Oral administration of [gamma] -aminobutyric acid and [gamma] -oryzanol prevents stress-induced hypoadiponectinemia.ARTICLE INFO
GABA GABA ?.
GABA (gamma-aminobutyric acid)
A neurotransmitter that slows down the activity of nerve cells in the brain.
Immobilization Immobilization Definition
Immobilization refers to the process of holding a joint or bone in place with a splint, cast, or brace. This is done to prevent an injured area from moving while it heals. stress
Metabolic syndrome is a cluster of risk factors including insulin resistance and type 2 diabetes type 2 diabetes
See diabetes mellitus. and is found to associate partly with chronic stress at work in human. Adiponectin circulates in mammal blood mainly as a low molecular weight (LMW LMW Low-Molecular-Weight
LMW Licensed Manufacturing Warehouse
LMW Littoral and Mine Warfare
LMW Lead Mobility Wing
LMW Limited Manufacturer Warranty
LMW Low-Water Mark
LMW LanMan Workstation
LMW Level of Maximum Wind
LMW Local Minority Woman Owned Company ) trimer, hexamer, and a high molecular weight (HMW HMW High Molecular Weight
HMW health, morale, and welfare (US DoD)
HMW Hazardous Material Warning
HMW How might we
HMW Highly Modular Workplan ) multimers. Low circulating levels of adiponectin are related to metabolic syndrome. We have then investigated the influence of immobilization stress on plasma adiponectin concentrations in mice. Relative LMW and HMW adiponectin levels were markedly reduced by immobilization stress (0.66 [+ or -] 0.07 and 0.59 [+ or -] 0.06 after 102 h, respectively), significantly different from the control values (p < 0.01 and 0.05, respectively). [gamma] - Aminobutyric acid (GABA) and [gamma] - oryzanol abundantly contained in germinated brown rice have some physiological functions. We further investigated the effect of GABA, [gamma] - oryzanol, GABA plus [gamma] - oryzanol on adiponectin levels in mice subjected to immobilization stress. GABA and [gamma] - oryzanol significantly increased the relative LMW and HMW adiponectin levels under immobilization stress (1.10 [+ or -] 0.11 and 0.99 [+ or -] 0.19 after 102 h, respectively, for GABA; 1.08 [+ or -] 0.17 and 1.15 [+ or -] 0.17 after 102 h, respectively, for [gamma] oryzanol). Additionally, the co-administration of GABA and [gamma] - oryzanol also increased both relative LMW and HMW adiponectin levels (1.02 [+ or -] 0.07 and 0.99 [+ or -] 0.10 after 102 h, respectively) and was effective in an earlier phase from 30 to 54 h. The results indicate that the co-administration of GABA and [gamma] -oryzanol might be effective in preventing stress-induced hypoadiponectinemia in mice and be also a promising tool for improving metabolic syndrome aggravated by chronic stress.
[c] 2011 Elsevier GmbH. All rights reserved.
The metabolic syndrome represents a cluster of metabolic risk factors, including central obesity, insulin resistance, dyslipidemia, hyperglycemia hyperglycemia: see diabetes. , and hypertension for type 2 diabetes and cardiovascular diseases. Metabolic syndrome is rapidly prevailing worldwide. Chronic stress, including psychological and physical stresses, is found to link with metabolic syndrome, and is believed an important risk factor for the development of metabolic syndrome (Vitaliano et al. 2002; Chandola et al. 2006).
Adiponectin is secreted from adipose tissue, exists in the circulation, and has been postulated to play an important role in the modulation of glucose and lipid metabolisms in insulin-sensitive tissues such as liver and skeletal muscle. Plasma adiponectin levels are decreased in the obese and insulin-resistant state (Arita et al. 1999; Yamauchi et al. 2001). Low adiponectin level is associated with metabolic syndrome (Saely et al. 2007; Devaraj et al. 2008). Plasma adiponectin consists of trimer (presented as a low molecular weight multimer, LMW, in the present study) and over-hexamer (presented as high molecular-weight multimers, HMW). Among them, the HMW multimers are believed the most bioactive forms (Pajvani et al. 2004; Waki et al. 2003). The reduced quantity of HMW adiponectin is in special associated with metabolic syndrome (Lera-Castro et al. 2006).
[gamma] - Aminobutyric acid (GABA) is one of the many nutritional components in brown rice and pre-germinated brown rice (PGBR) with slight germination germination, in a seed, process by which the plant embryo within the seed resumes growth after a period of dormancy and the seedling emerges. The length of dormancy varies; the seed of some plants (e.g. . It has been reported that PGBR rich in GABA effectively reduced glucose levels in diabetic rats (Hagiwara et al. 2004). We have also demonstrated that rice germ extract increased serum adiponectin levels of mouse and that its active compound was GABA (Uchida et al. 2008).
[FIGURE 1 OMITTED]
[gamma] - Oryzanol is one of major bioactive components in rice bran, and has been suggested to possess effects of lowering serum cholesterol levels (Rong et al. 1997; Wilson et al. 2007), anti-inflammatory effects (Akihisa et al. 2000), and an anti-cancer effect (Yasukawa et al. 1998) and to function as an antioxidant antioxidant, substance that prevents or slows the breakdown of another substance by oxygen. Synthetic and natural antioxidants are used to slow the deterioration of gasoline and rubber, and such antioxidants as vitamin C (ascorbic acid), butylated hydroxytoluene (isram et al. 2009; Xu et al. 2001). We have demonstrated that [gamma] - oryzanol suppressed NF-kB activation (Nagasaka et al. 2007; Islam et al. 2008) and directly induced the adiponectin secretion of adipocytes in part through inhibition of NF-kB activation (Ohara et al. 2009). We have recently demonstrated that cycloartenyl ferulate, one component of [gamma] - oryzanol, also prevents allergic inflammation (Oka et al. 2010).
In this study, we investigated the effect of immobilization stress on serum adiponectin levels in mice. We also evaluated the effects of GABA and [gamma] - oryzanol on serum adiponectin levels in mice under immobilization stress.
Materials and methods
GABA (> 98% in purity) was obtained from Wako (Osaka, Japan). 7-Oryzanol (> 99% in purity) was obtained from Oryza Oil & Fat Chemical Co., Ltd. (Aichi, Japan). The chemical structures of GABA and main components in [gamma] - oryzanol are shown in Fig. 1. Analytical grades were used for other chemicals.
Male C57BL/6J mice (9-10 weeks, an average weight of 25 g) were obtained from Clea Japan, Inc. (Tokyo, Japan). They were maintained at 50% relative humidity and a 12-h light/dark cycle at 20-22[degrees]C. Mice were ad libitum given water and the commercial diet type MF from Oriental Yeast Co., Ltd. (Tokyo, Japan). All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, Tokyo University of Marine Science and Technology and approved by the Committee for the Care and Use of Laboratory Animals, Tokyo University of Marine Science and Technology.
GABA and [gamma] - oryzanol administration
Each experiment was performed with 3-5 mice per group. Solutions of 30[micro]g/m1 GABA, 14.5[micro]g/m1 [gamma] - oryzanol, or the mixture of 30[micro]g/ml GABA and 14.5[micro]g/ml [gamma] - oryzanol in 0.0003% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS) (Dojindo, Kumamoto, Japan) were prepared. 0.5 ml of GABA (n =4), [gamma] - oryzanol (n = 3), GABA plus [gamma] - oryzanol (n = 5) or vehicle solution (0.0003% CHAPS, n = 5) was orally administered to each mouse after 24-h-fasting. After administration, about 30[micro]J of blood samples were immediately collected from tail vein (time 0). The mice were subjected to immobilization stress, widely used as one stress model as described in Pare and Glavin (1986). Mice were separately immobilized for 6 h in metal mesh cages.The blood samples (about 30[micro]l) were collected at 6, 30, 54, 78, and 102 h after administration. After centrifugation Centrifugation
A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal at 10,000 x g for 10min at 4[degrees]C, the resulting plasma was collected and stored at -80[degrees]C until use.
[FIGURE 2 OMITTED]
Measurement of plasma adiponectin
Plasma adiponectin levels were measured by Western blot analysis West·ern blot analysis
An electrophoretic procedure for separating proteins. . Sodium dodecyl sulfate Sodium dodecyl sulfate (or sulphate) (SDS or NaDS) (C12H25NaO4S),is an anionic surfactant that is used in household products such as toothpastes, shampoos, shaving foams and bubble baths for its thickening effect and its ability to (SDS 1. (company) SDS - Scientific Data Systems.
2. (tool) SDS - Schema Definition Set. )-polyacrylamide gel electrophoresis (PAGE) was performed according to the Laemmli method (1970). Plasma samples were subjected to SDS-PAGE SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. for non-reducing and non-heat-denaturing conditions in order to estimate plasma adiponectin multimer levels as reported by Waki et al. (2003). Briefly, plasma samples were diluted 1/500 with distilled water. The resulting samples were mixed with the same volume of 2 x Laemmli's sample buffer in the absence of 2-mercaptoethanol, and incubated for 1 h before electrophoresis. Ten microliters of samples (about 20[micro]g protein) were loaded in each lane of a commercial 2-15% gradient polyacrylamide gel (Cosmo Bio Co., Ltd., Tokyo, Japan) and electrophoresed. The separated proteins were then transferred onto polyvinylidene difluoride membranes (Millipore Co., Billerica, Mass., USA) as described by Towbin et al. (1979). The membranes were incubated in a Tris-buffered saline (TBS; pH 7.6 at 25[degrees]C, Takara, Shiga, Japan) containing 3% bovine serum albumin (Roche Diagnostics, Indianapolis, IN, USA) for 1 h at room temperature. After washing with TBS containing 0.5% Tween tween
A child between middle childhood and adolesence, usually between 8 and 12 years old.
[Blend of teen1 and between.] 20 (Wako, Osaka, Japan) for four times, the membranes were incubated with 1:2000 mouse anti-adiponectin, mouse monoclonal antibody (Millipore Co.). followed by the incubation with an Alexa Flour[R]680 conjugated goat anti-mouse IgG (H + L) (Molecular Probes, Inc., Eugene, OR, 1:20,000 in a dilution rate) for 1 h at room temperature. After washing, the immunoreactivity was observed and digitized using an Odyssey Infrared imaging system (LI-COR Biosciences). A typical Western blotting pattern of mouse adiponectin is shown in Fig. 2, which gave LMW adiponectin fractions with molecular mass of 80-150kDa and HMW adiponectin fractions with molecular mass of over 150 kDa. Signal intensity for adiponectin in digitized data was evaluated by Image-J (National institutes of Health, USA).
[FIGURE 3 OMITTED]
Results are shown as means [+ or -] SE. The data were analyzed by one-way ANOVA anova
see analysis of variance.
ANOVA Analysis of variance, see there . Differences with p values < 0.05 and 0.01 in Student's t-test between control and experimental values were considered significant.
Adiponectin levels after immobilization stress
We investigated the effects of immobilization stress on adiponectin levels in mice. 0.0003% CHAPS solution (as a vehicle of GABA or y-oryzanol) was orally administered to mice. They were then divided into two groups at random and one group was subjected to immobilization stress for 6 h before every blood samplings as described in Materials and methods section. The same handling at the same time was carried out for the control group expect for the immobilization stress procedure. The relative plasma LMW and HMW adiponectin levels drastically reduced in mice exposed to immobilization stress (Fig. 3). The control group not stressed did not exhibit the significant changes in relative plasma LMW and HMW adiponectin levels.
[FIGURE 4 OMITTED]
The effects of GABA on adiponectin levels in immobilization-stressed mice
We further evaluated the effects of GABA on adiponectin levels in the immobilization-stressed mice. The oral administration ofGABA significantly boosted the relative LMW adiponectin levels from 78 h and the relative HMW adiponectin levels at 102 h compared to the control group at each time (Fig. 4).
The effects of [gamma] - oryzanol on adiponectin levels immobilization-stressed mice
We previously reported that the relative adiponectin levels significantly increased through the oral administration of [gamma] - oryzanol (Ohara et al. 2009). In this study, therefore examined were the effects of [gamma] -oryzanol on plasma adiponectin levels in mice subjected to immobilization stress. The relative LMW adiponectin levels markedly increased at 102 h by the oral administration of [gamma] - oryzanol (Fig. 5A). The y-oryzanol administration significantly augmented the relative HMW adiponectin levels from 78 h to 102 h compared to the control group (Fig. 5B).
[FIGURE 5 OMITTED]
The effects of GABA plus [gamma] - oryzanol on adiponectin levels in immobilization-stressed mice
GABA and [gamma] - oryzanol showed each characteristic effect on adiponectin levels in stressed mice as stated above. The effects of GABA plus [gamma] - oryzanol on serum adiponectin levels were then evaluated. The oral administration of GABA plus [gamma] - oryzanol significantly increased the relative LMW and HMW adiponectin levels compared to the control group at each time after 30 h earlier than the cases of separate administrations (Fig. 6), where significant differences were observed from 30 to 102 h after administration for both adiponectin multimers. Thus, the co-administration of GABA and [gamma] -oryzanol might be effective for the regulations of adiponectin levels in mice subjected to immobilization stress.
[FIGURE 6 OMITTED]
In this study, we have evaluated the effects of immobilization stress on mouse plasma adiponectin levels. We have then successfully demonstrated that the immobilization stress markedly reduced serum adiponectin levels in mice and that oral administrations of GABA and [gamma] - oryzanol recovered the levels.
The metabolic syndrome is thought to arise from a combination of genetic, environmental, and lifestyle factors such as dietary habits and physical inactivity causing obesity (Grundy et al. 2005). High-fat diet induces obesity and insulin resistance, which is associated with decreasing adiponectin levels (Bullen et al. 2007). Adiponectin is one of the most important adipocy-tokines, and is positively correlated with insulin sensitivity, and low serum adiponectin is associated with metabolic syndrome (Saely et al. 2007; Devaraj et al. 2008). Stress has been thought an important risk factor for the metabolic syndrome (Vitaliano et al. 2002; Chandola et A1 2006). Stress-induced hypoadiponectinemia observed in the present study might be partly responsible for the risk for the metabolic syndrome.
Under the stress condition, the hypothalamic-pituitary-adrenal (HPA) axis is activated, increasing glucocorticoids secretion. It is generally accepted that glucocorticoids, such as cortisol cortisol (kôr`tĭsôl') or hydrocortisone, steroid hormone that in humans is the major circulating hormone of the cortex, or outer layer, of the adrenal gland. and corticosterone corticosterone (kôr'təkōstĕr`ōn), steroid hormone secreted by the outer layer, or cortex, of the adrenal gland. Classed as a glucocorticoid, corticosterone helps regulate the conversion of amino acids into carbohydrates and in rodents, are associated with the pathogenesis of metabolic syndrome (Anagnostis et al. 2009; Boullu-Ciocca et al. 2005). Boullu-Ciocca et al. (2008) reported that corticosterone levels were in inversely proportion to plasma adiponectin levels and adiponectin mRNA expression in adipose tissue in mice fed with high-fat diets. Increasing corticosterone levels might directly or indirectly reduce adiponectin levels in the stressed mice. Further studies should be performed to reveal details in mechanisms for the stress-induced hypoadiponectinemia.
GABA is the most ubiquitous inhibitory neurotransmitter in the mammalian central nervous system (Herman and Cullinan1997; Rosmond 2005). Since the GABA receptors are present also in the adipocytes (Nicolaysen et al. 2007), GABA might directly affect the adiponectin secretion from adipose tissue. On the other hand, GABA dose not cross the brain-blood barrier and the GABAergic system is observed also in peripheral tissues (Gilon et al. 1990). Peripheral GABA8 agonist stimulates gastric acid secretion also via the vagal vagal /va·gal/ (va´gal) pertaining to the vagus nerve.
Of or relating to the vagus nerve.
pertaining to the vagus nerve. systems (Piqueras and Martinez 2004). Adipose tissue receives both sympathetic and parasympathetic parasympathetic /para·sym·pa·thet·ic/ (-sim?pah-thet´ik) see under system.
Of, relating to, or affecting the parasympathetic nervous system. controls, similar to other endocrine organs (Kreier et al. 2002), Increase in adiponectin levels through the oral administration of GABA in the present study might be modulated also via vagal systems. However, the present study cannot specify the sites of action and further pharmacological studies are necessary to understand fully the GABA effect on adiponectin secretion.
[gamma] - Oryzanol, one of the major bioactive components in rice bran, was suggested to show a blood cholesterol lowering (Rong et al. 1997; Wilson et al. 2007) and an antioxidant effects (Isram et al. 2009; Xu et al. 2001). We have recently reported that [gamma] - oryzanol inhibited NF-kB activation (Nagasaka et al. 2007) and directly induced the adiponectin secretion of adipocytes in part via suppression of NF-kB activation (Ohara et al. 2009). In this study, the oral administration of GABA and [gamma] - oryzanol showed each characteristic effect that [gamma] - oryzanol and GABA conferred a gradual and a relatively rapid increases in the LMW adiponectin, respectively. Expectedly, GABA plus [gamma] - oryzanol likely acted co-additively on the increase in plasma adiponectin levels in stressed mice. These results suggest that GABA and [gamma] - oryzanol would be effective for adiponectin secretion via each different pathway as stated above.
Acute oral 50%-lethal doses of GABA and [gamma] -oryzanol, 12,680 mg/kg and 25,000 mg/kg for mouse, respectively, are much higher amounts than the doses in this study. Although toxicity of co-administration of GABA and [gamma] - oryzanol is unknown, in our preliminary co-administration test with a higher dose (4 mg GABA and 17 mg [gamma] - oryzanol per mouse), almost similar effects for adiponectin levels were observed without any pathological signs such as vomiting, diarrhea, and intestinal hemorrhage and abnormal behaviors such as tremor (data not shown).
In conclusion, the current study has demonstrated that GABA and [gamma] - oryzanol recover the adiponectin levels in stress-induced hypoadiponectinemia mice into the normal level. GABA and [gamma] oryzanol might prevent and/or ameliorate the stress-induced metabolic syndrome and the type 2 diabetes. Brown rice and pre-germinated brown rice are rich in both GABA and [gamma] - oryzanol, compared to other plants, and their extracts may be promising tools for improving metabolic syndrome aggravated by chronic stress.
This work is supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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* Corresponding author at: Laboratory of Marine Biochemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo “Todai” redirects here. For the restaurant called Todai, see Todai (restaurant).
The University of Tokyo (東京大学 , 1-1 Yayoi l, Bunkyo, Tokyo 113-8657, Japan. Tel.: +81 358415299; fax: +81 358418166.
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The production of cancer.
production of cancer.
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Kazuyuki Ohara (a), Yuka Kiyotani (a), Asako Uchida (a), Reiko Nagasaka (a), Hiroyuki Maehara (c), Shigeharu Kanemoto (c), Masatoshi Hori (d), Hideki Ushio (a), (b), *
(a) Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 5-7 Konan 4, Minato, Tokyo 108-8477, Japan
(b) Laboratory of Marine Biochemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1 Yayoi 1, Bunkyo, Tokyo 113-8657, Japan
(c) Satake Corporation, 2-30 Saijo Nishihonmachi, Higashi-Hiroshima, Hiroshima 739-8602, Japan
(d) Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1 Yayoi 1, Bunkyo, Tokyo 113-8657, Japan