Molecular Hydrogen Improves Obesity and Diabetes by Inducing Hepatic FGF21 and Stimulating Energy Metabolism in db/db Mice

Animals

Male Sprague–Dawley rats of 10 weeks of age and male C57BL/6 mice of 12 weeks of age were purchased from Nippon SLC (Hamamatsu, Shizuoka, Japan). Genetically diabetic male db/db mice (BKS.Cg +Lepr^db/+Lepr^db/Jcl) and their nondiabetic heterozygous db/+ littermates (BKS.Cg m+/+Lepr^db/Jcl) were purchased at 5 weeks of age from CLEA Japan, (Tokyo, Japan). Mice were given H₂-water from 6 weeks of age. For the diet-induced obesity study, C57BL/6 mice were given a high fat-diet (F2HFD1; Oriental Yeast, Tokyo, Japan) for 1 or 2 weeks. The care and use of laboratory animals were in accordance with the National Institutes of Health guidelines. This study was approved by the Animal Care and Use Committee of Nippon Medical School (Tokyo, Japan).

Hydrogen water

H₂-water was prepared as described previously (12). In brief, H₂ was dissolved in water under high pressure (0.4 MPa) to a supersaturated level and the saturated H₂-water was stored under atmospheric pressure in an aluminum bag with no dead volume. Saturated H₂-water was used as 100% H₂-water. H₂-water degassed by gentle stirring was used as control water. Saturated H₂-water was diluted with ninefold control water and used as 10% saturated H₂-water. Mice were given water freely using closed glass vessels equipped with an outlet line containing two ball bearings, which kept the water from being degassed. The vessel was freshly refilled with H₂-water at 2:00 pm every day.

Diet restriction

Diabetic mice were subjected to controlled dietary restriction, such that the daily diet consumption was equivalent to 80% or 50% of that of ad libitum diet consumption. Control groups were permitted free access to food and water throughout the experiment. In the diet-restricted group, mice were permitted ad libitum water, but food intake was restricted.

Measurement of H₂ concentration

H₂ concentration in rat liver tissue was measured using a needle-type hydrogen electrode (Unisense, Aarhus, Denmark). Rat received H₂-water orally by stomach gavage at 15 ml/kg. Throughout the experiment, the electrode current was measured with a picoammeter (Keithley, Cleveland, Ohio) and H₂ concentration was obtained from the calibration curve generated using known levels of H₂-saturated saline.

In an in vitro experiment, H₂ concentration in glycogen, glucose solutions, or drinking water in the glass vessel was measured using a needle-type hydrogen electrode as described above.

Sample collection and biochemical analysis

Mice were killed under anesthesia, blood was collected from the heart, and liver tissues were excised and frozen with liquid nitrogen or fixed with 4% paraformaldehyde for further analysis. The antioxidation effect of H₂ was determined by measuring lipid peroxides in the liver, using a malondialdehyde assay kit (Northwest Life Science Specialties, Vancouver, WA) and the level of lipid peroxides was expressed as nmol malondialdehyde (MDA)/mg protein. Plasma concentrations of total ketone bodies, triglyceride, and total cholesterol were determined with commercially available kits (Wako Pure Chemical Industries, Osaka, Japan). Plasma low-density lipoprotein-cholesterol and high-density lipoprotein-cholesterol were measured using kits (Sekisui Medical, Tokyo, Japan). Plasma glucose and nonesterified fatty acids were determined with kits available from Shino-Test (Tokyo, Japan) and Eiken Chemical (Tokyo, Japan), respectively. Plasma insulin was measured using an insulin ELISA kit (Morinaga Institute of Biological Science, Kanagawa, Japan).

Oil Red O staining

Mouse livers were fixed in 4% paraformaldehyde in phosphate-buffered saline, embedded, and cryosectioned 10 mm thick. The sections were rinsed with 60% isopropanol, stained with 0.25% Oil Red O solution, rinsed with 60% isopropanol, and mounted in aqueous mountant. The area of stained lipid (%) was calculated using the Image J program (ver 1.41; National Institutes of Health, Bethesda, MD) from four sections for each mouse.

Body fat composition analysis

For computed tomography analysis of body fat composition, mice were anesthetized with halothane in a mixture of nitrous oxide and oxygen (70%:30%, vol/vol) and then scanned using a LaTheta LCT-100, experimental animal computed tomography system (Aloka, Tokyo, Japan). Contiguous 1-mm slice images were used for quantitative assessment using LaTheta software (ver 1.00). Visceral fat, subcutaneous fat, and muscle were distinguished and evaluated quantitatively.

RNA isolation and reverse transcriptase-PCR

Total RNA was isolated from the liver tissue using an RNeasy Mini kit (QIAGEN, Valencia, CA). Complementary DNA generated by SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) was analyzed by quantitative PCR using Thermal Cycler Dice Real Time System TP800 (TAKARA BIO, Shiga, Japan). All samples were normalized to glyceraldehyde 3-phosphate dehydrogenase expression. Primer and probe sequences for each PCR are shown in Table 1.

Table 1. Primers and probes for reverse transcriptase-PCR

Behavioral analysis

Movement activities were recorded in db/+ and db/db mice automatically using a laboratory animal monitoring system (ACTIMO-100; Shinfactory, Fukuoka, Japan). Mice were housed individually and temperature was maintained at 22 °C. Food and water with or without hydrogen were available ad libitum. Mice were acclimatized to the chambers for 24 h before beginning recordings and then monitored for 48 h. Movement activity was measured as ambulatory counts from a record of consecutive adjacent infrared beam breaks. Cumulative ambulatory counts on the x- and y-axes were recorded every 10 min.

Indirect calorimetric analyses

Metabolic rate was measured by indirect calorimetric analysis in db/+ and db/dbmice using an open-circuit calorimeter (Oxymax; Columbus Instruments, Columbus, OH). Mice were housed individually in a chamber (20 × 10 × 12.7 cm) and temperature was maintained at 22 °C, with air flow of 0.5 l/min. Food and water with or without H₂ were available ad libitum. Mice were acclimatized to the chambers for 48 h before beginning recordings and then monitored for 48 h. VO₂and VCO₂ were measured every 10 min using an electrochemical O₂ analyzer and a CO₂ sensor (Oxymax), and the respiratory exchange ratio was calculated as VCO₂/VO₂ (volume of CO₂ produced per volume of O₂ consumed (ml/kg/h)).

Statistical analysis

We performed statistical analysis using StatView software (SAS Institute) by applying an unpaired two-tailed Student's t-test and ANOVA followed by Fisher's exact test, as described previously (7). Differences were considered statistically significant at P < 0.05.

Molecular hydrogen is accumulated in the liver with glycogen

We monitored the dynamic movement of H₂ in the liver after oral administration of H₂-water. Rat received H₂-water orally by stomach gavage, and levels of H₂ in the liver were monitored by directly inserting a needle-type hydrogen sensor into the liver for an hour (Figure 1a). The H₂ concentration profile gave a peak 5 min after administration of H₂-water in both a fed and fasted liver; however, a great difference was found between the fed and fasted liver; the maximum H₂ level in the fed liver was twofold that of the fasted liver. Moreover, the fed liver maintained a considerable H₂ level for an hour, while that in the fasted liver returned to the basal level after 25 min.

Since a fed liver is rich in glycogen, we speculated that higher polymers of carbohydrates including glycogen have the capacity to maintain H₂. To verify this speculation, we examined saturated solubilities of H₂ in glycogen and glucose solutions by a hydrogen sensor. H₂ was dissolved in a glycogen or glucose solution by bubbling H₂ gas up to a saturated level (Figure 1b). Compared to the glucose solution and water, the glycogen solution dissolved a significantly higher amount of H₂. The hydrogen sensor was not influenced by glycogen and glucose (data not shown). In addition, H₂ was maintained for a longer time in saturated glycogen solution than in saturated glucose solution or water in a plastic tube with the lid off under atmospheric pressure (Figure 1c). The half-life was 19.1 ± 2.3, 30.9 ± 3.0, and 20.3 ± 0.4 min (mean ± s.d., n = 3) in water, 100 mg/ml glycogen and glucose solutions, respectively. The half-life of dissolving H₂ in the glycogen solution was prolonged 1.6-fold. Thus, it is concluded that H₂ can be accumulated and reserved in the liver with glycogen, suggesting that expenditure of glycogen should accompany the release of H₂.

Consuming hydrogen water reduces oxidative stress in the liver and improves fatty liver

Since obesity is a proinflammatory disease, consumption of H₂-water may suppress obesity by acting as an anti-inflammatory. To examine the antioxidation effect of H₂ on the liver, we used obesity and type 2 diabetes model mice db/dblacking functional leptin receptors, because oxidative stress is accumulated in the liver. Db/db mice and their lean littermates drank H₂-water ad libitum for 3 months. H₂-water was exchanged for fresh saturated or 10% saturated water at 1400 h every day. The concentration of dissolved H₂ was measured as described in Methods and Procedures (Figure 2a). Since db/db mice drank much more water than wild-type mice, H₂ was degassed much faster into the air phase in the vessel used for db/db mice; however, a considerable amount of H₂ was maintained by this method.

The effect of H₂ on oxidative stress in the liver was examined as judged by the level of malondialdehyde (MDA), an oxidative stress marker derived from lipid peroxides. The MDA level in the liver of H₂-administered mice significantly fell to nearly the level in nondiabetes control mice, indicating that consumption of H₂-water ad libitum markedly suppressed oxidative stress (Figure 2b). A dotted pattern caused by the accumulation of fat disappeared in the liver of db/db mice with H₂, compared to control water administered db/db mice (Figure 2c, insets of upper panels). Oil red O staining indicated that H₂ administration significantly reduced neutral lipid accumulation in the livers of db/db mice (Figure 2b, lower panels and Figure 2d, left graph). Furthermore, even with short-term administration (1–2 weeks), H₂ significantly reduced fat accumulation in the liver of high fat-diet-induced obesity mice using the wild-type (Figure 2d, middle and right graph). These data clearly indicate that consumption of H₂ markedly reduces hepatic oxidative stress levels and improves fatty liver in db/db as well as diet-induced obesity mice.

Consuming hydrogen water suppressed body-weight gain and reduced plasma glucose and triglyceride levels

To investigate the effect of H₂ on the obesity of db/db mice, body-weight was monitored throughout the experimental period, and body fat mass at 18 weeks old was measured by computerized tomography. Mice were divided randomly into 3 groups. Group I (control) was allowed to freely drink water without H₂. Group II and Group III were given H₂-water with 0.8 mmol/l (saturated H₂-water; 100%) and 0.08 mmol/l (10% saturated level of H₂), respectively. In the initial phase of the experiment, the weight gains of all group animals were similar, suggesting no toxic effect of H₂-water; however, while the control mice exhibited a progressive weight increase, the mice in both 100% and 10% H₂-administered groups achieved a modest weight increase (Figure 3b). Group II mice (100% H₂) were slimmer and their body-weight was significantly lower than that of Group I mice (control) at 18 weeks of age (Figure 3a,b). The suppression of body-weight gain was observed in mice drinking 10% H₂-water (Group III) (Figure 3b). Body fat was also substantially lower in 100% H₂-consuming mice (Figure 3c,d). Since the consumed amounts and volumes of diet and water did not differ among groups (Figure 3e,f), it is suggested that H₂ consumption stimulates energy metabolism to suppress the gain of fat and body weights.

Next, we performed biochemical examinations of blood. Plasma levels of glucose and insulin were significantly reduced in the 100% H₂-administered group and triglycerides were significantly lowered in both 100% and 10% H₂-administered groups (Figure 4a–c). Plasma total ketone bodies tended to increase in the 100% H₂-administered group (Figure 4d), while no change in plasma levels of free fatty acid, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol-cholesterol, total cholesterol, and adiponectin was found (Figure 4e–i). These data demonstrate that the consumption of H₂ markedly improves obesity, hyperglycemia, and the plasma triglycerides of diabetic db/db mice.

Consuming hydrogen water shows a similar effect to diet restriction

Since db/db mice cannot regulate their appetite to eat excess diet by a deficient leptin receptor gene, diet restriction should be effective to improve obesity and diabetes (30). When db/db mice were subjected to controlled dietary restriction, the levels of plasma glucose, insulin, and triglycerides significantly fell (Figure 5a–c). When comparing the effects by drinking H₂-water and dietary restriction, the plasma glucose level of mice given 80% of ad libitum diet consumption was the same as that of H₂-administered mice (Figure 5a). Furthermore, an additive effect was observed when mice were given both H₂-water and a restricted diet (Figure 5a). A similar additive effect was seen in plasma triglyceride levels (Figure 5c). In particular, when both H₂-water and 80% diet restriction were given to db/db mice, the plasma triglyceride reached the level of control db/+ mice (Figure 5c).

Consuming hydrogen water increases hepatic mRNA level of FGF21

The liver plays an essential role in controlling blood glucose levels by modulating glucose catabolism and gluconeogenesis. To understand the mechanism of how H₂ regulates glucose and triglycerides, db/db mice given H₂-water for 3 months were subjected to expression analyses of genes related with the regulation of gluconeogenesis. Phosphoenolpyruvate carboxykinase catalyzes the initial step of gluconeogenesis, and glucose-6-phosphatase catalyzes the last committed step of this process (31). Data from real-time PCR revealed that H₂ administration had no influence on hepatic mRNA levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, catalytic subunit (G6PC) (Figure 6c,d).

Since FGF21, an atypical member of the fibroblast growth factor family, contributes to energy metabolism, we focused on the gene expression of FGF21 after integrated study of the gene expression. H₂ administration induced hepatic mRNA levels of FGF21, regardless of a fed or fasted liver (Figure 6a,b). These results indicate that, at least in part, the induction of hepatic FGF21 contributes to the lowering effect on plasma glucose and triglyceride levels.

Drinking hydrogen water stimulates energy metabolism

To verify whether drinking H₂-water stimulates energy metabolism, we examined oxygen (O₂) consumption and carbon dioxide (CO₂) production. First, we compared physical ability by detecting the movement of mice with infrared beams. Although db/db mice was apparently less active in light and dark than control db/+ mice, no difference was found between db/db mice with and without H₂-water (Figure 7a,b).

Db/db mice with or without H₂-water consumed less O₂ and produced less CO₂than db/+ control mice (Figure 7c–f). Moreover, it is significant that H₂-drinking db/db mice consumed more O₂, 10%, and produced more CO₂, 10%, than db/dbmice without H₂-water during both night and day (Figure 7c–f). Since respiratory exchange rates (VCO₂/VO₂) did not differ between db/db mice with and without H₂-water, the carbon source for energy production was not changed to stimulate energy metabolism (Figure 7g,h).

Thus, drinking H₂-water suppresses the gain of fat and body weights and improves metabolic parameters by stimulating energy metabolism.