發炎環境下下視丘POMC神經元的細胞分子變化之研究

Abstract

周邊或中樞系統的發炎已經被認為是許多神經系統疾病的重要成因。發炎對神經系統的影響可透免疫細胞分泌的細胞素影響神經細胞的功能。近年雖然有許多細胞素對神經細胞的研究,但神經細胞在發炎環境中產生的改變仍不完全清楚。大腦下視丘弓形核(arcuate nucleus, ARC)區域中會分泌POMC/CART神經元抑制食慾並提高能量消耗,是代謝調控中樞的重要成員。當下視丘長期發炎時,動物會出現食量增加以及代謝調控異常的現象,但其神經機制仍未完全清楚。本研究旨在探討發炎環境中POMC神經元中對代謝調控訊號的反應及粒線體的改變。這些改變在生理上會影響食物攝入調節、能量消耗、葡萄糖代謝與脂肪組織的分解或褐變(browning)。本研究主要分為四個部分,第一部分我們探討在mHypo-POMC/GFP1神經元中瘦素和胰島素信號傳導途徑,實驗顯示POMC神經元通過PI3K/AKT/mTOR路徑可整合來自瘦素和胰島素訊號,在刺激短時間內會激活AKT-mTOR路徑並利用mTOR來抑制AMPK路徑蛋白,也會提高POMC轉錄來維持代謝平衡,而長期則使負調節基因Socs3及Atg7表達上升,使細胞產生自噬作用來維持身體穩態。第二部分則探討在發炎因子的環境下是否會影響mHypo-POMC/GFP1神經元調控代謝的機制,實驗發現在長期發炎的環境下,POMC基因表現被抑制。另外,瘦素和胰島素刺激引起POMC神經元的PI3K-AKT路徑活化與AMPK的抑制,會在含有細胞因子及趨化因子環境下受到干擾,因此在低度發炎的環境下會干擾瘦素與胰島素調節POMC神經元的傳遞訊號。第三部分是探討在發炎環境下POMC神經元的粒線體有何改變,在含有細胞因子及趨化因子的發炎環境下,POMC神經元中的粒線體並未破壞,電子傳遞鍊基因表現及質子滲漏的結果並沒有差異,但細胞中ROS上升、呼吸速率下降、粒線體整體功能下降及解偶聯下降,粒線體的動力學變慢,裂變與融合下降,整體造成粒線體變大,粒線體變化會使POMC神經元的代謝調控能力受破壞。第四部分以高脂食物飼養小鼠做為肥胖及代謝疾病的模式,觀察肥胖下視丘中是否有發炎傾向,以及探討下視丘中的神經元粒線體調控,實驗餵食老鼠一個月的高脂肪飲食,使老鼠產生肥胖並引發全身性發炎,但血脂並未受影響,而在下視丘中也發現促發炎因子上升,這種體內環境會刺激POMC神經元的作用,並促進粒線體動力,包括生成、分裂與融合,試圖維持代謝的調控與平衡,但尚不了解更長時間的餵食對此代謝調控系統有何影響。總而言之我們的實驗結果顯示在發炎的環境下,對POMC神經元中的代謝調控與粒線體有所影響,至於對代謝調控的影響仍有待進一步研究。

Accumulating evidences have revealed modulatory roles of immune activities on normal physiology of central nervous system. Also, inflammation in the periphery is associated with neurological diseases through the functions of cytokines. The studies of cytokine actions on neurons were mainly focused on single specific cytokines, such as TNF-α, IL-6, etc, the influence of inflammatory environment, which contained mixtures of numerous cytokines, chemokines, and pro-inflammatory molecules secreted by innate immune cells, are not fully understood. POMC/CART neurons in the arcuate nucleus of the hypothalamus play crucial roles of regulating appetite, energy expenditure, and central control of metabolism. Chronic inflammation in the hypothalamus induced food intake dysregulation and abnormal metabolism. This study aimed to investigate of neural alterations induced by the inflammatory milieu. We conducted cellular studies by culturing hypothalamic POMC neurons with conditioned medium from LPS stimulated bone marrow derived macrophages, whereas high fat diet-fed mice were chosen for examining physiological impacts. We first examine the leptin and insulin signaling in mHypo-POMC/GFP cells. Short-term stimulation of leptin or insulin activates the PI3K-AKT-mTOR pathway downregulating AMPK and increases the expression of POMC gene, while long-term incubation upregulates the expression of negative-regulatory gene SOCS3 and autophagy gene ATG7 to maintain body homeostasis. The leptin and insulin signaling in POMC neurons were altered under inflammatory conditions. POMC gene expression is inhibited after long-term incubation with pro-inflammation cytokines. Also, and the activation of AKT and mTOR is relatively weakened after leptin and insulin stimulation. Furthermore, the metabolism-related cellular responses, such as mitochondrial functions and dynamics in POMC neurons, against proinflammatory stimuli, will be elucidated. Although intracellular ROS/RNS levels were elevated, the results of expressions of the electron transport chain genes and proton leakage suggested that mitochondria in POMC neurons remained undamaged. In addition, respiratory rate and overall mitochondrial function revealed a trend of decrease in the inflammatory environment. Inflammatory milieu induced significant decrease of mitochondrial uncoupling and slowed down mitochondrial dynamics by downregulating the expression of both fission and fusion genes. The balance of mitochondrial fission and fusion was also changed resulting in the enlargement of overall mitochondrial size. In summary, the mitochondrial alterations induced by inflammatory environment suggested mitochondrial functions and metabolic regulation abilities are disrupted in hypothalamic POMC neurons. Finally, we examined the metabolic regulations, inflammation status, arcuate functions and mitochondrial status in the high-fat diet-fed mice. Our data revealed that after feeding high-fat diet fed to C57BL/6 mice for four weeks, the testing developed obesity and systemic inflammation, but not metabolic dysregulation such as elevated blood triglycerides. The expression of metabolic regulating genes of arcuate POMC neurons indicated that the energy balance machinery in the arcuate nucleus remained functioning after 4-weeks of high fed diet feeding. Mitochondrial dynamics was promoted in the hypothalamus, suggesting the mitochondrial regulation in the calorie excessive conditions was not disrupted. These regulations could be altered in longer feeding of high fat diet for the development of further metabolic dysregulation, such as misbalance of glucose or lipid metabolic homeostasis. In conclusion, our results displayed the effects of inflammation milieu on metabolic regulation and mitochondrial functions in hypothalamic POMC neurons, but the physiological significance of these alterations needs further investigation.