| 摘要: | 背景：生物能源，基本上是單個細胞的重要維持整個代謝功能的核心。藉由線粒體ATP的合成，可以使得維持生物體分解及合成養分，例如代謝物轉換，神經傳導和許多其他生物能源需要處理的過程。當微環境改變、基因突變或是外來損傷時，導致生物能源轉換功能失衡，是否會引發器官和生物個體病理變化，進而產生疾病呢？吾人乃以三大主題：營養因素、粒線體功能失衡、及損傷因素，來探討在生物能源失衡時可能產生之病理變化並探討其可能機轉及藥物治療之可能性。
方法及範圍：首先，探討於過多營養微環境下對細胞代謝之影響：利用HepG2細胞於高糖及高脂肪的培養環境中，利用油紅O染色觀察所產生之脂肪小滴(Lipid droplets，LD)，探討對細胞存活率、脂肪甘油三酯脂肪酶(ATGL)活性之表現，並嘗試利用硫辛酸(α-lipoic acid)來影響脂肪小滴堆積。第二主題，在脂質代謝步驟中與基因之缺損之相關性：吾人利用一家族肌肉病變患者，我們針對樣本進行組織病理染色檢查、電子顯微鏡的超微結構檢查、血漿中脂肪代謝物分析, 以及基因突變分子診斷。利用基因定序確立突變位點並利用週邊血液所製成淋巴母細胞，偵測其細胞內ATP之產生及粒線體膜電位之改變。 最後，探討神經與肌肉細胞之間對生物能量的交互影響：吾人利用老鼠小腿之腓腸肌及比目魚肌在切斷坐骨神經後之去神經損害時，利用組織化學染色、蛋白質定量、海馬生物能量測定器，探討共同活化因子PGC-1α(peroxisome proliferators-activated receptor γcoactivator 1α)在肌肉內之表現，其肌肉內粒線體內糖解及呼吸鏈改變，並試由PGC-1α活化物（pyrroloquinoline quinone, PQQ）矯正，看是否會減緩去神經時造成肌肉萎縮程度。
結果摘要：
第一主題：高糖高脂肪微環境下確實可以增加細胞內脂肪小滴數目，硫辛酸不僅可抑制細胞內的脂肪合成途徑，亦可強化ATGL活性，經由AMPK 途徑來分解LDs。說明ATGL不僅在脂肪組織LDs降解中發揮重要的作用，同時在非脂肪細胞的脂肪代謝中扮演著重要角色。第二主題：病理檢驗發現肌肉細胞中有過量脂肪小滴(lipid droplets)堆積，基因定序證實為多發性醯基輔酶A去氫酶缺乏症 (Multiple Acyl-CoA Dehydrogenase Deficiency, MADD) 。我們發現點位異常 c.250G>A於ETFDH基因的第三外顯子。此淋巴母細胞經過脂肪酸的添加培養，會造成ETFDH表現量的下降、ATP生成的不足和粒線體膜電位的減低。第三主題：去坐骨神經損傷時，對小鼠之後腿肌肉可造成之肌纖維型態之轉型(fiber-type shifting)，及肌細胞氧化磷酸化能力受損；投與PQQ後，可刺激PGC-1α生成、活化粒線體內電子傳遞（electron transport chain, ETC）複合體活性、並進而減緩肌肉細胞之萎縮。
結論：
經由吾人對營養因素、粒線體功能失衡及損傷因素之實驗探討得知：生物能量學可以幫助我們對疾病的病理機制有新的理解，提供新的治療策略的發展，並作為疾病生物標誌物的進展。
Background: The term bioenergy is basically the important critical role to maintain the entire single cell metabolic function. With mitochondrial ATP synthesis, it can make the maintenance of biological decomposition and synthesis of nutrients, such as metabolites conversion, nerve conduction and many other bioenergy processes. Whether will they lead to pathological changes in individual organism to produce disease when the micro-environmental changes, mitochondrial gene mutations or external damage resulting in biological energy conversion function imbalance? We denominated three themes: nutritional factors, mitochondrial function imbalance and injury factors, to explore the pathological changes may occur when the bioenergetic imbalances and to explore the possible mechanisms of pathophysiology and the possibility of drug treatment.
Materials & Methods: First, investigate under the influence of excessive nutrition in the micro-environment for the cell metabolism. The HepG2 cells were cultured in high glucose and high fat environment. The Oil-red O staining was for observing the lipid droplet formation. The cell viability and adipose triglyceride lipase (ATGL) activity were observed. And try to affect the accumulation of lipid droplets via α-lipoic acid (α-LA). The second theme, we explored the metabolic dysfunction for the lipid droplets due to the gene defect. The tissue samples were conducted histochemical stain, western blot, electromicroscopic examination and blood fatty metabolite analysis. Using gene sequencing to establish the mutation site and examined the ATP production and mitochondrial membrane potential in the peripheral blood leukocytes culture system. Finally, we explored the interconnection between nerve and muscle cells in bioenergetics. We designed the injury model via cutting the sciatic nerve. The hindlimbs (gastrocnemius and soleus muscles) were sampled for histochemical staining, protein quantification, and measured the energetic usage via Seahorse XF-24-Analyzer. The co-master factors, peroxisome proliferators-activated receptor γ coactivator 1α (PGC-1α) expression and the glycolytic function and respiratory chain changes within the mitochondria were evaluated. We administered of PGC-1α activator (pyrroloquinoline quinone, PQQ) via subcutaneous osmotic pump continuously to examine the extent of muscle atrophy after denervation.
Results: The first theme demonstrated that high-sugar high-fat microenvironment could indeed increase the number of lipid droplets within cells. α-lipoic acid not only inhibited fat synthesis pathway inside the cell, but also could strengthen ATGL activity via AMPK pathway to decompose LDs. ATGL plays an important role in the degradation of LDs, in adipose tissue and in non-adipose cells. The second theme showed excess LDs accumulation in the skeletal muscle cells. According the gene sequencing confirmed the diagnosis, multiple acyl-coenzyme A dehydrogenase deficiency (Multiple Acyl-CoA Dehydrogenase Deficiency, MADD). We found the homozygous mutation c.250G>A on the exon 3. The decline in the expression of ETFDH, the decrease of ATP production, and reduction the mitochondrial membrane potential were found in the lymphoblastoid cell culture. The third theme was explored the sciatic nerve injury model. The hindlimbs’ muscle fiber-type patterns were showed on the transition (fiber-type shifting), and impaired oxidative phosphorylation. After administration of PQQ, PGC-1α generation was restored; mitochondrial electron transfer (electron transport chain, ETC) complex activity was activated in vivo; thus, slowed down the atrophy of skeletal muscle.
Conclusion: Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression. |
