D-乳酸在糖尿病腎臟中之研究

The Study of D-Lactate in Diabetic Rat Kidney

廖紫歆(Liao Tzu-Hsin)

指導教授 ： 李仁愛

臺北醫學大學/藥學院/藥學研究所/碩士(2003年)

摘要

中文摘要 動物體內僅有肝臟與腎臟可進行糖質新生的作用，在糖尿病下，腎臟糖質新生的增加使葡萄糖的釋放大量增加近300%，被認為是造成高血糖的主要原因。乳酸為腎臟糖質新生的主要來源，其含有一不對稱碳，故具有D-、L-乳酸兩種鏡相異構物，而D-、L-乳酸兩者之生成相當不同，L-乳酸是糖解作用之終產物，D-乳酸為體內一醣化終產物 (advanced glycation end-products) ─甲基乙二醛 (methylglyoxal) 進行去毒化反應所生成，目前缺乏對乳酸鏡像異構物與腎臟糖質新生間相關的探討。 為瞭解D-乳酸在糖尿病腎臟中之含量，以及對於糖質新生的影響，故利用已建立之管柱切換高效液相層析法，檢測腎臟均質液中D-乳酸之濃度，並利用一葡萄糖試劑組套，探討腎臟均質液中額外加入之D-、L-乳酸對葡萄糖釋放的影響，以及腎臟中D-乳酸與自由基的關係。 實驗結果顯示，以HPLC分析糖尿病大鼠腎臟中之D-乳酸，發現會隨著誘導時間1、2、3、4個月而累積，且呈現一線性增加 (2.99, 13.11, 18.19, 23.23 ?mol/mg vs. 0.79 ?mol/mg as control groups)；在糖質新生方面，於腎臟均質液中添加之D-乳酸 (3.47 ?g/ml) 可抑制L-乳酸所釋放之葡萄糖(17.24 ?g/ml)，推測D-乳酸在糖質新生作用上可與L-乳酸拮抗；同時利用能捕捉自由基之活性氧原子的細胞色素c (cytochrome c)，檢測含有外加D-乳酸之腎臟均質液與控制組相較，發現有大量自由基的生成 (145% vs. 100% as control)，進而推論糖尿病中，堆積在腎臟無法排除之D-乳酸會使氧壓 (oxidative stress) 增加，漸進對腎臟造成傷害，造成糖尿病的腎性病變 (diabetic nephropathy)。

關鍵字

糖尿病 ； 腎臟 ； D-乳酸 ； 糖質新生 ； 細胞色素 c ； 自由基

並列摘要

Abstract Renal gluconeogenesis has been stressed in diabetes mellitus, as excess glucose is released into the circulation and hyperglycemia is intensified. The main gluconeogenic precursor in kidney is thought to be lactate; however, less is emphasized enantiomerically. L-lactate is a glycolysis end-product, but D-lactate is formed after detoxification of methylglyoxal, which is the main source of advanced glycation end-products. For investigating the complete metabolism and the physiological role of D-lactate, we measured D-lactate levels in normal and diabetic rat kidney homogenates by our established column-switching HPLC method with fluorescence detection. The influence of D-lactate on gluconeogenesis was also estimated by determining the glucose concentrations in D- or L-lactate added in kidney homogenates. Furthermore, the relation between D-lactate and free radicals was determined by cytochrome c with a UV spectrophotometer at 550 nm. This study indicated that D-lactate concentrations in rat kidney were significantly and time-dependently accumulated in diabetic groups after induced for 1, 2, 3, 4 months (2.99, 13.11, 18.19, 23.23 ?mol/mg, respectively), as comparing that in normal groups (0.79 ?mol/mg). In addition, the histology of induced 3-month diabetic rat renal showed some structural changes of progressive diabetic nephropathy. Moreover, 80% of glucose released by addition of 6.0 mM of L-lactate (17.24 ?g/ml) was suppressed when 6.0 mM of D-lactate (3.47 ?g/ml) was supplied into rat kidney homogenates. It was supposed that D-lactate inhibited gluconeogenesis as an antagonist of L-lactate in rat kidney. On the other hand, the accumulation of D-lactate maybe damage the renal by generating the reactive oxygen species, in which it was determined by cytochrome c with external 6.0 mM D-lactate in rat kidney homogenates.

並列關鍵字

diabetus mellitus ； kidney ； D-lactate ； gluconegenesis ； cytochrome c ； free radicals

參考文獻

Ahmed MU, Frye EB, Degenhardt TP, Thorpe SR, and Baynes JW. (1997) N?-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochemistry Journal, 324: 565-570.

Allen DA, Harwood SM, Varagunam M, Raftery MJ, and Yaqoob MM. (2003) High glucose-induced oxidative stress causes apoptosis in proximal tubular epithelial cells and is mediated by multiple caspases. The FASEB Journal, 17:908-910.

Bari L, Atlante A, Guaragnella N, Principato G, and Passarella S. (2002) D-lactate transport and metabolism in rat liver mitochondria. Biochemistry Journal, 365:391-403.

Baynes JW and Thorpe SR. (1999) Role of Oxidative Stress in Diabetic Complications. Diabetes, 48:1-9.

Bongaerts G, bakkeren j, Severijnen R, Sperl W, Willems HM, Naber T, Wevers R, Meurs A, and Tolboom J. (2000) Lactobacilli and acidosis in children with short small bowel. Journal of Pediatric Gastroenterology and Nutrition, 30: 288-293.