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Effects of Hydrogenized Water on Intracellular Biomarkers for Antioxidants, Glucose Uptake, Insulin Signaling and SIRT 1 and Telomerase Activity

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  • Sierra Productions Research, LLC
Article

Effects of Hydrogenized Water on Intracellular Biomarkers for Antioxidants, Glucose Uptake, Insulin Signaling and SIRT 1 and Telomerase Activity

Abstract and Figures

Hydrogen has been shown in several clinical trials to be completely safe without adverse events and there are no warnings in the literature of its toxicity or adverse effects during long-term exposure. Molecular hydrogen has proven useful and convenient as a novel antioxidant and modifier of gene expression in many conditions where oxidative stress and changes in gene expression result in cellular damage. Our intracellular biomarker studies have shown that a hydrogenized water drink formula containing 2.6 ppm dissolved hydrogen was able to penetrate cellular membranes and function as an antioxidant in human liver cells (HePG2) utilizing the Cellular Antioxidant Assay (CAA). This assay uses the protection of a florescent probe as a marker for cellular damage by reactive oxygen species (ROS), such as peroxyl radical, and compares this to the known antioxidant standard, Quercetin. Using this system oxidative damage was reduced in a dose-dependent manner. One ml of hydrogenized water was found to possess antioxidant capacity equivalent to 0.05 µmole of quercetin. When examined in a human colon cell line (Caco-2 cells), hydrogenized water demonstrated a dose-and time-dependent permeability inhibition of an intracellular fluorescent glucose derivative (2-NBDG), indicating decreased glucose uptake. In another study, the impact of hydrogenized water on Akt phosphorylation (Ser473), a biomarker for insulin signaling, was monitored in human skeletal muscle cells. The hydrogenized water treatment markedly elevated the level of phosphorylation of Akt (Ser473) in a dose-dependent manner. The anti-aging effects of hydrogenized water were examined utilizing SIRT1 expression as a biomarker of aging in human umbilical cells (HUVECs). Hydrogenized water increased dose-dependent SIRT1 gene expression. Hydrogenized water also increased telomerase activity (an anti-aging biomarker in HUVEC cells) up to 148% when cells were treated with media containing 25% hydrogenized water formula. Increased telomerase activity caused by hydrogenized water may be able to protect telomeres from degradation, suggesting the possible use of hydrogenized water in therapeutic interventions of age-related diseases. These studies show that commercial hydrogenized water improved the levels or activities of a few intracellular biomarkers specific for antioxidant activity, glucose uptake, insulin signaling and SIRT 1 and telomerase activities. Industrial Relevance: The molecular hydrogen used in this study indicates that certain commercial sources of hydrogenized water can provide similar antioxidant and gene expression modifications seen in other sources of molecular hydrogen. The biomarkers evaluated here lend well to hydrogenized water's biological activity relating to health conditions and aging.
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American Journal of Food and Nutrition, 2016, Vol. 4, No. 6, 161-168
Available online at http://pubs.sciepub.com/ajfn/4/6/4
©Science and Education Publishing
DOI:10.12691/ajfn-4-6-4
Effects of Hydrogenized Water on Intracellular
Biomarkers for Antioxidants, Glucose Uptake, Insulin
Signaling and SIRT 1 and Telomerase Activity
Robert Settineri1,*, Jin Ji2, Chunlan Luo2, Rita R. Ellithorpe3, Gonzalo Ferreira de Mattos4, Steven Rosenblatt5,
James LaValle6, Antonio Jinenez7, Shigeo Ohta8, Garth L. Nicolson9
1Sierra Productions Research, Irvine, USA
2Brunswick Laboratories, Inc., Southborough, USA
3Tustin Longevity Center, Tustin, USA
4Laboratory of Ion Channels, School of Medicine, Universidad de la República, Montevideo, Uruguay
5Saint John’s Health Center, Santa Monica, USA
6Progressive Medical Center, Orange, USA
7Hope4Cancer Institute, Baja California
8Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nippon Medical School, Japan
9Department of Molecular Pathology, The Institute for Molecular Medicine, Huntington Beach, USA
*Corresponding author: sierraprod@aol.com
Abstract Hydrogen has been shown in several clinical trials to be completely safe without adverse events and
there are no warnings in the literature of its toxicity or adverse effects during long-term exposure. Molecular
hydrogen has proven useful and convenient as a novel antioxidant and modifier of gene expression in many
conditions where oxidative stress and changes in gene expression result in cellular damage. Our intracellular
biomarker studies have shown that a hydrogenized water drink formula containing 2.6 ppm dissolved hydrogen was
able to penetrate cellular membranes and function as an antioxidant in human liver cells (HePG2) utilizing the
Cellular Antioxidant Assay (CAA). This assay uses the protection of a florescent probe as a marker for cellular
damage by reactive oxygen species (ROS), such as peroxyl radical, and compares this to the known antioxidant
standard, Quercetin. Using this system oxidative damage was reduced in a dose-dependent manner. One ml of
hydrogenized water was found to possess antioxidant capacity equivalent to 0.05 µmole of quercetin. When
examined in a human colon cell line (Caco-2 cells), hydrogenized water demonstrated a dose- and time-dependent
permeability inhibition of an intracellular fluorescent glucose derivative (2-NBDG), indicating decreased glucose
uptake. In another study, the impact of hydrogenized water on Akt phosphorylation (Ser473), a biomarker for insulin
signaling, was monitored in human skeletal muscle cells. The hydrogenized water treatment markedly elevated the
level of phosphorylation of Akt (Ser473) in a dose-dependent manner. The anti-aging effects of hydrogenized water
were examined utilizing SIRT1 expression as a biomarker of aging in human umbilical cells (HUVECs).
Hydrogenized water increased dose-dependent SIRT1 gene expression. Hydrogenized water also increased
telomerase activity (an anti-aging biomarker in HUVEC cells) up to 148% when cells were treated with media
containing 25% hydrogenized water formula. Increased telomerase activity caused by hydrogenized water may be
able to protect telomeres from degradation, suggesting the possible use of hydrogenized water in therapeutic
interventions of age-related diseases. These studies show that commercial hydrogenized water improved the levels
or activities of a few intracellular biomarkers specific for antioxidant activity, glucose uptake, insulin signaling and
SIRT 1 and telomerase activities. Industrial Relevance: The molecular hydrogen used in this study indicates that
certain commercial sources of hydrogenized water can provide similar antioxidant and gene expression
modifications seen in other sources of molecular hydrogen. The biomarkers evaluated here lend well to
hydrogenized water’s biological activity relating to health conditions and aging.
Keywords: Hydrogenized water, bioassays, antioxidant, Caco-2 permeability assay, glucose uptake, insulin
receptor, SIRT 1, telomerase activity
Cite This Article: Robert Settineri, Jin Ji, Chunlan Luo, Rita R. Ellithorpe, Gonzalo Ferreira de Mattos,
Steven Rosenblatt, James LaValle, Antonio Jinenez, Shigeo Ohta, and Garth L. Nicolson, Effects of
Hydrogenized Water on Intracellular Biomarkers for Antioxidants, Glucose Uptake, Insulin Signaling and SIRT 1
and Telomerase Activity.” American Journal of Food and Nutrition, vol. 4, no. 6 (2016): 161-168.
doi: 10.12691/ajfn-4-6-4.
American Journal of Food and Nutrition 162
1. Introduction
Hydrogen is the smallest and most abundant element,
and in its molecular form H2 is colorless, odorless, tasteless
and virtually non-toxic. For some time H2 was not considered
a bioactive molecule, but a recent study found that inhaled
hydrogen gas protects the brain against ischemia reperfusion
injury and stroke, and this was likely due to an antioxidant
mechanism that reduces free radical damage [1].
Hydrogen can be delivered by gas inhalation, injection
of hydrogen-rich saline solutions or by drinking hydrogen-
rich water (hydrogenized water). Using these methods of
administration H2 has been shown to have various
preventive and therapeutic effects useful in emergency
and critical care medicine [2]. In animal model studies
hydrogen gas has been shown to ameliorate intracerebral
hemorrhage [3] and reduce hyperoxic lung injury in vivo
[4]. Hydrogen gas has also been useful in diagnostic
medicine. For example, a hydrogen breath test has been
used in clinical practice to detect abnormal bacterial
overgrowth in the small intestine, or to diagnose the
malabsorption of lactose or fructose [5].
It was reported that molecular hydrogen could improve
the damage caused by cerebral ischemia reperfusion injury
and selectively reduce strong cytotoxic oxygen radicals,
including hydroxyl radical (•OH) and peroxynitrite
(ONOO) [2,5]. Molecular hydrogen has also proven to be
useful in tissue injury and aging [1,2,5,7,8,9,10,11]. In this
process, free radicals, such as reactive oxygen species
(ROS) and reactive nitrogen species (RNS), are generated
as by-products of oxidative metabolism. Since H2 can
rapidly diffuse across cell membranes, it is an
effective ·OH radical scavenger in cultured cells.
ROS/RNS can induce cumulative oxidative damage to
cellular macromolecules, eventually resulting in cellular
dysfunction, cell death and in some cases, leading to or
assisting in the development of various diseases.
It has also been demonstrated that H2 rapidly diffuses
across cell membranes and is an effective ·OH radical
scavenger in cultured cells and this has been related to its
ability to rapidly diffuse across cell membranes. Since
then, numerous studies have focused on understanding the
potential therapeutic value of hydrogen and hydrogen-rich
water in various disease models [12,13].
A comprehensive review the literature on the effects of
molecular hydrogen (H2) on animals and human subjects
and patients with a variety of diagnoses, such as metabolic,
rheumatic, cardiovascular, neurodegenerative disease,
infections, radiation damage, exercise as well as effects on
aging has recently been published. [12].
Here we investigated the effects of hydrogenized water
on several intracellular biomarkers related to human
health, metabolism and longevity, such as those specific
for antioxidant activity weight management/diabetic
functional improvement insulin receptor signaling and
anti-aging indicators [17,18].
2. Materials and Methods
Hydrogenized water identified as “Izumio Water” was
acquired from Naturally Plus Co. Ltd. Ropongi, Minato-
ku, Tokyo, Japan. At the time of initial packaging, the
infused hydrogen within the test water was analyzed by
both manometric and diaphragm polarographic electrode
measurements at final concentration of 2.6 ppm and pH
7.0. The human Caco-2 cell line and mouse C2C12
myoblast cell line were obtained from American Type
Culture Collection (ATCC, Manassas, USA). HG-DMEM
(Dulbecco's modified Eagle's medium with high glucose)
was from GIBCO™ (Grand Island, NY, USA). Fetal
bovine serum (FBS) and horse serum were purchased
from Hyclone (Pittsburg, PA, USA). Insulin, fatty acid-
free bovine serum albumin (BSA) was obtained from
(Sigma, St. Louis, MO, USA). The sources of other
materials are indicated in individual method sections.
2.1. Cellular Antioxidant Assay
The Cellular Antioxidant Analysis (CAA) analyzes the
capacity of hydrogenized water to protect a fluorescent
probe (as a marker) from damage by reactive oxygen
species (ROS) in intracellular environments. The assay
was carried out as previously described [19]. First, the
probe was incubated with representative human liver cells
(HePG2 cells obtained from ATCC, Manassas, VA, USA).
Once inside cells the loss of probe fluorescence functions
as an indication of the extent of damage the probe
experiences from the oxygen radicals. Various concentrations
of hydrogenized water were incubated with the cells to
allow its natural absorption. Then an oxygen radical
inducer, 2,2'-Azobis(2-amidinopropane) dihydrochloride
(AAPH) from Sigma-Aldrich, (St. Louis, MO, USA), was
introduced into cellular environment, which triggers the
release of peroxyl radicals. Without excess antioxidants
present outside or inside the cells, oxygen radicals
permeate through cellular membranes and oxidize the
marker probe, and when in excess the oxygen radicals
damage the cells. In contrast, when excess antioxidant
material is present inside cells, damage does not occur.
The cellular antioxidant effect of the hydrogenized water
can be measured by assessing the preservation effect of
hydrogenized water absorbed inside of cells. quercetin, a
known bioeffective antioxidant, is used as the positive
control. The cellular antioxidant effect of the test material
is compared with that of quercetin, and the comparison
result is then expressed as µmole quercetin equivalency
(QE) per gram (or milliliter) of a tested material.
2.2. Glucose uptake in Caco-2 human Cells
This study investigates the effect of hydrogenized water
on the permeability of glucose across a Caco-2 cell
monolayer. In this study, Caco-2 human cells were grown
at 37°C in 5% CO2 and at 95% relative humidity using
Dulbecco’s Modified Essential Medium (DMEM)
supplemented with 10% fetal bovine serum, 1% non-essential
amino acids, and 0.05% penicillin/streptomycin/amphotericin.
Cells were removed at 8090% confluence using a 0.25%
trypsin/0.20% ethylene diamine teraacetic acid (EDTA)
solution and replated. Media were changed approximately
every 48 h. For the transport experiments Caco-2 cells
(passages 2640) were seeded at 6.5×104 cells/cm2 into
polycarbonate 12-well Transwell® plates (Corning Costar
Corporation, Cambridge, MA, USA) (0.4μm mean pore
size) and used 2128 days after seeding. 2-NBDG (2-(N-
(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose),
163 American Journal of Food and Nutrition
a fluorescently-labeled deoxy glucose analog, was used as
a probe for the detection of glucose movement across
Caco-2 cell monolayers. Six concentrations of hydrogenized
water (0, 3.125, 6.25, 12.5, 25 or 50% v/v hydrogenized
water in ionic and osmotic balanced DMEM), along with
200 μg/ml 2-NBDG were added to the apical side of
confluent monolayers of Caco-2 cells, and the monolayer
cultures were then incubated for 30 min and 1 hr.,
respectively. At each time point, the receiver (basolateral)
sides of the cell media were removed for analysis. The
degree of glucose permeation across the cell monolayer
was monitored using changes in the fluorescent intensity
of 2-NBDG [15]. Statistical assessments were performed
by t-test analyses.
2.3. Cellular Insulin Receptor Signaling
Assessment Using Akt phosphorylation
The effects of hydrogen water on insulin signaling in
human skeletal muscle cells was studied using Akt
phosphorylation as the cellular biomarker. C2C12 murine
myoblasts (ATCC, Manassas, VA, USA, CRL-1772) were
cultivated in DMEM (Lonza, Portsmouth, NH, USA)
containing 4.5 g/L glucose, 2 mM glutamine and 10%
FCS (Thermo Scientific). Cells were plated and grown to
approximately 8090% confluence. The cells were serum
starved (DMEM 0.5% FCS) overnight, then treated with
hydrogenized water at 0, 3.125, 6.25, 12.5, 25 or 50% v/v
hydrogenized water in ionic and osmotic balanced DMEM
for 60 min. Cells were also treated with concentrations of
30 nM insulin (Sigma, Aldrich, St. Louis, MO, USA) for
15 min as a control. After the hydrogenized water
treatment, cells were then washed in PBS and lysed in
lysis buffer (Thermo Fisher Scientific, Fremont, CA,
USA). After protein measurement, SDS loading buffer
(3% (w/v) SDS, 3% (v/v) β-mercaptoethanol, 10 mM
EDTA (pH 8), 20% (v/v) glycerol, and 0.05% (w/v)
bromphenol blue) was added, and the samples were boiled
for 5 min at 95 °C. Proteins were separated by SDS-
polyacrylamide gel electrophoresis (PAGE) and
transferred to a nitrocellulose filter. The membranes were
blocked for minimum of 1 h in Net-G [150 mM NaCl, 5
mM EDTA (pH 8), 50 mM TrisHCl (pH 7.5), 0.05% (v/v)
Triton X-100, 0.25% (w/v) gelatin] and incubated with
primary antibody overnight at 4 °C. Proteins were
detected by horseradish peroxidase-conjugated secondary
antibody using an ECL reagent as described by the
manufacturer (Thermo Scientific, Fremont, CA, USA).
2.4. Cellular Aging Assessment
The impact of hydrogenized water on the cellular aging
process was carried out via cellular assessment of two
aging biomarkers: SIRT1 and telomerase activity,
respectively.
2.4.1. SIRT1 as Biomarker for Aging
Cellular aging via SIRT1 assessment measures the
ability to prevent or augment cellular SIRT1 production.
In this assay, human umbilical vein endothelial cells
(HUVECs) were treated with 0, 3.125, 6.25, 12.5, 25 or 50%
v/v hydrogenized water in buffer and in duplicate and
incubated continued for up to 24 hr. One group of cells
was treated with 100 µM of Resveratrol (Sigma Aldrich,
St. Louis, MO, USA) for 24 hr as control. Subsequently,
the cells were lysed, and SIRT1 expression levels were
analyzed via Western blot.
2.4.2. Telomerase Activity as an Aging Biomarker
Cellular aging via telomerase activity assessment
investigates the effect of hydrogenized water on aging via
changes in telomerase activities in human umbilical vein
endothelial cells (HUVECs). Here, HUVECs were treated
with 0, 3.125, 6.25, 12.5, 25 or 50% v/v hydrogenized
water in buffer and in duplicate for 24 hr. One cell sample
was treated with 10 nM raloxifene (Sigma, Aldrich, St.
Louis, MO, USA) for 24 hr as positive control.
Telomerase activity was determined using the TRAPeze®
XL Telomerase Detection Kit (Millipore, Billerica, MA,
USA) according to the manufacturer's instructions.
3. Results and Discussion
3.1. Cellular Antioxidant Assay
CAA analyzes the capacity of a material to protect a
fluorescent probe (the biomarker) from damage by
reactive oxygen species (ROS) in the intracellular
environment. In this assay, peroxyl is used as the free
radical (ROS), and human liver cells are used as the
cellular model [19]. Quercetin is used as the standard, and
the results are expressed as μmole quercetin equivalency
(QE) per gram (or milliliter) of the tested material. Our
analysis determined that the CAA value of the
hydrogenized water was 0.05 µmole QE per mL, which
indicates that one ml of hydrogenized water possesses
antioxidant capacity equivalent to 0.05 µmole of quercetin.
This analysis indicates that commercial hydrogenized
water can permeate cellular membranes and function as an
in vivo antioxidant in human cells. An example of CAA
value of a fruit rich in antioxidants, e.g., cranberries, has
CAA value of ~ 12.5 µmole QE per gram of fresh fruit
[20]. Compared with cranberries, the CAA value
of hydrogenized water is relatively low. However, the
usual daily intake of water is much higher than fruit alone.
Another point that must be taken into consideration is that
the active evaporation of hydrogen from hydrogenized
water can significantly impact the actual amount of
hydrogen that can permeate into the cells and function as a
cellular antioxidant. In human bodies, such hydrogen
evaporation may be significantly reduced or the effect of
evaporation may be different.
3.2. Impact of Hydrogenized Water on
Glucose Uptake Using the Caco-2 Membrane
Model
Differentiated polarized human Caco-2 colon epithelial
cells express a wide range of transporter proteins on their
cell membranes that mimic human intestinal epithelia [20].
The Caco-2 absorption study is considered to be the
reference standard in pharmaceutical and nutraceutical
industries for in vitro prediction of in vivo human
intestinal absorption and bioavailability of orally
administered substances. In the drug discovery field, the
Caco-2 cell permeability assay is also used to predict
drug-drug interactions (DDIs) of orally administered drugs
American Journal of Food and Nutrition 164
[21,22,23]. Here, we used caco-2 cells as a monolayer
epithelial membrane to investigate the bioabsorption of
glucose, and how hydrogenized water can impact the
degree of glucose absorption.
To study the effect of hydrogenized water on glucose
permeation we measured the permeability of 2-NBDG, a
fluorescent derivative of glucose, across Caco-2 cell
monolayers after incubation with various concentrations
of hydrogenized water (30 or 60 min incubation). The
results in Figures 1 and Figure 2 show the ability of
hydrogenized water to modulate the permeability of a
fluorescent derivative of glucose across Caco-2 cell mono-
layers in a concentration- and time-dependent fashion,
suggesting the potential of molecular hydrogen to function
as a glucose permeation inhibitor.
Figure 1. Permeability of glucose (monitored via the fluorescent derivative of glucose 2-NBDG) across Caco-2 cell monolayers upon incubation with
HBSS (control) or 0.25, 0.5, 6.25, 12.5, 25 or 50% v/v hydrogenized water in HBSS in an incubation time of 30 min. *P<0.05
Figure 2. Permeability of glucose (monitored via a fluorescent derivative of glucose) across Caco-2 cell monolayers upon incubation with HBSS
(control) and 3.125%, 6.25%, 12.5%, 25% and 50% v/v hydrogenized water in HBSS in an incubation time (60 min). *P<0.05
The results show that the decrease in apical-to-
basolateral permeability of glucose in Caco-2 cells
(monitored via a fluorescent derivative of glucose) was
associated with an increase in hydrogenized water
concentration and incubation time (Figure 1 and Figure 2).
Incubation of Caco-2 cell monolayers with higher
concentrations (50% v/v) of hydrogenized water in HBSS
caused a time-dependent decrease in glucose permeability
but the effect was less compared to lower concentrations.
3.3. Impact of Hydrogenized Water on
Cellular Insulin Signaling Activity
Skeletal muscle is the primary site of glucose uptake,
disposal, and storage, accounting for approximately 75%
of the entire body's glucose uptake under insulin
stimulation. The insulin receptor in this tissue is a
transmembrane receptor that is activated by insulin and
insulin-like growth factors. Metabolically, the insulin
receptor plays a key role in glucose regulation, a
functional process that under degenerate conditions may
result in various health issues including obesity, metabolic
syndrome, diabetes, and cancer. [13]
The main consequence of activation of the insulin
receptor is inducing glucose uptake. Under "insulin
insensitivity", or a decrease in insulin receptor signaling
conditions, cells are unable to take up glucose, resulting in
hyperglycemia (an increase in circulating glucose).
Eventually this can lead to diabetes mellitus type 2. For
this reason, materials that are able to enhance the
activation of insulin receptor are helpful in improving
insulin sensitivity. When insulin receptor is activated, it
165 American Journal of Food and Nutrition
indirectly activates the phosphorylation of a protein kinase
B (PKB), also known as Akt. Therefore, the measurement
of Akt phosphorylation has been used as a surrogate
biomarker for insulin receptor activation [24]. In our study,
the impact of hydrogenized water on Akt phosphorylation
was monitored after cellular treatment with a series of six
concentrations of hydrogenized water. Akt is an insulin-
signaling transduction molecule existing downstream of
the insulin receptor, and its serine residue (Ser473) is
phosphorylated by insulin signaling. As shown in Figure 3a
and Figure 3b, the treatment of hydrogenized water
markedly elevated the level of phosphorylation of Akt
(Ser473) in skeletal muscle cells in a dose-dependent
manner. The level of total Akt has also been measured in a
number of cellular studies and shown remaining
unchanged throughout the study (Data not shown). A
previous study reported that H2 suppressed lipid
peroxidation, resulting in a decrease in its end-product, 4-
hydroxy-2noneal (4HNE), and that 4-HNE modified Akt
activation [26]. Thus it is possible the H2 indirectly
contributes to insulin signaling [25].
Figure 3(a). Effects of Hydrogenized water on the Akt insulin signaling pathway in skeletal muscle cells. Skeletal muscle cells were incubated with 0%,
3.125%, 6.25%, 12.5%, 25% and 50% v/v hydrogenized water in buffer for 60 min. Cells were treated with concentrations of 30 nM insulin (Sigma,
Aldrich, St. Louis, MO) for 15 min as control and lysed for phospho-Akt (Ser473) and GAPDH Western blotting. (b) The intensity of the Western
blotting images shown in Figure 3(b)
Figure 3(b). The intensity of the optical density of the Western blotting images shown in Figure 3(a)
3.4. Impact of Hydrogenized Water on
Cellular Aging Processes
Aging is a complex biological process that involves
various changes in biochemical activities, among which
SIRT1 expression and telomerase activity have been
identified as key biomarkers of the aging process. Here,
we studied the impact of hydrogenized water on these two
key markers to assess the potential anti-aging properties of
hydrogenized water.
3.4.1. SIRT1 as biomarker for aging
SIRT1 is a protein that is believed to play important
roles in longevity and reduction of age-related diseases.
Previous studies have shown that when mammals age,
SIRT1 expression decreases, whereas induction and
activation of SIRT1 has been associated with extended
lifespan. These studies have triggered the search for
SIRT1 activators that may be used as dietary supplements
to promote health and longevity [27].
American Journal of Food and Nutrition 166
Here, we studied the anti-aging effect of hydrogenized
water on SIRT1 expression. We compared SIRT1
expression levels of human cells treated with or without
test materials. As shown in Figure 4 and Figure 5,
hydrogenized water appeared to increase SIRT1 gene
expression in a dose-dependent manner until the effect
plateaued at 50% hydrogenized water.
Figure 4. Effects of hydrogenized water on SIRT1 expression in human umbilical vein endothelial cells. Human umbilical vein endothelial cells were
incubated with hydrogenized water at 0, 3.125, 6.25, 12.5, 25 and 50% v/v for 24 hour. Cell were treated with concentrations of 100 μM Resveratrol for
24 hr as a control. Cells were lysed and lysates prepared for SIRT1 and GAPDH Western blotting
The Western blotting images shown in Figure 4 were analyzed and the intensity of the images was graphed as Figure 5
below:
Figure 5. The effect of hydrogenized water on SIRT1 expression in human umbilical endothelial cells. The results are expressed as relative densities of
the western blot images in Figure 4
To demonstrate the effect of hydrogenated water on
SIRT1 expression, we conducted single run study per
concentration for a series of six concentrations of
hydrogen water instead of replicates of a single
concentration of the hydrogen water. The results indicated
that hydrogenized water increased SIRT1 gene expression
in a dose-dependent manner in umbilical vein endothelial
cells (HUVECs) up to 25% hydrogenized water. The
results also suggested that hydrogenized water may
contribute to improvements in regulation of stress
response, metabolic homeostasis, and aging. Increased
expression of SIRT1 by hydrogenized water treatment
could also result in delayed cellular senescence and
enhanced endothelial function.
3.4.2. Telomerase Activity as an Aging Biomarker
Telomerase is a ribonucleoprotein complex that
contributes to the maintenance of telomeric structure and
length. The relationship between expression of telomerase,
telomere length stabilization or shortening and the
extension of the life span of the human cell has recently
been reported, making telomerase activity a useful
biomarker for anti-aging materials [28]. To assess the
impact of hydrogenized water on cellular aging, we
studied the effects of hydrogenized water on telomerase
167 American Journal of Food and Nutrition
activity. As shown in Figure 6, hydrogenized water
increased telomerase activity in HUVECs when the cells
were treated with media containing more than 25% of
hydrogenized water. The results indicate that
hydrogenized water may contribute to the improvement of
age regulation. Thus increasing telomerase activity by
hydrogenized water may be able to protect telomeres from
degradation, suggesting a possible utility of hydrogenized
water in therapeutic intervention of age-related diseases.
Hydrogenized water increased telomerase activity in
representative human cells when cells were treated with
media containing 25% of hydrogenized water. The
enhancement of the telomerase biomarker by
hydrogenized water was 148.4% when treated with 25%
hydrogenized water, suggesting that hydrogenized water
may be able to protect telomeres from degradation during
age-related diseases.
Telomerase activity (normalized to control cells)
Figure 6. Effects of Hydrogenized water on Telomerase activity in
human umbilical vein endothelial cells (HUVECs). Human umbilical
vein endothelial cells (HUVECs) were exposed to 0%, 3.125%, 6.25%,
12.5%, 25%, 50% v/v hydrogenized water in bufer, and 10 nM
raloxifene for 24 hr. 10 nM raloxifene, a known telomerase activity up-
regulator, is used as a reference material and positive control. The
telomerase activity data are normalized against that of the control cells.
4. Conclusion
Molecular hydrogen has proven to be a safe and useful
approach to provide antioxidant and gene- and activity-
regulating activities to cells, tissues and whole organisms.
[12,13] Here we found that a commercial hydrogenized
water product was effective in providing a selection of
these activities to cell systems used for testing the effects
of materials as antioxidants and gene- and activity-
regulating molecules. Although the in vitro systems used
here for testing commercial hydrogenized water did not
comprise a comprehensive list of all possible test systems,
they did show that the commercial hydrogenized water
product was active and effective in a few selective tests
that have been used previously in product development.
Thus we can conclude that the commercial hydrogenized
water product tested by us here appears to be bioactive
and capable of various activities attributed to molecular
hydrogen, such as its antioxidant properties, as well as
others that have not been tested previously.
Acknowledgement
This work is supported by funding provided by
Naturally Plus USA.
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... Hydrogenized water has been shown to act as a cellular antioxidant and gene regulator [22,23]. Previously we found that hydrogenized water was an effective cellular antioxidant and could decrease transport of glucose in a dose-and time-dependent manner and elevate the levels of phosphorylation of Akt, a protein kinase B biomarker for insulin signaling [24]. We also found that hydrogenized water could increase the levels of age-related SIRT1 gene expression and increase the expression of telomerase activity in a dose-dependent process [24]. ...
... Previously we found that hydrogenized water was an effective cellular antioxidant and could decrease transport of glucose in a dose-and time-dependent manner and elevate the levels of phosphorylation of Akt, a protein kinase B biomarker for insulin signaling [24]. We also found that hydrogenized water could increase the levels of age-related SIRT1 gene expression and increase the expression of telomerase activity in a dose-dependent process [24]. Here we examined the ability of hydrogenized water in vitro to protect brain cells from oxidative damage by increasing glutathione levels and reducing glutamate toxicity. ...
... At the time of initial packaging, the infused hydrogen within the test water was analyzed by both manometric and diaphragm polarographic electrode measurements at final concentration of 2.6 ppm (1.3 mM H 2 ) and pH 7.0. The Izumio hydrogenized water was used in all of the assays [24]. ...
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... Much of the clinical research to date has focused on the effects of HRW on oxidative stress in conditions ranging from age-related diseases to exercise fatigue [52][53][54][55]. HRW is versatile option for the treatment of many ailments, as it is portable and can be produced in a number of ways. ...
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Coffee has an anti-diabetic effect, specifically the amelioration of both hyperglycemia and insulin resistance, in KK-A(y) mice, a type 2 diabetes animal model. To investigate coffee's effect on insulin signaling in liver, skeletal muscle, and adipose tissue (epididymal fat), we assayed the tyrosine phosphorylation of insulin receptor (IR) and serine phosphorylation of Akt. In Expt. 1, we assayed insulin signaling under nonfasting conditions in KK-A(y) mice that ingested water or coffee for 4 wk. Coffee ingestion ameliorated the development of hyperglycemia but did not affect insulin signaling in liver or skeletal muscle under such conditions. In Expt. 2, we assayed insulin signaling under basal and insulin-stimulated conditions in KK-A(y) mice that ingested water or coffee for 3 wk. The levels of tyrosine phosphorylation of insulin receptor in response to insulin injection in insulin-sensitive tissues were not different between mice that drank water and those that drank coffee. Coffee ingestion significantly increased the insulin-induced serine phosphorylation of Akt in liver and skeletal muscle, but not in epididymal fat, of KK-A(y) mice. Our results also indicated that coffee ingestion may contribute to the improvement of insulin resistance and hyperglycemia in KK-A(y) mice via the activation of Akt in insulin signaling in liver and skeletal muscle.
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Objective: Hydrogen inhalation was neuroprotective in several brain injury models. Its mechanisms are believed to be related to antioxidative stress. We investigated the potential neurovascular protective effect of hydrogen inhalation especially effect on mast cell activation in a mouse model of intracerebral hemorrhage. Design: Controlled in vivo laboratory study. Setting: Animal research laboratory. Subjects: One hundred seventy-one 8-week-old male CD-1 mice were used. Interventions: Collagenase-induced intracerebral hemorrhage model in 8-week-old male CD-1 mice was used. Hydrogen was administrated via spontaneous inhalation. The blood-brain barrier permeability and neurologic deficits were investigated at 24 and 72 hours after intracerebral hemorrhage. Mast cell activation was evaluated by Western blot and immuno-staining. The effects of hydrogen inhalation on mast cell activation were confirmed in an autologous blood injection model intracerebral hemorrhage. Measurement and main results: At 24 and 72 hours post intracerebral hemorrhage, animals showed blood-brain barrier disruption, brain edema, and neurologic deficits, accompanied with phosphorylation of Lyn kinase and release of tryptase, indicating mast cell activation. Hydrogen treatment diminished phosphorylation of Lyn kinase and release of tryptase, decreased accumulation and degranulation of mast cells, attenuated blood-brain barrier disruption, and improved neurobehavioral function. Conclusion: Activation of mast cells following intracerebral hemorrhage contributed to increase of blood-brain barrier permeability and brain edema. Hydrogen inhalation preserved blood-brain barrier disruption by prevention of mast cell activation after intracerebral hemorrhage.