Recent extensive studies have revealed that molecular hydrogen (H2) has great potential for improving oxidative stress-related diseases by inhaling H2 gas, injecting saline with dissolved H2, or drinking water with dissolved H2 (H2-water); however, little is known about the dynamic movement of H2 in a body. First, we show that hepatic glycogen accumulates H2 after oral administration of H2-water, explaining why consumption of even a small amount of H2 over a short span time efficiently improves various disease models. This finding was supported by an in vitro experiment in which glycogen solution maintained H2. Next, we examined the benefit of ad libitum drinking H2-water to type 2 diabetes using db/db obesity model mice lacking the functional leptin receptor. Drinking H2-water reduced hepatic oxidative stress, and significantly alleviated fatty liver in db/db mice as well as high fat-diet-induced fatty liver in wild-type mice. Long-term drinking H2-water significantly controlled fat and body weights, despite no increase in consumption of diet and water. Moreover, drinking H2-water decreased levels of plasma glucose, insulin, and triglyceride, the effect of which on hyperglycemia was similar to diet restriction. To examine how drinking H2-water improves obesity and metabolic parameters at the molecular level, we examined gene-expression profiles, and found enhanced expression of a hepatic hormone, fibroblast growth factor 21 (FGF21), which functions to enhance fatty acid and glucose expenditure. Indeed, H2 stimulated energy metabolism as measured by oxygen consumption. The present results suggest the potential benefit of H2 in improving obesity, diabetes, and metabolic syndrome.
Oxidative stress is involved in many lifestyle-related diseases, including diabetes, atherosclerosis, heart failure, Alzheimer's disease, and Parkinson diseases (1,2,3,4,5,6). Recent studies have revealed that molecular hydrogen (H2) acts as a novel antioxidant and prevents or ameliorates diseases associated with oxidative stress in animal experiments (7,8,9,10,11,12,13,14,15,16,17,18) and clinical tests (19,20,21,22). The brain, heart, liver, and intestine were protected from oxidative stress by inhalation of 1–2% H2 gas (7,8,9,10,11). Interestingly, instead of inhaling H2 gas, drinking water with dissolved H2 (H2-water) protected the brain and kidney from oxidative stress (12,13,14,15). These studies strongly suggest the potential of H2 as an effective therapeutic and preventive antioxidant; however, water dissolves H2 at 0.8 mmol/l at saturated level. Thus, it has been an open question why consumption of even a small amount of H2 is effective for various disease models.
Oxidative stress is one of the causes of type 2 diabetes (1,2,3). To examine whether H2 has benefits on type 2 diabetes, we used db/db mice, in which oxidative stress is accumulated in the liver and leads to hyperglycemia and hyperlipidemia (23). Db/db mice lack a functional leptin receptor, and have been extensively studied as a model for type 2 diabetes (24,25).
In this study, we showed that H2 can be accumulated in the liver with glycogen after oral administration. Next, chronic consumption of H2-water reduced oxidative stress in the liver of db/db mice, and improved obesity and diabetes. As a mechanistic study, we showed that long-term consumption of H2-water enhanced the expression of a hepatic hormone, fibroblast growth factor 21 (FGF21), which is a regulator of energy expenditure (26,27,28,29). These findings suggest the great potential for hydrogen therapy and prevention of metabolic syndrome.
Methods and Procedures
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 +Leprdb/+Leprdb/Jcl) and their nondiabetic heterozygous db/+ littermates (BKS.Cg m+/+Leprdb/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).
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.
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
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/db mice 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. VO2 and 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)).
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/db lacking 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 CO2 than 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/db mice 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.
In this study, we show a novel benefit of H2, it may be useful on therapy for and prevention of obesity and diabetes. So far, many reports have confirmed that consumption of H2 reduces oxidative stress in various disease models and clinical tests (7,8,10,11,12,13,14,15,16,19). Clinical tests revealed that drinking H2-water reduced oxidative stress makers in patients with type 2 diabetes (19) or subjects with potential metabolic syndrome (20), and influenced glucose (19) and cholesterol metabolism (20). The mitochondria are a major source of reactive oxygen species during energy production metabolism and H2 directly protects mitochondria that are exposed to reactive oxygen species (7). Thus, it may be reasonable that mitochondrial energy metabolism, especially fatty acid metabolism, functions against oxidative stress to efficiently expend glucose and fatty acid.
Initially, H2 was dissolved in culture media and shown to protect cells and organelles by directly reacting highly active reactive oxygen species (7). Next, the brain, heart, liver, and intestine were protected from oxidative stress by inhalation of H2 gas (7,8,9,10,11). Interestingly, instead of inhaling H2 gas, H2-water was effective in protecting the brain and kidney from oxidative stress (12,13,14,15) and decreased oxidative stress markers of patients with diabetes and potential metabolic syndrome (19,20). Drinking H2-water is the most convenient method to consume H2; however, when H2 is consumed from H2-water, a very limited amount of H2 can be consumed, because H2 is dissolved in water at a saturated level of only 0.8 mmol/l under atmospheric pressure. After H2-water is consumed, most H2 in blood becomes undetectable within 30 min (14), probably via expiration from the lungs; thus, it has been an open question why H2-water is effective despite its small amount and short exposure. In this study, it was found that H2 can be accumulated and reserved in the liver with glycogen, which at least partly explains this question.
Moreover, it has been reported that H2 acts as an anti-inflammatory and antiallergic regulator by inducing inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6 and some phosphorylating signal factors (9,10,14,32). On the other hand, the concept that obesity is a proinflammatory disease has been accepted (33); thus, consumption of H2-water may suppress obesity by acting as an anti-inflammatory.
Alternatively, we found that consumption of H2-water enhanced the expression of FGF21. Since it has not been shown that H2 directly regulates transcription, H2 is indirectly involved in FGF21 expression. Since FGF21 is a metabolic hormone that improves insulin sensitivity and glucose clearance, reduces plasma triglyceride concentrations and suppresses weight gain when fed a high-fat diet (26,27,28,29), all the findings shown in this study were elucidated by the enhanced expression of FGF21.
Indeed, we revealed that drinking H2-water stimulates energy metabolism as measured by O2 consumption and CO2 production. The enhancement of energy metabolism may fully elucidate why consumption of H2-water suppresses the gain of fat and body weights and improves metabolic parameters; however, it remains unknown whether the induction of FGF21 fully elucidates the enhancement of energy metabolism. Moreover, the relationships among the reduction of oxidative stress, induction of FGF21 expression and stimulation of energy metabolism are still unclear. It should be analyzed whether these relationships are direct or indirect. It might be valuable to examine the relationship of obesity with oxidative stress. Although the primary target of H2 essentially remains unknown, these findings provide a clue to understand the mechanism of chronic treatment with H2-water.
Finally, we would like to emphasize that the novel benefit of H2 in therapeutic and preventive applications for metabolic syndrome could be achieved by the most convenient way, H2-water.
We thank Blue Mercury (Tokyo, Japan) for the generous gift of hydrogen water and M. Takeda for technical assistance. This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No.20300230 for S.O. and No.20500723 for N.K.) and the Ministry of Health, Labour and Welfare for S.O. (Intramural Research Grant (20B-13) for Neurological and Psychiatric Disorders of NCNP).