β₁-adrenergic receptor signaling mediates enhancement of ghrelin secretion by oral hydrogen water

It has been shown that gastric secretion of ghrelin is regulated by local environmental cues including blood glucose, estrogen, insulin and catecholamines^20,21,22. In particular, it has been reported that β₁-adrenergic receptor stimulation increases ghrelin secretion in vitro and in vivo^22,23. We verified the expression of β₁– and β₂– adrenergic receptors in stomach tissue samples by real-time PCR method, and determined that there were no significant changes in the levels of expression after administration of oral H₂-water for 4 days (data not shown). The increase in plasma ghrelin levels by oral hydrogen water was eliminated by administration of the β₁-adrenergic receptor-specific blocker, atenolol (10 mg/kg i.p.) injected 30 min prior to H₂ water administration on each of four days (Figure 3). Thus, activation of β₁-adrenergic receptors is required for hydrogen water-induced enhancement of circulating ghrelin.

Open in a separate window

Figure 3

H₂ water increases plasma ghrelin levels in a β₁-adrenergic receptor-dependent manner.

Plasma ghrelin levels were measured on the last day of experiment. Each group of mice (11–13 weeks of age; n = 4–5 for each group) was administered control water or H₂ water (made with an open-air water electrolysis system) once a day for 4 d with or without i.p. injection of atenolol (10 mg/kg) prior to water ingestion. The H₂ water group showed a significant increase in plasma ghrelin level compared to the control group (* p = 0.038), which was abrogated by pretreatment with atenolol (** p = 0.039). Datafare represented as mean ± SEM.

Preparation and administration of H₂– water

H₂ water was prepared by one of three methods. The first method utilized the spontaneous ionization reaction of magnesium in water¹⁴. A magnesium stick (AZ31, Nakagawa Metal, Japan, composed of 96% magnesium, 3% aluminum, and 1% zinc) was polished to remove the oxidized surface and wiped clean with 1 N hydrogen chloride before dipping into drinking water for 15–20 min at 25°C. The second method was based on the electrolysis of water with platinum electrodes at 150 V DC for 20 min (open-air water electrolysis method). The anode was placed inside a drinking straw to vent generated oxygen. In the third method (employed in the experiments with MPTP-induced PD model mice), H₂ gas was produced by solid polymer electrolyte water electrolysis method at 5 V DC in which the cathode and anode were separated by a solid polymer electrolyte membrane (Nafion® E.I., du Pont de Nemours & Co., Inc.) and collected through a polyethylene tube. Saturated H₂ water was then generated by bubbling water in a drinking bottle with H₂ gas such that H₂ gas filled the headspace. H₂ water made with this method and kept in sealed drinking bottles remained saturated with H₂ for at least 24 hr.

In most experiments, each mouse received 0.8 ml of H₂ water (administered within 30 min of preparation) or control water (obtained by boiling H₂ water to degas) once every morning via feeding needle according to the experimental schedule. In experiments with MPTP-induced PD model mice, fresh H₂ water was supplied every 24 hr and water intake was ad libitum for the 7 days prior to MPTP administration.

Measurements of ghrelin expression

Male and female mice aged between 41 and 48 weeks were chosen for the initial screening for the altered expression of gastric enzymes (n = 4 for each group), as they are relatively stable in dietary intake, which may influence mostly on the digestive enzyme expressions. Since the previous study was done with male mice¹⁴, the following experiments chose male mice. For the time-course analysis of ghrelin induction by hydrogen water, male mice (16–21 weeks, average 17.9 weeks, n = 5–10 in each group) were selected. Adrenoceptor mediated changes of ghrelin secretion was analyzed in male mice (age between 11–13 weeks, n = 4–5 for each group). Of note, there was no body weight change more than one gram during the period of experiment (4 days). These results are in line with the previous report with a recombinant ghrelin injection to mice, where the body weight change was below one gram in three days³⁴.

Mice were administrated H₂ water or control water for 4 days according to the experimental schedule. At the end of the experiment, mice were sacrificed by cervical dislocation and blood was collected by cardiopuncture with EDTA as anti-coagulant. Blood was centrifuged at 2000 g for 5 min at 4°C and the plasma was collected and mixed with 1 N hydrogen chloride (10% of plasma volume) to avoid inactivating deacylation of ghrelin²⁰. Samples were stored at −80°C until analysis. Following blood collection, the stomach was removed intact and snap-frozen in liquid nitrogen for further analysis. For consistency, mice were killed 4–5 hours after the final administration of H₂ water and at the same time of day. At the time of blood sampling the stomach was almost full of chow, which suggests that the water administration at a time (0.8 ml/time) through the feeding needle did not cause the sustained reduction on appetite.

The active form of ghrelin was measured in plasma by ELISA (Active Ghrelin ELISA kit, Sceti, Japan) according to the manufacturer’s instructions. For semi-quantitative PCR, stomach tissue frozen in liquid nitrogen was crushed into fine granules and total RNA was extracted with RNAiso (Takara, Japan) according to the manufacturer’s instructions. Two μg of total RNA was then used to synthesize first strand cDNA with a SuperScript VILO cDNA Synthesis Kit (Invitrogen, USA). mRNA expression levels were quantified by real-time PCR with SYBR green dye (Thunderbird Sybr qPCR Mix, Toyobo, Japan) with the specific primer sets shown in Table 1, and normalized to ribosomal protein L4 (RPL4) mRNA^35,36,37.

Administration of ghrelin receptor antagonist, β₁-adrenoceptor blocker, and MPTP

The ghrelin receptor antagonist, D-Lys³ GHRP-6 (Sigma-Aldrich, USA; 100 nmol/day), β₁-adrenoceptor blocker, atenolol (ICI, USA; 10 mg/kg), or saline was administered by i.p. injection daily for 7 days in conjunction with supplying fresh control or H₂ water. On day 7, MPTP-HCl (Sigma-Aldrich, USA; 15 mg/kg in 0.9% NaCl per injection) or saline as control was administrated by i.p. injection four times at 2 hr intervals. Mice were supplied with untreated tap water for the next 7 days before removal of brains under deep anesthesia (pentobarbital, 50 mg/kg i.p.).

Stereological and immunological analyses of nigral dopaminergic neurons

Stereological analysis was carried out as described^14,38,39,40. In brief, coronal sections (30 μm thickness) were obtained through SNpc (−2.70 mm to −3.80 mm relative to bregma)⁴¹ with a MICROM cryostat. Free-floating sections were incubated with Block Ace (Dainippon Pharmaceutics, Japan) for 30 min followed by incubation with anti-tyrosine hydroxylase (TH) antibody (Chemicon, USA, 1:3000 in 10% Block Ace) for 2 days at 4°C. After rinsing, sections were immersed in a solution of 3% H₂O₂ in methanol/PBS (1:1) for 10 min at room temperature, followed by incubation for 2 hr in biotinylated goat anti-rabbit IgG (1:400, Vector, USA) and processing with a Vectastain ABC kit (Vector, USA) using 3′3′-diaminobenzidinetetrahydrochloride (DAB, Vector, USA) as peroxidase chromogen.

Unbiased stereological counts of TH-immunoreactive cell bodies in the SNpc were obtained using an optical fractionator method⁴² and Stereo Investigator software (Stereo Investigator 8, MicroBrightField Inc., USA). The boundary of SNpc was delineated under 100 × magnification and immunopositive neurons were counted in every third section (eight sections per brain) at 400 × magnification on a Nikon ECLIPSE 80 i microscope using a grid of 70 × 70 μm on a counting grid (75 × 100 × 12 μm) with 2 μm upper and lower guard zones. Gundersen’s coefficient of error in all samples was < 0.07.

Immunological detection and quantification of TH expression in the substantia nigra tissue were performed by western blotting with actin as control. Animals were prepared identically to those of the stereological analysis. The substantia nigra was removed and stored at −80°C until use. Samples were lysed on ice in hypotonic buffer (20 mM Hepes pH 7.6, 10 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 0.1% Triton X-100) with protease inhibitor cocktail (Roche, USA), and cleared by a centrifugation at 20 k G for 10 min at 4°C. Following quantification of protein concentration using the BCA method with BSA as control, five μg of tissue lysate reduced in sample buffer was resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-TH antibody (1:2000) overnight at 4°C. Proteins were visualized using anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system (Immobilon Western Chemiluminescent HRP Substrate, Millipore, USA). Chemiluminescence image was quantified by Gauge application (Fuji Film, Japan) and the value was normalized to the level of actin band on the same sample specified by anti-Actin monoclonal antibody (Clone C4, Millipore, USA).

The authors are grateful to Dr. Douglas T. Hess (Case Western Reserve Univ.) for valuable advice and critical reading of the manuscript, and Dr. Yoshinori Tanaka (Corporate Engineering Division, Appliances Company, Panasonic Corporation, Japan) for help with hydrogen measurement. The authors also acknowledge the technical support of Mr. Yuichiro Kojima (Kyushu Univ.). This work was partly performed in the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University. This work is supported in part by Grant-in-Aid for Scientific Research on Innovative Areas (MEXT 20117008 to A.M.), Grant-in-Aid for Exploratory Research (JSPS 24659111 to A.M.), Grant-in-Aid for Scientific Research (B) (JSPS 23390053 to H.N.), Grant-in-Aid for Scientific Research (S) (JSPS 22221004 to Y.N.), Grant-in-Aid for Scientific Research (C) (JSPS 22590084 to M.N.) and Academic Challenge in Robert T. Huang Entrepreneurship of Kyushu University (to M.Y.).