臺灣博碩士論文加值系統

糖尿病是一種常見代謝性疾病，其病理特徵為高血糖。而胰島素為人體維持血糖恆定的主要介質。傳統中醫藥用於治療糖尿病已有多年的經驗，但其機轉及有效成分仍不是很清楚。在本研究中，我們藉由臨床經驗和文獻搜尋確認胡蘆科要物或作物皆有降血糖的潛能，因此，我們萃取苦瓜種子成分(簡稱MCSE)，並確認其具有降血糖的活性，經由機轉探究，發現MCSE主要是透過影響胰島素訊息傳遞路徑(insulin signaling transduction pathway)。進一步，發現一具有抵抗胰蛋白酶的酵素分解作用(胰蛋白酶抑制物; trypsin inhibitor)，會與胰島素接受器結合，於本研究中我們將其命名為Momordica charantia insulin receptor (IR)-binding protein (簡稱mcIRBP)。利用電偶交鏈串聯式質譜儀的分析結果，發現mcIRBP有三個區域(17-21, 34-40, and 59-66 residues)，可能與胰島素接受器上的leucine-rich repeat domain與cystein-rich region有交互作用。經由 胰島素接受器結合試驗(IR-binding assay)，也顯示mcIRBP會與胰島素呈現協同作用(cooperative manner)。而且在mcIRBP與胰島素接受器結合後， mcIRBP活化胰島素接受器上的酪胺酸激酶活性為5.87+/-0.45倍，phospho-IR protein的增加量為1.31 +/-0.03倍，且影響phosphoinositide-3-kinase/Akt 訊號途徑，並使得3T3-L1細胞株的葡萄糖攝入增加，5 uM mcIRBP可達1.25+/-0.07倍。藉由腹腔注射2.5 nmol/kg mcIRBP 可以顯著降低正常小鼠的血糖至20.9+/-3.2%，糖尿病小鼠的血糖至10.8+/-3.6%。基因表達微陣列晶片的分析顯示mcIRBP影響的基因，含括小鼠的大部分胰島素訊息傳遞路徑。本研究的實驗，證實mcIRBP是新穎的胰島素接受器結合蛋白，並結合至胰島素與胰島素接受器所結合之不同結合位置，且可同時促進細胞的葡萄糖攝入與活體的醣類代謝。

Diabetes mellitus, a common metabolic disorder, is characterized by hyperglycemia. Insulin is the principle mediator of glucose homeostasis. Traditional Chinese medicine have been used in clinics for the treatment of diabetes for years. We surveyed literatures and experiences of Traditional Chinese medicine on diabetes and found that herbs belonging to family Curcubitaceae showed hypoglycemic potentials. Therefore, we analyzed hypoglycemic effects and mechanisms of Momordica charantia and its components in this study. Extract of Momordica charantia seeds (MCSE) exhibited hypoglycemic activity mainly via the insulin signaling transduction pathway. Moreover, we newly identified a trypsin inhibitor, named as MC insulin receptor (IR)-binding protein (mcIRBP), that might interact with IR. The physical and functional interactions between mcIRBP and IR were clearly analyzed in the present study. Photo-crosslinking coupled with mass spectrometry showed that three regions (17-21, 34-40, and 59-66 residues) located on mcIRBP physically interacted with leucine-rich repeat domain and cystein-rich region of IR. IR-binding assay showed that the binding behavior of mcIRBP and insulin displayed a cooperative manner. After binding to IR, mcIRBP activated the kinase activity of IR by 5.87 +/- 0.45-fold, increased the amount of phospho-IR protein by 1.31+/-0.03-fold, affected phosphoinositide-3-kinase/Akt pathways, and consequently stimulated the uptake of glucose in 3T3-L1 cells by 1.25 +/- 0.07-fold in the presence of 5 uM mcIRBP. Intraperitoneal injection of 2.5 nmol/kg mcIRBP significantly decreased the blood glucose levels by 20.9+/- 3.2% and 10.8+/-3.6% in normal and diabetic mice, respectively. Microarray analysis showed that mcIRBP affected genes involved in insulin signaling transduction pathway in mice. In conclusion, our findings suggested that mcIRBP was a novel IRBP that bound to the sites different from the insulin-binding sites on IR and stimulated both the glucose uptake in cells and the glucose clearance in mice.

Table of Contents
Chapter 1 Introduction--------------------------------------------------------1
1.1 Research Motivation ------------------------------------------------------1
1.2 Research Purpose-----------------------------------------------------------3
Chapter 2 Literature Review-------------------------------------------------4
2.1 Prevalence and economic cost of diabetes------------------------------4
2.2 Exploration of the therapeutic potential Chinese herbal medicine of
acute thirst disease in Chinese ancient literatures----------------------4
2.3 Exploration of the bioactivity component of Cucurbitaceae family-8
Chapter 3 Materials and Methods------------------------------------------11
3.1 Chemicals-------------------------------------------------------------------11
3.2 Investigation using PubMatrix------------------------------------------12
3.3 Preparation of MCSE and TI---------------------------------------------12
3.4 Animal experiment and glucose tolerance test------------------------13
3.5 Microarray analysis ------------------------------------------------------14
3.6 2-dimensional electrophoresis (2-DE)----------------------------------15
3.7 Liquid chromatography coupled with tandem MS (LC-MS/MS) analysis----------------------------------------------------------------------16
3.8 Docking calculations------------------------------------------------------17
3.9 Cloning of mcIRBP gene-------------------------------------------------17
3.10 Expression and purification of recombinant mcIRBP --------------18
3.11 Photo-crosslinking coupled with mass spectrometry----------------18
3.12 Cell culture ---------------------------------------------------------------19
3.13 Insulin receptor-binding assays-----------------------------------------19
3.14 Western blot analysis ----------------------------------------------------21
3.15 Adipocyte differentiation and glucose uptake assay-----------------21
3.16 Streptozotocin (STZ)-induced type 1 diabetic mice and glucose
tolerance assay------------------------------------------------------------22
3.17 Statistical analysis -------------------------------------------------------23
Chapter 4 Results---------------------------------------------------------------24
4.1 Hypoglycemic effect-related available articles in Asia by PubMatrix -------------------------------------------------------------------------------24
4.2 Hypoglycemic effect of Cucurbitaceae family in mice --------------26
4.3 Signal transduction pathways of MCSE on glucose metabolism in
diabetic mice ---------------------------------------------------------------28
4.4 Analysis of Protein Constituents in MCSE by 2-DE and LC-MS/MS --------------------------------------------------------------------------------32

4.5 Identification of IR-binding protein of MCSE by docking and IR
kinase activity assays-------------------------------------------------------36
4.6 Expression and purification of mcIRBP ---------------------------------39
4.7 Analysis of the physical interaction between IR and mcIRBP--------41
4.8 mcIRBP enhanced the kinase activity and autophosphorylation of IR
---------------------------------------------------------------------------------45
4.9 mcIRBP affected IR/PI3K/Akt signaling pathway --------------------47
4.10 mcIRBP stimulated the uptake of glucose in 3T3-L1 adipocytes
-------------------------------------------------------------------------------49
4.11 mcIRBP displayed the hypoglycemic effect in normal and diabetic mice -------------------------------------------------------------------------51
4.12 Signal transduction pathways of mcIRBP on glucose metabolism in
normal and diabetic mice------------------------------------------------53
Chapter 5 Discussion------------------------------------------------------------60
Chapter 6 Conclusion-----------------------------------------------------------73
References-------------------------------------------------------------------------75
Chinese abstract------------------------------------------------------------------91
Acknowledgement----------------------------------------------------------------93


List of Figures
Figure 4.1 Hypoglycemic effect-related available articles in Asia by PubMatrix---------------------------------------------------------25
Figure 4.2 Hypoglycemic effect of MCSE in mice by glucose tolerance
test-----------------------------------------------------------------27
Figure 4.3 Signal transduction pathways contributing to the modulatory effect of MCSE on glucose metabolism in diabetic mice---30
Figure 4.4 Analysis of constituents in MCSE-----------------------------34
Figure 4.5 Physical interaction between TI and IR. Docking structure between IR and TI-----------------------------------------------37
Figure 4.6 Cloning, expression, and purification of recombinant
mcIRBP ------------------------------------------------------40
Figure 4.7 Docking structure of IR and mcIRBP-------------------------42
Figure 4.8 Physical interaction between mcIRBP and IR---------------43
Figure 4.9 Competitive binding assay of mcIRBP-----------------------44
Figure 4.10 Effect of mcIRBP on the kinase activity and autophosohorylation of IR--------------------------------------46
Figure 4.11 Effect of mcIRBP on the insulin signaling pathway in
HepG2 cells------------------------------------------------------48
Figure 4.12 Effect of mcIRBP on the glucose uptake of 3T3-L1
adipocytes-------------------------------------------------------50
Figure 4.13 Hypoglycemic effects of mcIRBP in normal and type 1
diabetic mice by glucose tolerance assay-------------------52

List of Tables
Table 2.1 The top ten most commonly used Chinese medicinal herbs in Taiwan----------------------------------------------------------------6
Table 4.1 P - value of pathways associated with glucose
and lipid metabolism in MCSE-treated ob/ob mice------------29
Table 4.2 Expression levels of MCSE-regulated genes in adipose
tissue-------------------------------------------------------------------31
Table 4.3 MCSE proteins identified by LC-MS/MS------------------------35
Table 4.4 Intramolecular Interaction between IR and TI-------------------38
Table 4.5 The top 20 up-regulated and down-regulated genes by mcIRBP
in normal mice -------------------------------------------------------54
Table 4.6 The top 20 up-regulated and down-regulated genes by mcIRBP
in diabetic mice ------------------------------------------------------56
Table 4.7 Pathways significantly affected by mcIRBP in normal mice---59

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