第二章 文獻回顧
2.3 胰島素阻抗
2.3.2 PI3K(Phosphatidylinositol-3-kinase)pathway
胰島素主要的標的器官(target tissue)為肝臟、肌肉、脂肪。胰島素藉由 I3K 的訊息傳遞路徑促使這些組織將葡萄糖攝入。組織的細胞表面佈滿著二聚體 (dimer)型式的胰島素接受體(insulin receptor; IR)，當 insulin 與 receptor 結合後，
會使 receptor β 次單元 C 端的 tyrosine 自磷酸化(autophosphorylation)，並使下游
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的 (insulin receptor substrate; IRS)及(growth factor receptor-binding protein; Grb1) 活化。IRS 分為 IRS1 及 IRS2，在脂肪與肌肉組織中以 IRS1 為主，在肝臟組織 中 則 是 兩 者 並 存 。 活 化 型 的 IRS (tyrosine 被 磷 酸 化 ) 及 Grb1 ， 能 夠 活 化 (phosphatidylinositol-3-kinase; PI3K) ， 使 (phosphatidylinositol 4,5-bisphosphate;
PIP2) 磷 酸 化 成 (phosphatidylinositol 3,4,5-triphosphate; PIP3) ， PIP3 將 與 (3-phosphoinositide-dependent protein kinase-1; PDK1)結合，使下游的 AKT (PKB, protein kinase B)和 Protein kinase C zeta (PKCζ)活化。藉由以上訊息傳遞促使 (glucose transporter 4; GLUT4)轉位(translocation)至細胞膜上，使細胞得以攝入葡 萄糖(glucose uptake) (Eriksson, 2007)。
圖2-2、胰島素訊息傳遞路徑。
Insulin signalling via the PI3K-dependent pathway in adipocytes and skeletal muscle.
Insulin binding to its receptor will lead to glucose transport activation and other metabolic effects and this is exerted via a cascade of signaling proteins. GLUT4, glucose transporter 4; IR, insulin receptor; IRS1/2, insulin receptor substrates 1 and 2;
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PDK1, phosphatidylinositol dependent protein kinase 1, PI3K, phosphatidylinositol 3-kinase;PIP2,phosphatidylinositol-3,4-phosphate;PIP3,phosphatidylinositol-3,4,5-ph osphate; aPKC, atypical protein kinase C; PKB, protein kinase B. (Eriksson, 2007)
2.3.3 葡萄糖轉運蛋白(glucose transporter; GLUT)
目前已知人類的葡萄糖轉運蛋白家族中有 13 種，其共同特徵為跨膜蛋白，
均跨越細胞膜 12 次，序列的 N 端及 C 端均位於細胞質內。根據序列同質性與結 構相似度，可分為三群：第一群為 GLUT1-4 ，主司葡萄糖運輸；第二群為 GLUT5,7,9,11，主司果糖運輸；第三群為 GLUT6,8,10。第一群中 GLUT1-3 主要 負責細胞基礎狀態下對葡萄糖的攝取，會持續表現，不受胰島素刺激而改變細胞 膜上的數目。GLUT-2 主要維持細胞的促進擴散作用(facilitated diffusion)來運輸 glucose，亦不受胰島素影響。而 GLUT4 是唯一能受 insulin 刺激而增加 glucose uptake 的轉運蛋白，以骨骼肌及脂肪組織的表現量最高。(張, 2010; 李, 2008)
圖 2-3、葡萄糖轉運蛋白跨膜蛋白構型。
GLUT family of proteins comprises 13 members at present, which are predicted to span the membrane 12 times with both amino- and carboxyl-termini located in the cytosol. On the basis of sequence homology and structural similarity, three subclasses of sugar transporters have been defined: Class I (GLUTs 1–4) are glucose transporters;
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Class II (GLUTs 5, 7, 9 and 11) are fructose transporters; and Class III (GLUTs 6, 8, 10, 12 and HMIT1) are structurally atypical members of the GLUT family, which are poorly defined at present. The diagram shows a homology plot between GLUT1 and GLUT4. Residues that are unique to GLUT4 are shown in red. (Bryant, 2002)
圖 2-4 葡萄糖轉運蛋白家族。
Dendrogram of the glucose transporter (GLUT) family. An unrooted radial phylogram was drawn from a multiple sequence alignment of the thirteen members of the human GLUT family. The tree was constructed using neighbour-joining analysis of a distance matrix generated with PHYLIP software (Felsenstein, 1989). The three classes of GLUT proteins are colour-coded as: blue, class I; red, class II; green, class III. The scale bar represents 0·1 substitutions per amino acid position. HMIT, H+-coupled myo-inositol transporter. (Wood and Trayhurn, 2003)
22 表、2-2 葡萄糖轉運蛋白家族
Glucose transporter (GLUT) family
23 2.3.4 高果糖與胰島素阻抗
果 糖 (fructose) 於 果 糖代 謝路 徑 中 經 由 果 糖激 酶 (fructokinase) 、 醛 縮酶 B (aldolase B) 、 三 碳 糖 激 酶 (triosekinase) 作 用 產 生 甘 油 醛 -3- 磷 酸 (glyceraldehyde-3-phosphate, GA3P) ， GA3P 可 直 接 進 入 糖 解 代 謝 (glycolysis pathway)，因此相較於 glucose 而言，fructose 能夠更快速的併入 glycolysis 進行 醣 類 代 謝 。 glycolysis 最 終 產 物 為 pyruvate 再 經 由 丙 酮 酸 脫 氫 酶 (pyruvate dehydrogenase; PDH)複合體作用生成 Acetyl-CoA，Acetyl-CoA 藉由檸檬酸合成 酶(citrate synthase)的作用，得以通過粒線體膜至細胞質，並於細胞質內經由乙醯 輔酶 A 羧化酶(Acetyl-CoA carboxylase; ACC)、脂肪酸合成酶 (Fatty acid synthase;
FAS)進行脂肪酸的生合成(de novo synthesis) (黃, 2009)。三酸甘油酯(triglycerol;
TG) 的 前 驅 物 為 帶 有輔 酶 A 的 脂 肪 酸 以 及 甘 油 -3-磷 酸 (gycerol-3-phosphate;
G3P)，當 1 分子的 fructose 經果糖代謝後會生成一分子的 GA3P 及一分子的磷酸 雙羫丙酮(dihtdroxyacetone pgosphate; DHAP)，GA3P 可再轉換成 DHAP，而 DHAP 經由甘油磷酸脫氫酶(glycerolphosphate dehydrogenase)的還原作用可生成 G3P。
高果糖飲食會導致內生性的脂質增加，G3P 與 fatty acid 於肝中酯化成 TG，再經 由極低密度脂蛋白(very low density lipoprotein; VLDL)運送至血液循環，造成高 血脂症(hyperlipidemia)，當攜帶大量 TG 的 VLDL 會轉變成(intermediate density lipoprotein; IDL)，會被血管壁中的脂蛋白脂解酶(lipoprotein lipase; LPL)分解成低 密度脂蛋白(low density lipoprotein; LDL)，並將 TG 分解成 Free fatty acid 及 DG、
MG，進入脂肪組織後再酯化成 TG 儲存，導致體脂肪堆積。由於體脂肪質量增 加，肥胖者位於細胞表面的胰島素接受器(insulin receptor)數目減少，影響胰島素 訊息傳遞，insulin 無法抑制脂肪細胞中的荷爾蒙敏感性脂解酶(hormone-sensitive lipase; HSL)，造成脂肪細胞分解 TG 並釋出大量 free fatty acids 至血液循環，造
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成 insulin resistence (謝, 2005; Johnson et al., 2008)。
2.3.5 脂肪組織與胰島素阻抗
脂肪組織除了以三酸甘油脂型式儲存能量的生理功能外，同時具有內分泌的 功能，能夠分泌多種細胞激素，統稱脂肪細胞激素(adipocytokine)，例如: 脂聯 素(adiponectin; ApN)、瘦體素(leptin)、腫瘤壞死因子-α (TNF-α)、介白素-6 (IL-6)、
纖維蛋白溶解酶原激活抑制素(plasminogen activator inhibitor-1; PAI-1) 血清張力 素原(angiotensinogen; AGT)、視網醇結合蛋白(retinol-binding protein-4; RBP-4)、
單核球趨化蛋白-1(monocyte chemoattractant protein-1; MCP-1)，將會影響各組織 器官的胰島素敏感性(Maury and Brichard, 2010)。
25 圖2-5、果糖代謝。
Fructose metabolism.
Fructose enters cells via a transporter (typically Glut 5, Glut 2, or SLC2A9) where it is preferentially metabolized by fructokinase (KHK) to generate fructose-1-phosphate.
Unlike phosphofructokinase, which is involved in glucose metabolism, fructokinase has no negative feedback system to prevent it from continuing to phosphorylate substrate, and as a consequence ATP can be depleted, causing intracellular phosphate depletion, activation of AMP deaminase, and uric acid generation. In addition, fructose is lipogenic and can generate both glycerol phosphate and acyl coenzyme A, resulting in triglyceride formation that is both secreted and stored in hepatocytes. IMP, Inosine monophosphate; TCA, trichloroacetic acid. (Johnson et al., 2008)
26 圖 2-6、果糖對各組織器官影響。
Effect of fructose on various organ systems.
Table sugar, HFCS, and natural sources provide fructose, which in excess has numerous effects on the brain, liver, vasculature, kidney, and adipocyte. The net effects induce all features of the metabolic syndrome and ultimately type 2 diabetes.
(Johnson et al., 2008)
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圖2-7、脂肪細胞激素引發代謝症候群之病理機轉。
Adipokines involved in the pathogenesis of the metabolic syndrome
Adipokines may modulate insulin sensitivity, cardiovascular homeostasis, inflammation and fat mass. Adiponectin (ApN) and omentin are downregulated in obesity, while most adipokines are overproduced. ApN, leptin and possibly vaspin are adipocyte-specific, while other adipokines may also be produced by non-adipose cells (including macrophages) of adipose tissue and by non-adipose tissues as well. PAI-1, plasminogen activator inhibitor type 1; AGT, angiotensinogen; TNF-α, tumor necrosis factor-α; RBP-4, retinol-binding protein-4; MCP-1, monocyte chemoattractant protein-1; IL-6, interleukin-6. (Maury and Brichard, 2010)
28 圖2-8、胰島素阻抗病理機轉。
Pathophysiology of insulin resistance.
Free fatty acids (FFAs) released from adipose tissue increase production of glucose and triglycerides (TGs) and secretion of very-low-density lipoprotein(VLDL) in the liver. Associated lipid/lipoprotein abnormalities include decreased levels of high-density lipoprotein cholesterol (HDL-C) and increased levels of small dense low-density lipoprotein (LDL) particles. FFAs also reduce insulin sensitivity in muscle by inhibiting insulin mediated glucose uptake. Increases in circulating glucose increase pancreatic insulin secretion, resulting in hyperinsulinemia. Hyperinsulinemia may result in enhanced sodium reabsorption and increased sympathetic nervous system activity. It also may contribute to the development of hypertension. (Jellinger, 2007)
29 2.4 參與醣類代謝與脂質代謝之關鍵酵素
2.4.1 Glycolysis pathway
糖解作用代謝路徑關鍵酵素如下：
(一)六碳醣激酶(Hexokinase; HK)
Glycolysis 第一步驟，需鎂離子(
Mg
2+)作為輔因子，將 glucose 轉變成 6-磷 酸 葡 萄 糖 (glucose-6-phosphate; G-6-P) ， 並 消 耗 1 分 子 的 腺 核 苷 三 磷 酸 (adenosine-5'-triphosphate; ATP) ， 生 成 1 分 子 的 腺 核 苷 二 磷 酸 ( adenosine diphosphate; ADP)，反應會受到 G-6-P 的負向調控。(二)磷酸果糖激酶(Phosphofructokinase; PFK)
Glycolysis 第二步驟，將 6-磷酸果糖(fructose-6-phosphate; F-6-P)轉變為 1,6 雙磷酸果糖(fructose-1,6-bisphosphate; F-1,6BP)，消耗 1 分子 ATP，生成 1 分子 ADP，此步為 glycolysis 的限速步驟，被檸檬酸(citrate)、ATP 負向調節，2,6 雙 磷酸果糖(fructose-2,6-bisphosphate)、ADP、AMP 會促進此步反應。
(三)丙酮酸激酶 (Pyruvate kinase; PK)
Glycolysis 第十步驟，將磷酸烯醇丙酮酸(phosphoenolpyruvate; PEP)轉變為丙 酮酸(pyruvate)，反應過程中將磷酸根轉移給 ADP，生成 1 分子的 ATP，此步為 受質磷酸化作用。
2.4.2 De novo lipogenesis pathway
(一)乙醯輔酶 A 羧化酶 (Acetyl-CoA carboxylase; ACC)
脂 肪 酸 的 合 成 需 要 起 始 分 子 Acetyl-CoA 及 數 個 丙 二 醯 輔 酶 A (Malonyl-CoA)，連續性的組合。此步為脂肪酸合成的第一步驟及關鍵調控步驟，
作用於 cytosol、需要生物素(biotin)當作輔成基(prosthetic group)，為羧化作用
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(carboxylation)，將一分子的 Acetyl-CoA 與
CO
2羧化成 Malonyl-CoA，並消耗一 分子的 ATP 及 H2O，受 Acetyl-CoA 負向調控、citrate 會促進此反應。(二)脂肪酸合成酶 (Fatty acid synthase; FAS)
脂肪酸合成酶為複合體，是含有 7 種酵素活性的 polypeptide，須以 dimer 形 式存在才具有催化活性，並同時合成 2 分子 Fatty acid。每一回合反應依序為縮 合、還原、脫水、還原。合成方向為甲基端往羧端。複合酶組成如下：
a.醯基攜帶蛋白(Acyl carrier protein; ACP)：於複合酶的中心，能與醯基中間代謝 物共價結合。
b.ACP-乙醯基轉移酶(ACP-acetyltransferase; AT)：為第一步驟，將 Acetyl-CoA 的 乙醯基轉移給 ACP。
c.ACP-丙二醯基轉移酶(ACP-malonyltransferase; MT)：將 Malonyl-CoA 的丙二醯 轉移給 ACP。
d.β-酮基-醯基-ACP-合成酶(β-ketoacyl-ACP-synthase; KS)：將乙醯基與丙二醯進 行縮合反應，釋放出一分子
CO
2，形成乙醯乙醯基(acetoacetyl group)。e.β-酮基 -醯基 -ACP-還原酶 (β-ketoacyl-ACP-reductase; KR)： 於乙醯乙醯 ACP (acetoacetyl-S-ACP)上之羰基(carbonyl group)：產生還原反應，形成 D-β-羥丁 醯-ACP (D-β-hydroxybutyryl-S-ACP)。需消耗 NADPH 提供還原力。
f.β- 羥 化 醯 基 -ACP- 脫 水 酶 (β-Hydroxyacyl-ACP dehydratase; HD) ： 將 D-β-hydroxybutyryl-S-ACP ， 脫 水 形 成 反 式 -Δ2 - 丁 基 烯 醯 ACP (trans-Δ2 -butenoyl-S-ACP)。
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g.烯醯-ACP 還原酶(Enoyl-ACP reductase; ER)：將 trans-delta ²-butenoyl-S-ACP 的 雙鍵還原成丁醯-ACP (butyryl-S-ACP)，需 NADPH 提供還原力。
2.4.3 Lipolysis pathway
脂解作用代謝路徑關鍵酵素如下：
(一) 脂肪細胞三酸甘油酯脂解酶(adipose triglyceride lipase; ATGL)
存在於脂肪組織，能將 TG 分解成雙醯甘油酯(diglycerides; DG)並釋放出 free fatty acid，但不具有分解 DG 能力(Braseamle,2010;Lass et al., 2006)，不會受體內 中 cAMP-dependent protein kinase 的磷酸化調控改變其活性(Langin, 2006)。ATGL 能催化三酸甘油酯的脂解反應，研究指出 ATGL 對三酸甘油酯的第一個酯鍵，專 一性比起 HSL 更高。動物實驗中將 HSL 基因剔除老鼠體內，仍可發現受 ATGL 所分解的 DG 堆積。(Braseamle, 2010)
(二)荷爾蒙敏感性脂解酶(hormone sensitive triacylglycerol lipase; HSL)
存在於脂肪組織，將TG分解成DG並釋放出free fatty acid，脂解作用的限速 步驟，細胞中cAMP為關鍵的第二信息傳遞者(second messenger)能啟動細胞內數 種訊息傳遞路徑(Chen et al., 2010)，cAMP可使cAMP-dependent protein kinase活 化，而讓HSL磷酸化而活化。昇糖激素(glucagon)藉由促進腺苷環化酶(adenylate cyclase; AC)來活化cAMP促使HSL磷酸化而活化。而胰島素及甲基次黃嘌呤類物 質(methyl xanthines)如：caffeine，將藉由促進磷酸二酯酶(phosphodiesterase; PDE) 來抑制cAMP促使HSL去磷酸化而失活(Cluas et al., 2005)。
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圖2-9、人類具有功能性區塊的脂肪酸合成酶二元體構型
The structural and functional organization of animal and human FAS dimer. The linear arrangement of the component activities and their domains are indicated in the subunits of FAS. The dimer formation results in two active centers. The functional division indicates the participation of DI of one subunit and DII and DIII of the second subunit in generating the active center. The ID regions (ID) have no known catalytic activities but play an essential role in the structural organization of catalytically active FAS. AT, acetyl transacylase; MT, malonyl transacylase; ER, enoyl reductase; KR, b-ketoacyl reductase; DH, b-hydroxyacyl dehydratase.(Chirala et al., 2000)
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圖2-10、脂肪細胞中調控三酸甘油酯同化及異化作用之代謝路徑
Model of anabolic and catabolic pathways that regulate triglyceride (TG) levels in adipocytes.
The catabolic pathways involve ATGL, HSL, and monoacylglycerol lipases, which act on TG in a series of reactions to yield glycerol and fatty acids. CGI-58, which binds to perilipin and localizes to adiposomes, is an activator of ATGL. The nature of this activation is unknown. Intermediates of this catabolic pathway may also be recycled for phospholipids and TG synthesis. The anabolic pathways involve the esterification of fatty acids to glycerol. Fatty acids, derived from cellular uptake, de novo synthesis, or lipolytic breakdown of intracellular TG, are ‘‘activated’’ by the addition of a CoA moiety (via the action of acyl CoA synthetases, not shown for simplicity), and are subsequently esterified to glycerol through the actions of enzymes of the glycerolipid synthesis pathways. Significant amounts of hydrolysis products are normally reesterified (Leibel and Hirsch, 1985), a process that may be regulated. The model proposes that MGAT and DGAT enzymes are involved in this reesterification.
TG, triacylglycerol; DG, diacylglycerol; MG, monacylglycerol; FA, fatty acid; ATGL, adipose triglyceride lipase; HSL, hormone-sensitive lipase; MGL, monoacylglycerol lipase; DGAT, acyl CoA:diacylglycerol acyltransferase; MGAT, acyl
CoA:monacylglycerol acyltransferase. (Yen and Farese, 2006)
34 圖2-11、肝臟組織調控葡萄糖代謝。
Important pathways regulating glucose metabolism in the liver.
Excessive hepatic glucose output occurs in diabetes through increases in glycogenolysis and/or gluconeogenesis. Inhibitors of glycogen phosphorylase inhibit glucose output by decreasing hepatic glycogen catabolism. Other relevant targets include fructose-1,6- bisphosphatase, which controls a rate-limiting step in gluconeogenesis, and glucose-6-phosphatase, which catalyses the final common step required for release of glucose from the liver. NEFA, non-essential fatty acids; PEP, phosphoenolpyruvate. (Moller, 2001)
35 2.5 樣品與抗糖尿病用藥
2.5.1 沒食子酸
沒食子酸(gallic acid; GA)為酚酸(phenolic acids)化合物，屬於苯甲酸(benzoic acis)類衍生物，化學名 3,4,5-三羥基苯甲酸(3,4,5-trihydroxybenzoic acid)，分子式 C6H2(OH)3COOH ， 分 子 量 170.12 g/nmol ， 外 觀 為 白 色 粉 末 ， 屬 厭 水 性 (hydrophobic)，常用於多酚含量試驗的定量標準品。(Jang et al., 2008)
2.5.2 愛妥糖
愛妥糖(ACTOS)為口服抗糖尿病藥物，主要作用為降低 insulin resistance，主 治第 2 型糖尿病，其功效成分為 pioglitazone hydrochloride 能改善肌肉及脂肪組 織的 insulin sensitivity，且抑制肝臟 gluconeogenesis，同時能降低血中的 insulin 濃度。
(一)Pioglitazone hydrochloride 的理化特性
Pioglitazone hydrochloride 為白色結晶狀粉末，分子式 C19H20N2O3SHC1、分 子量 392.9，可溶於 N,N-dimethyl-formamide，微溶於無水酒精，極易溶於 acetone 及 acetonitrile，不溶於水、乙醚。
(二)Pioglitazone hydrochloride 的藥理機轉
(二)Pioglitazone hydrochloride 的藥理機轉