1、 S36 中西医结合学报2010年6月第8卷第6期Journal of Chinese Integrative Medicine，June 2010，Vo18，No6 目的：观察中药复方糖耐康对肥胖Zucker大鼠糖代谢和胰岛素抵抗的影响。 方法：6周龄雄性肥胖Zucker大鼠12只，适应性喂养2周后，随机分为对照组和糖耐康组(324 gkg)，所有 大鼠给予高脂饲料喂养，疗程为4周。每周检测体质量和血糖；入组前和治疗l4、28 d时行口服葡萄糖耐量 实验(oral glucose tolerance test，OGTT)并检测空腹血清胰岛素水平；第28天时检测空腹血脂4项和血浆 游离脂肪酸(
2、free fatty acids，FFA)水平；第29天时进行高胰岛素正葡萄糖钳夹实验检测平均葡萄糖输注率 (glucose infusion rate，GIR)；实验结束后处死大鼠并取材，检测骨骼肌中蛋白激酶B(protein kinase B， PKBAkt)、磷酸化蛋白激酶B(phosphoAkt，p-AktThr308)、葡萄糖转运蛋白4(glucose transporter protein 4，GLUT4)的表达和脂肪组织GLUT4的蛋白表达。 结果：与对照组相比，治疗4周后，糖耐康组血清胰岛素水平没有变化，餐后血糖和OGTT中120 min时的 血糖水平均显著下降；糖耐康组高胰岛
3、素正葡萄糖钳夹实验后GIR显著提高；糖耐康能显著增加骨骼肌 Akt、p-Akt(Thr308)的蛋白表达，减少脂肪组织GLUT4的蛋白表达；糖耐康有降低体质量、血脂(三酰甘 油、胆固醇、低密度脂蛋白)和FFA含量的趋势，但两组比较差异无统计学意义。 结论：糖耐康可能是通过增加骨骼肌Akt和p-Akt(Thr308)的表达，增强GLUT4的葡萄糖转运能力来实现 降低血糖，改善外周胰岛素抵抗的功效。 关键词：中药复方；糖尿病，2型；胰岛素抵抗；葡萄糖转运蛋白4；葡萄糖钳制技术；肥胖Zucker大鼠 Diabetes is a serious health problem around the wo
4、rldOver 9Oof patients with diabetes have type 2 diabetest Islet dysfunction and peripheral insulin resistance are both present in type 2 diabetes and are both necessary for the development of hyper glycemia 1It has already been demonstrated by epidemiological studies that hyperglycemia is one of the
5、 principal causes of complications as cardio vascular and cerebrovascular diseasesI 3-5 JCompli cations lcad to poor lire qualityas well as high morbidity and mortalityEffective blood glucose control 1s the key to preventing or reversing type 2 diabetes Diabetes is called wastingthirst disease in tr
6、adi tional Chinese medicine(TCM)，characterized by polydipsia，polyphagia，polyuria，and emacia tionIn TCM theory，wastingthirst is considered a result of yin deficiency with drynessheatThe treatment of diabetes should focus on nourishing yin to clear away heat from the body JThis study sought to determi
7、ne whether the mixture of aqueous and ethanol soluble extract of Tangnai kang(TNK)，a compound traditiona1 Chinese medicine，could normalize hyperglycemia in an animal model with gene mutation，obese Zucker rat mode1The model exhibits obesity and high insulin resistance，such as hyperglycemia，glucose in
8、tolerance，and hyperinsulinemia，which resembles human type 2 diabetesWe also explored the mechanisms responsible for glucose transporter 4 (GLUT4)translocation by measuring upstream protein expression level of signal transduction pathway 1 Materials and methods 11 Materials TNK was composed of ica Pr
9、unellae Vulgaris(Xiakucao)，Folium Psidii Grajavae(Fanshiliuye)，Fructus Ligustri Lucidi (Nuzhenzi)，Herba Saururi(Sanbaicao)and Radix to the herbal Ginseng(Renshen)and prepared proportion of 2：2：1：1： drugs were obtained from according 12A11 Shenyang Pharmaceutica1 Group CorporationThe process was as f
10、ollowsFirstly，the first four plants were mixed and then extracted by 8 times amount of 75ethanol twice(1 h each time)using reflux condenserThe recovered ethanol solution(6O to 70)was lyophilized which resulted in 1 383 of the original extractSecondly，6 times amount of water was added into the residu
11、e and the mixture was filtrated for 1 hproducing 549 extractLastlyP臼nax Ginseng fine powder，the two extracts，and excipients were mixed together to produce dried granule for future usePrepara tion process was performed by Sichuan Medico Pharmaceutica1It is confirmed in Pharmacopoeia of the Peoples Re
12、public of China(2000 ed)with high-performance liquid chromatography (HPLC)analysis that the total concentration of Re and Rgl in the ginsenoside was 035while spectrophotometry analysis confirmed that the total flavonoids in the extract was 42 3the total triterpene acids was 19 9and the total triterp
13、enoid saponin was 36 7 Beforc each experiment，TNK(324 gkg，about 45 g crude herbal drugs)was suspended in distilled water (1 0 mL)for oral administrationRats in control group were administered with distilled waterNo noticeable adverse effects were observed in any rats after the treatment 12 Animals T
14、he study protocol was approved by the Institutional Animal Care and Usc Committee of the Beijing University of Chinese MedicineTwelve male obese Zucker rats， 6-week old，weighing(188981786)g，were obtained from Professor Wang Heyao of Shanghai Institute of Materia MedicaA11 rats were housed in environ
15、mentally controlled conditions with a 中西医结合学报2O1O年6月第8卷第6期Journal of Chinese Integrative Medicine，June 2010，Vo18，No6 537 1 2h 1ightdark cycle and they had free access to rodent pellet highfat dietincluding 685basic diet，15pork fat，15sucrose，1cholesterol and 05cholateexcept when fasted before some ex
16、perimentsAfter being fed for 2 weeks， all rats were randomly divided into TNK group and control group by body weight and received oral administration once a day for 4 weeks 13 Measurements of fed glucose leve1fasting serum insulin，oral glucose tolerance test，blood lipid，free fatty acids，and body wei
17、ght Fed blood glucose level in tail blood samples(about 5 L each)was measured at 4：00 PMon days 714， 21and 28 Fasting serum insulin level test and oral glucose tolerance test(OGTT)were performed at 9：OO AMon days 014 and 28Blood lipid and free fatty acids(FFA)were only detected on day 2 8 On test da
18、ys，blood samples(about 5O0 L each) of rats(fasted for 1 2 hours)were taken for later tests before they received oral administration of glucose(2 gkg)for 3O，6O，and 120 minutes of OGTT(about 5 L for each blood sample from tail) Body weight was measured every week during the test 14 Biochemical assays
19、Blood glucose level was determined by a glucose analyzer(ACCUCheck， Roche，Switzerland)， serum insulin level was assayed with radioimmunoassay (RIA) for human insulin(Furui Bioengineering Company， Beijing)，lipid concentrations，including triglyc eridecholesterol。lowdensity lipoprotein choles terol(LDL
20、C)，and highdensity lipoprotein cholesterol(HDLC)were assayed with an auto matic biochemistry analyzer(AU400 Biochemical Analyzer，Olympus，Japan)，and FFA was assayed with enzyme-linked immunosorbent assay kit(ADL， USA)All detection items were measured according to the manufacturersprocedures 15 Hyperi
21、nsulinemic-euglycemic clamp test The clamp technique was performed as developed by Attele et al 1Rats received TNK or distilled water for 2 8 days，as described aboveOn day 24rats were anesthetized with hydral at a dose of 350 mgkgA catheter f PE50)was jnserted into the left internal iugular vein，and
22、 then fixed at the vertex of the headThe catheterized rats were allowed to recover for 4 days(from day 2 5 to day 2 8)while they continued to receive the treatment until day 28Clamp study was performed at 9：00 AMon day 29Before the 240minute hyperinsulinemiceuglycemic clamp test，rats were fasted for
23、 1 2 hDuring the clamp test，rats were awake and unrestrainedBoth insulin(1 0 mU (kgmin)，Novolin，NOVO Nordisk，Denmark) and 2 0glucose were administered into the same catheter implanted in the iugular vein by a two channel microdialysis syringe pump(WZS50F6， Smith Medical， Zhejiang)， and glucose was i
24、nfused at variable rates to maintain a plasma glucose at approximately 66 mmolL Blood samples(about 5 L each)were collected from the tail every 1 5 minutes during the clamp test to measure the glucose level and to adjust glucose infusion rateThe average glucose infusion rate was tested at 210， 220，
25、225， 230，235 and 240 minutes of clamp period for the determina tion of wholebody glucose disposa1At the end of the clamp study，rats were sacrificed under anes thesiaGastrocnemius muscle from hindlimbs and epididymal white adipose tissue were removed and dissected as soon as possiblethen frozen immed
26、i ately with liquid N cooled aluminum blocks and stored at一8Ofor later analysis 16 Western_blOttin2 To check the total amount of protein kinase B (Akt)，phosphoAkt (Thr308)， and glucose transporter protein 4 (GLUT4) in skeletal muscle as well as the GLUT4 in white adipose tissuethe extracted protein
27、samples were subiected to sodium dodecyl sulfatepolyacrylamide gel electroDhoresis and Western blottingBlots were probed with anti Akt， antiphosphoAkt (Thr308)， and anti GLUT4(Cell Signaling Technology，Beverly， MA)Detection of immunoreactive band was performed by using the electrochemillumines cence
28、 kit(Amersham Biosciences，UK)Densi tometry was performed by scanning the radio graphs using the Image J 70 system(Media Cybernetics) 17 Statistical analysis Values were expressed as sData from each set of observations were compared by using Students test and ANOVA for repeated measures with SAS 80 s
29、oftware(SAS Institute IncCary，NC)Differences were considered significant if P value was less than 005 2 Results 21 Effects of TNK on fed blood glucose level Fed blood glucose level was shown in Table 1 Fed blood glucose level in TNKtreated obese Zucker rats was significantly lower than vehicle treat
30、ed rats on day 28(P=0039) Table 1 Effects of TNK on fed blood glucose level in obese Zucker rats ( s，mmolL) PO05“us control group 538 中鹾医结合学报2010年6月第8卷第6期Journal of Chinese Integrative Medicine，June 2010，Vo18，No6 22 Effects of TNK on glucose tolerance test Glucose tolerance was evaluated by OGTTOral
31、 glucose load failed to return to the fasting level at 1 20 minutes in TNK-treated rats on days 0 and 1 4On day 28，the glucose level decreased signifi- cantly in TNK-treated rats at 1 20 minutes，indiea ting a noticeable improvement of glucose disposal (P一0046，Table 2) Table 2 Effects of TNK on gluco
32、se tolerance test in obese Zueker rats (is，mmolL) P005“os control group 23 Efleets 0f TNK on Fasting serum insulin fasting serum insulin level level was shown in Table 3No significant difference was found between the two groups on days 0，14 or 28 Table 3 Effects of TNK on fasting serum insulin level
33、 in obese Zueker rats ( s，mUL) 24 Effects of rINK on body-wide insulin-stimulated glucose disposal rate Bodywide insulinstimulated glucose disposal rate was tested by hyperinsulinemic euglycemic clamp as shown in Table 4After the 28 day treatment of TNK，the rate of insulinstimulated glucose disposaI
34、 in obese Zucker rats was significantly higher than vehicletreated rats(P一0025) Table 4 Effects of TNK on body-wide insulin-stimulated glucose disposal rate in obese Zucker rats (js) P005 control group 25 Efleets of TNK on body weight and blood lipid and FFA contents There was a limited average body
35、 weight reduction in the TNK group over 4 weeks as compared with the vehicle(P一0070， Table 5)In parallel，FFA level also decreased slightly(P一0】1)in the TNK group after 28一day treatment as compared with the control group The levels of serum triglyceride， cholesterol， LDLCand HDLC in the TNK group did
36、 not change significantly as compared with the control group(Table 6) Table 5 Effects of TNK on body weight change of obese Zueker rats ( s，g) Table 6 Effects of TNK on blood lipid and FFA levels in obese Zucker rats ( s，retoolL) 26 Effects of TNK on protein expressions in skeletal muscle and white
37、adipose tissue We next measured the protein levels of Akt，phosphoAkt (Thr308)， and GLUT4 in skeletal muscle (Figure 1)as well as protein level of GLUT4 in white adipose tissue(Figure 1)by Western blottingTNK did not alter protein expression of GLUT4(094014 vs 111024，P一0as)in 中西医结合学报2010年6月第8卷第6期Jour
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