEPCK; 5′-CGCAAGCTGAAGAAATATGACAA-3′ and 5′-TCGATCCTGGCCACATCTC-3′, G6P; 5′-TGGGCAAAATGGCAAGGA-3′ and 5′-TCTGCCCCAGGAATCAAAAAT-3′, NPY; 5′-CTACTCCGCTCTGCGACACT-3′ and 5′-AGTGTCTCAGGGCTGGATCT-3′, AgRP; 5′-CGGCCACGAACCTCTGTAG-3′ and 5′-CTCATCCCCTGCCTTTGC-3′, POMC; 5′-CAGGTCCTGGAGTCCGAC-3′ and 5′-CATGAAGCCACCGTAACG-3′, CART; 5′-AGAAGAAGTACGGCCAAGTC-3′ and 5′-GGACAGTCACACAGCTTCC-3′. The human Q-PCR primers sequences are as follows, PEPCK; 5′-CAGGCGGCTGAAGAAGTATGA-3′ and 5′-AACCGTCTTGCTTTCGATC-3′, G6P; 5′-GCCACATCCACAGCATCTATAA-3′ and 5′-CCAGAGTCCACAGGAGGTCTAC-3′.

Western blot

Protein was isolated from cells and tissue samples using radioimmunoprecipitation assay buffer enriched with protease and phosphatase inhibitors (Roche, Basel, Switzerland). Equal amounts of protein were loaded and separated on sodium dodecyl sulphate-acrylamide gels and then incubated with specific antibodies. The following primary antibodies were used: pAKT (4060S), AKT (2967S), pFOXO1 (9461S), FOXO1 (2880S), Lamin A/C (2032S) and PEPCK (12940S) from Cell Signaling Technology (Danvers, MA, USA). GAPDH (SC-25778) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and G6P (ab83690) was from Abcam (Cambridge, UK). The protein levels were detected using Pierce ECL western blotting substrate (Waltham, MA, USA) following the standard western blot procedure, as described previously.10 Images were captured with a UVP Bio-Spectrum 600 imaging system (Ultra-Violet Products Ltd. Cambridge, UK).

Cell fractionation

HEK 293 cells were harvested and the cell membranes lysed using ice-cold buffer A containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.9), 10 mM KCL, 0.1 mM EDTA (pH 8.0), 0.1 mM EGTA (pH 8.0), 1 mM DTT, and protease and phosphates inhibitors. 10% Triton X-100 was added to the samples followed by 1 min centrifugation. The cytoplasmic supernatant was then collected. The pellet was then lysed with ice cold buffer C containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.9), 400 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 1 mM DTT and protease and phosphatase inhibitors. The supernatant containing the nuclear extract was collected. Protein levels in the cytoplasmic and nuclear extracts were detected following the standard western blot procedure. GAPDH and Lamin A were used as internal controls for the cytoplasmic and nuclear extracts, respectively.

FoxO1 localization assay

FoxO1 localization was assayed by transfecting HEK 293 cells with FoxO1-GFP using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA), followed by 6 h FA treatment. The cells were fixed in 4% paraformaldehyde for 10 min and washed three times with phosphate-buffered salinePBS. Slides were mounted using VECTASHIELD mounting medium with DAPI (Vector Laboratories, Inc. Burlingame, CA, USA) and visualized using the Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Luciferase assay

PEPCK and G6P luciferase constructs were kindly provided by Dr Hueng-Sik Choi, as previously described.31 AgRP, NPY and POMC luciferase constructs were kindly provided by Dr Min-Seon Kim, as previously described.19 HEK 293 cells were transfected with specific DNAs using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s protocol, and renilla (100 ng) was co-transfected as an internal control. Cells were lysed using luciferase lysis buffer composed of 25 mM Tris phosphate pH 7.8, 2 mM DTT, 2 mM EDTA, 10% glycerol and 1% Triton X-100. The cell lysate samples were assayed in triplicate after injection of luciferase and renilla substrates using the BioTek Synergy TM 2 machine (BioTek Instruments Inc., Winooski, VT, USA). The luciferase substrate (pH 8.0) was composed of 200 mM Tris-HCL, 15 mM MgSO4, 0.1 mM EDTA, 25 mM DTT, 1 mM ATP, 0.2 mM coenzyme A and 200 μM luciferin. The renilla substrate (pH 5.0) was composed of 1.1 M NaCl, 2.2 mM Na2EDTA, 0.22 M KH2PO4 (pH 5.1), 0.44 mg ml−1 BSA, 1.3 mM sodium azide, 1.43 μM coelenterazine.32 The relative luciferase activity of the FA treatment was calculated as a ratio of luciferase and renilla activity normalized to vehicle treatment.

Histological analysis

White adipose tissue and liver samples were fixed in 4% neutral buffered formalin. The tissue samples were then paraffin embedded and cut into 5 μm slices. The slices were stained with hematoxylin and eosin (H&E) following the standard H&E procedure. The stained slides were visualized by a Nikon Digital Camera DXM1200 microscope system (Nikon Corporation, Tokyo, Japan).

Statistical analysis

The data were analyzed using GraphPad Prism 5.0 software. Statistical comparisons were performed using Student’s t-test or ANOVA, and results with a P-value <0.05 were considered to be statistically significant. All results are expressed as the mean±s.e.m. as indicated in the figure legends.

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