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1.1 Reagents
1.4 Animal grouping and drug delivery
weight 18 ~ 22 g,[link widoczny dla zalogowanych], 6 weeks old SPF level of 32 male Kunming mice, purchased from Guangdong Province Medicine Experimental Animal Center, Permit No. SCXK (Guangdong) 2008  0002. Rearing temperature (23 ± 2) ℃, illumination time 12 h / d (7:00 ~ 19:00), after 1 week feeding experiment.
HPLC system (Japan HITACHI Corporation); MK3 type microplate reader (Labsystem, Finland); 3  18K desktop-type high-speed refrigerated centrifuge (Sartorius Company, Germany); 2720-type PCR instrument (Applied Biosystems U.S. companies ); electrophoretic imaging system (U.S. Bio  Rad Laboratories Company); Milli Q Plus device super-pure water (Millipore Company USA); electrophoresis and horizontal electrophoresis tank for the Japanese Mupid Company.
Key words
Effect of carnosine on glycometabolism in restraint  stressed mice
1.2 experimental equipment
Key words: carnosine; restraint stress; liver glycogen; glucocorticoids
carnosine; restraint stress; liver glycogen; glucocorticoids
carnosine (carnosine), corticosterone (corticoste  rone) standard and hydrocortisone (cortisol) standard for the American Sigma products; acetonitrile (HPLC grade) for the Fisher Scientific company's products; ethyl acetate and NaOH as Guangzhou Chemical Reagent Factory; glucose was purchased from Tianjin Pharmaceutical; liver glycogen kit purchased from Nanjing Jiancheng technology companies; glucose assay kit purchased from Shanghai Rongsheng Sheng-pharmaceutical company; Trizol was purchased from Guangzhou Genewindows biotechnology company; reversed record system for PCR kits were purchased from Beijing Tian root biotechnology company; PCR primers based on GenBank to provide gene sequences using the Primer Design software to design primers by Genewindows companies synthesis, glycogen synthase  2 (Gy  2) primers : 5 ' AACGAC AGCCGATGAGT  3' (upstream), 5 ' CTGGCACG AGAAAGGAT  3' (downstream), the amplified product length 423 bp; the 18s as the internal reference: 5 ' GGGAGA GCGGGTAAGAGA  3' (upstream), 5 ' ACAGGACTAGGC GGAACA  3' (downstream), the amplified product length 241 bp.
Stress load of carnosine on glucose metabolism in mice



Abstract Objective To study the stress load of carnosine on glucose metabolism in mice. Methods Male KM mice were divided into normal control, stress control group, 150 mg / kg and 300 mg / kg carnosine treated group 1 day intragastric administration, continuous administration of load restraint stress after 7 d 20 h intragastric glucose 2 g / kg, blood glucose clearance, hepatic glycogen synthesis, glycogen synthase gene expression and corticosterone levels. The results with the stress load of the control group, carnosine can increase blood glucose elimination rate of stress, promote glycogen synthase gene expression, and enhanced glycogen synthesis. In addition, carnosine can reduce plasma corticosterone levels. Conclusion Carnosine increased stress load capacity of glycogen synthesis in mice, and its mechanism may be related to carnosine reduced glucocorticoid levels in vivo, activation of glycogen synthase related.
The mice were randomly divided into normal control group, restraint stress control group,[link widoczny dla zalogowanych], 150 mg / kg and 300 mg / kg carnosine treated group,[link widoczny dla zalogowanych], a total of 4 groups of 8. Carnosine treatment group to 0.1 mL/10 g dose of oral administration, the normal control group and the restraint stress model group were fed the same amount of water blank, 1 day consecutive administration of 7 d. In addition to the normal control group, all mice were fed in the last 30 min after the restraint stress experiment, mice restraint device with reference to the literature [4] made for a well-ventilated 50 mL polypropylene centrifuge tube tip at the end, to give a one-time binding 20 h (12:00 ~ 8:00 the next day), mice can not eat during the water binding. Normal control group of mice was water fasting mice.
the animals in each group bound end of the fasting and fed glucose after 30 min 2 g / kg, oral glucose 0,[link widoczny dla zalogowanych],20,40,60 and 80 min after tail vein blood was collected in heparin, a small centrifuge tube 5 000 r / min 5 min centrifugation, the plasma glucose assay kit with blood glucose concentrations.
(Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, Guangdong 510632, China) Abstract: Objective To study the effect of carnosine on glycometabolism in restraint  stressed mice. Methods Male KM mice were randomized into normal control group , stress control group, 150 and 300 mg / kg carnosine groups, which were intragastrically administered once a day consecutively for 7 days, and then intragastrically administered with glucose at 2 g / kg restraint stress for 20 hours; the glucose elimination rate,[link widoczny dla zalogowanych], hepatic glycogen synthesis, gene expression of glycogen synthetase, and corticosterone level were determined.Results Compared with the control group, carnosine accelerated the basic glucose metabolism, up  regulated the mRNA expression of glycogen synthase, elevated the liver glycogen synthesis in restraint  stressed mice. And it also reduced the plasma corticosterone level.Conclusion Carnosine could improve hepatic glycogen synthesis in restraint  stressed mice. The mechanism might be related to the degression of glucocorticoids and the activation of glycogen synthase.

1.6 HPLC determination of plasma corticosterone levels in mice <span style=
placed in whole blood of mice treated with heparin tube, to 5 000 r / min 5 min centrifugation separation of plasma. Take 0.5 mL of plasma, and 2 mL ethyl acetate added to shaking extraction, separation by the upper ethyl acetate solution, the lower liquid water and then ethyl acetate 1 times, after the combined ethyl acetate solution washed with 0.1 mL NaOH 1 , and then distilled water 2 times. Ethyl acetate solution is dried, placed under nitrogen by adding 100 μL of volatile mobile phase (acetonitrile: water = 38:72, volume ratio) solution of the sample solution was corticosterone. Woodward and others by the method, using HPLC internal standard method (internal standard as cortisol) in plasma corticosterone levels [6]. Detection conditions: UV detection, the detection wavelength was 254 nm; column: 5C18 (4.6 mm × 150 mm, 5 μm); mobile phase: acetonitrile  water (38%: 72%); flow rate: 1 mL / min .

1 Materials and methods
1.3 experimental animals
recent years with the social environment and changes in lifestyle, living and working under increasing pressure, caused by a variety of stressors are increasing year by year the number of sub-health state. People under prolonged stress load easily lead to decreased immunity, leading to a variety of diseases. Carnosine is a natural with high biological activity of two peptides, widely exist in mammalian muscle tissue and nerve tissue [1]. The results show that carnosine has antioxidant anti-aging functions, nervous system protection, anti-glycosylation effect on kidney toxicity and liver injury by improvements to prevent arteriosclerosis and other physical activity [2,3]. However, carnosine has not been loaded state of stress the role of glucose metabolism to improve coverage. This experiment observed restraint load of carnosine on blood glucose concentration and hepatic glycogen synthesis, and plasma corticosterone changes in mice to explore the stress caused by carnosine on body glucose metabolism improvement.
YANG Dong  hui, LI Yi  fang, YAO Nan, HE Rong  rong, KURIHARA Hiroshi
1.5 glucose tolerance test in mice [5]
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