第二章 台灣金線連降血糖機轉之研究
第三節 ：討論
本篇研究顯示口服投與 AFEF 不會影響正常小鼠血糖，而胰島
素對正常小鼠降血糖效果明顯。腹腔葡萄糖耐受性試驗中，單一劑量
AFEF 及胰島素投與可以減少葡萄糖誘導之血糖上升，同樣也可以減 少 STZ 誘導糖尿病空腹高血糖。長時間投與 AFEF 可以減少糖尿
病小鼠升高的血糖、三酸甘油脂、膽固醇、及糖化血紅素。長時間投
與胰島素可減少糖尿病小鼠升高的血糖，糖化血紅素，三酸甘油脂，
但是無法減少升高的膽固醇，這些結果顯示 AFEF 對糖尿病小鼠具
有降血糖作用，但是藥效強度遠小於胰島素。
第 1 型糖尿病中有 3 個主要降血糖機轉，其分別為 (1). 延緩小
腸葡萄糖吸收，(2). 減少肝糖輸出量，或是 (3). 促進骨骼肌利用葡
萄糖。延緩小腸葡萄糖吸收的藥物如 -glucosidase ，抑制酵素可以
延緩碳水化合物分解成葡萄糖而減低飯後血糖的上升； Acarbose 抑 制胰臟及唾液腺alpha-amylase 及 disaceharidases 活性 阻礙碳水
化合物分解成葡萄糖而減低飯後血糖的急遽上升(86)。投與 AFEF，
在 糖 尿 病 小 鼠 小 腸 三 種 雙 糖 酵 素 (sucrase, lactase 及 galactase activity) 並未受影響。
不論在胰島素缺乏及胰島素阻抗的糖尿病(87)，肝臟葡萄糖的產生
主要是造成糖尿病高血糖最重要的因素，糖質新生及肝糖分解是肝臟
葡萄糖的產生主要的來源，荷爾蒙如 Glucagon、 catecholaimnes
(epinephrine and norepinephrine)、 cortisol 及 growth hormone 均 能 刺 激 肝 醣 分 解 及 糖 質 新 生 作 用 使 肝 臟 葡 萄 醣 產 量 增 加(88-89)，
PEPCK 是肝中糖質新生作用控制速率的重要酵素 (90)， PEPCK 基 因的轉錄可受 glucagon (via cAMP) 及 glucocorticoids 刺激增加，
受到胰島素抑制，在一定的條件下葡萄糖也可減少 PEPCK 基因表
現。
口服 AFEF 1.5 g/kg body weight 並不影響 epinephrine 誘導正
常小鼠上升的血糖。在 STZ 誘導的糖尿病小鼠，長期投與 AFEF 1.5
g/kg body weight 明顯減少肝糖含量及有意義增加肝中 PEPCK mRNA 表現量，些結果顯示 AFEF 不會影響糖尿病小鼠肝臟中葡萄 糖產生，但是胰島素減少 epinephrine 誘導正常病小鼠上升的血糖及
回復肝醣的減少，並且不增加肝中 PEPCK mRNA 表現量，這些結
果顯示 AFEF 對肝臟中降血糖機轉不同於胰島素。
葡萄糖由血中運送至骨骼肌細胞中依賴 GLUT4 在細胞膜上產
生的運糖作用，不論在胰島素缺乏及胰島素阻抗的骨骼肌中有 2 種
訊息機轉導致 GLUT4 (91) 由細胞質胞器中轉移到細胞膜上，(1). 與 胰島素無關的路徑是活化 AMPK， 促進細胞質中的 GLUT4 胞器
轉移到細胞膜而增加葡萄糖被吸收 (uptake) (92)，(2). 則是藉由 phosphorylating histone deacetylase 使 GLUT4 基 因 表 現 增 加
(93-94)，此兩種方式皆可以增加細胞膜上 GLUT4 的量，進而增加葡
萄糖由血中運送至骨骼肌細胞中的量。長期投與 AFEF (0.5, 1.0, 及
1.5 g/kg body weight) 及 胰 島 素 明 顯 增 加 糖 尿 病 小 鼠 骨 骼 肌 中 GLUT4 蛋白表現量，長期投與 AFEF 1.5 g/kg body weight 及胰島 素明顯增加糖尿病小鼠 AMPK 及GLUT4 mRNA 的表現量，結果顯
示 AFEF 降血糖機轉主要在促進骨骼肌利用葡萄糖。
結論， AFEF 呈現明顯降血糖效果，其機轉不同於胰島素作用。
AFEF 明顯增加糖尿病小鼠調降 GLUT4 和 AMPK 基因表現量，
顯示降血糖機轉可能是經由活化骨骼肌中 GLUT4 和 AMPK 基因
表現途徑達成降血糖作用。
Table 2-1. Primer sequences of PCR amplification.
mRNA Primer sequences Length
(bp) GLUT4: Sense 5' ACC CTG GGC TCT GTA TCCC 3' 202 GLUT4 Antisense 5' GGT TCG TCA TTG AGC GAG GAG 3'
PEPCK: Sense 5' GTC TAT GAA GCC CTC AGC T 3' 127 PEPCK: Antisense 5' AAG AAG GGT CGC ATG GCAA 3'
AMPK: Sense 5' ATT GAA ATC ACC GAA AAC AC 3' 266 AMPK: Antisense 5'CCT AAC AGC TGC TGA AGT TT 3'
GAPDH: Sense 5'TGT GTC CGT CGT GGA TCT GA3' 76 GAPDH: Antisense 5' CCT GCT TCA CCA CCT TCT TGA 3'
Table 2-2. Effect of AFEF on blood glucose in mice Blood glucose (mg/dl) Treatment Dose
0 hr. 1 hrs. 2 hrs. 3 hrs. 4 hrs.
Control 109±7 118±12 122±14 109±13 110±5 AFEF 0.5 g/kg 111±12 125±7 124±13 104±11 103±12
1.0 g/kg 105±5 115±6 1174±15 107±10 99±10 1.5 g/kg 111±19 111±13 102±6 107±10 97±6 Insulin 1 U/kg 109±14 50±8** 70±12* 85±2** 83±8**
All values are means ± S.D. (N=6). * p<0.05, **p<0.01 as compared with the control group.
Table 2-3. Effect of AFEF on intraperitoneal glucose tolerance test in mice
Blood glucose (mg/dl) Treatment Dose
0 min. 30 mins. 60 mins. 90 mins. 120 mins. 180 mins.
Control 123±11 284±49 265±31 222±29 213±13 164±7 AFEF 0.5 g/kg 124±7 276±2 247±21 231±12 191±26 162±118
1.0 g/kg 123±13 164±45** 152±14** 164±17** 164±14** 144±17
1.5 g/kg 121±4 202±49.3* 176±15** 173±8** 179±15* 149±9 Insulin 1 U/kg 127±9 56±7*** 67±10*** 94±15*** 123±9*** 144±19
All values are means ± S.D. (N=6). * p<0.05, **p<0.01, ***p<0.001 as compared with the control group.
Table 2-4. Effect of AFEF on blood glucose in epinephrine-induced hyperglycemic mice
Blood glucose (mg/dl) Treatment Dose
0 hr. 1 hrs. 2 hrs.
EPI + H2O 115±11 337±14 190±29 EPI+AFEF 0.5 g/kg 116±17 353±74 212±87
1.0 g/kg 113±11 330±17 180±30
1.5 g/kg 115±68 341±58 219±10
EPI+ Insulin 1 U/kg 130±28 48±15** 89±39**
All values are means ± S.D. (N=6). * p<0.05, **p<0.01 compared with the EPI + H2O group.
EPI: epinephrine
Table 2-5. Effect of single dose administration of AFEF on fasting blood glucose in streptozotocin-induced hyperglycemic mice
Blood glucose (mg/dl) Treatment Dose
0 hr. 1 hrs. 2 hrs. 3 hrs. 4 hrs.
STZ + H2O 285±25 293±12 295±40 278±27 291±59 STZ+AFEF 0.5 g/kg 292±38 279±24 269±18 272±42 268±56 1.0 g/kg 278±34 246±32** 252±6* 242±25 253±35 1.5 g/kg 299±63 237±40** 238±50** 237±38 241±35 STZ+Insulin 3 U/kg 319±33 46±18*** 71±15*** 136±70*** 194±54**
All values are means ± S.D. (N=8). * p<0.05, **p<0.01, ***p<0.001 as compared with the STZ+ H2O group.
STZ: Streptozotocin
Table 2-6. Chronic effect of AFEF on fasting blood glucose in streptozotocin-induced hyperglycemic mice
Blood glucose (mg/dl) Treatment Dose
Week 0 Weeks 1 Weeks 2 Weeks 4 Weeks 6 control 72±17 69± 17 77±24 76 ±9 89±22 STZ+ H2O 268±12 298 ±21### 277±17### 293±27### 284±19###
STZ+AFEF 0.5 g/kg 266.0±39 271 ±13 252±13 258±4 269±12 1.0 g/kg 262±18 249 ±20** 224±23* 201±75** 210±81**
1.5 g/kg 263±18 249 ±44** 240±27* 204±52** 212±55**
STZ+Insulin 5 U/kg 264±20 190 ±35*** 195±40*** 134±53*** 103±30***
All values are means ± S.D. (N=8). ### p<0.001 as compared with the control group. *p<0.05, **p<0.01, ***p<0.001 as compared with the STZ+
H2O group.
STZ: Streptozotocin
Table 2-7 Chronic effect of AFEF on intestinal activities of disaccharides enzymes in streptozotocin-induced hyperglycemic mice
sucrase lactase galactase Treatment Dose μM glucose/mins.
/mg protein
μM glucose/mins.
/mg protein
μM glucose/mins.
/mg protein
control 401±59 31±7 46±23
STZ+ H2O 453±52 34±5 46±7
STZ+AFEF 0.5 g/kg 541±74 43±5 55±15
1.5 g/kg 476±81 40±3 50±5
STZ+Insulin 5 U/kg 392±86 26±3* 41±8
All values are means ± S.D. (N=8). *p<0.05 as compared with the STZ+ H2O group.
STZ: Streptozotocin
0
Fig. 2-1 Chronic effect of AFEF on plasma triglycerides in streptozotocin-induced hyperglycemic mice. All values are means ± S.D. (N=8). ### p<0.001 as compared with the control group. * p<0.05,
**p<0.01, ***p<0.001 as compared with the STZ+ H2O group.
CON: control; STZ: Streptozotocin
0 20 40 60 80
100 ###
*
CON H2O 0.5 1.5 5
Plasma cholesterol (mg/dl) *
1.0
STZ
AFEF(g/kg) Insulin(U/kg)
Fig. 2-2 Chronic effect of AFEF on plasma cholesterol in streptozotocin-induced hyperglycemic mice. All values are means ± S.D. (N=8). ###
p<0.001as compared with the control group. * p<0.05,
as compared with the STZ+ H2O group.CON: control; STZ: Streptozotocin
0 2 4 6 8 10
12 ###
***
CON H2O 0.5 1.5 5
Glycated hemoglobin (mg/dl)
1.0
STZ
AFEF(g/kg) Insulin(U/kg)
***
***
Fig. 2-3 Chronic effect of AFEF on glycated hemoglobin in streptozotocin-induced hyperglycemic mice. All values are means ± S.D. (N=8). ### p<0.001 as compared with the control group.
***p<0.001 as compared with the STZ+ H2O group.
CON: control; STZ: Streptozotocin
0.0
Liver triglycerides (mg/g wet tissue)
1.0
STZ
AFEF(g/kg) Insulin(U/kg)
**
Fig. 2-4 Chronic effect of AFEF on liver triglycerides in streptozotocin-induced hyperglycemic mice. All values are means ± S.D. (N=8). ## p<0.01 as compared with the control group. **p<0.01 as compared with the STZ+ H2O group.
CON: control; STZ: Streptozotocin
0 10 20 30 40 50 60
CON H2O 0.5 1.5 5
Liver glycogen (mg/g wet tissue)
1.0
STZ
AFEF(g/kg) Insulin(U/kg)
**
***
***
Fig. 2-5 Chronic effect of AFEF on liver glycogen in streptozotocin-induced hyperglycemic mice. All values are means ± S.D. (N=8). **p<0.01, ***p<0.001 as compared with the STZ+ H2O group.
CON: control; STZ: Streptozotocin
Fig. 2-6 Chronic effect of AFEF on the GLUT4 protein expression in skeletal muscular in streptozotocin-induced hyperglycemic mice.
All values are means ± S.D. (N=8). ###
p<0.001 as compared with the
control group. **p<0.01, ***p<0.001 as compared with the STZ+H2O group.
CON: control; STZ: Streptozotocin
Fig. 2-7 Chronic effect of AFEF on the AMPK and GLUT4 mRNA expression in skeletal muscular in streptozotocin-induced hyperglycemic mice. A, Fragments were amplified by RT-PCR. The expression levels of AMPK (B), and GLUT4 (C) mRNA were measured and quantified densitometrically. Values were normalized to GAPDH mRNA expression. All values are means ± S.D. (N=8). # p
< 0.05, ### p<0.001 as compared with the control group. *p<0.05,
**p<0.01 as compared with the STZ+ H2O group.
CON: control; STZ: Streptozotocin
Fig. 2-8 Chronic effect of AFEF on the PEPCK mRNA expression in liver in streptozotocin-induced hyperglycemic mice. A, Fragments were amplified by RT-PCR. The expression levels of PEPCK (B) mRNA were measured and quantified densitometrically. Values were normalized to GAPDH mRNA expression. All values are means ± S.D.
(N=8). **p<0.01 as compared with the STZ+ H2O group.
CON: control; STZ: Streptozotocin