探討miR-137和高脂飲食對食慾的影響

Abstract

肥胖的根本原因是卡路里的攝入超過消耗，使能量以脂質的形式在脂肪細胞中堆積。控制食慾使身體消耗量大於攝入量，是緩解肥胖的方式之一。本次研究以食慾為出發點，探討高脂肪食物（High-fat diet; HFD）與基因改變對小鼠食慾的影響。 小分子核醣核酸（Micro RNA, miRNA）是一段長度約為22個核苷酸的RNA分子，目前已被證實可以透過和目標基因的結合影響其表達。本次研究所涉及的miR-137在腦部具有很高的表現量，在先前實驗室探討miR-137全身剔除小鼠（whole-body homozygous miR-137 knockout mice）代謝的過程中，觀察到在給予小鼠HFD後，miR-137+/-小鼠體重上升較多，其進食量也比對照組（miR-137+/+）更高，由此我們猜測miR-137與食慾有著一定的關聯性。由於miR-137-/-小鼠具有早夭的特性，實驗以miR-137+/-小鼠為主，以一般飼料（Regular diet; RD）飼養到六周大成鼠時，再依據實驗設計給予HFD餵食三天，觀察到miR-137+/-小鼠相較於對照組（miR-137+/+）體重上升更快，進食量更多，其腦中的促食慾基因神经肽Y （Neuropeptide Y; NPY）也有上升。初步證實了miR-137與食慾的關聯性。 另一方面，我們試圖探討高脂肪飲食對食慾的影響。首先我們將野生型（wild-type; WT）小鼠分為兩組，分別用RD和HFD餵養，共餵養六週。觀察發現長期食用HFD的組別體重增長較多，進食量較少，但其食慾相關基因並無明顯變化。我們推測可能是因為少量的HFD即可滿足WT小鼠的日常能量需求，所以進食量減少，由此將後續實驗時間改為三天。 接下來我們將WT小鼠分為四組，第一組給予三天RD，第二組給予三天HFD，第三組給予14天HFD，第四組先用HFD飼養三天，後改用RD再飼養三天。記錄小鼠實驗前後的進食量對比發現在換HFD的當天小鼠進食量會明顯增加，隔天則下降至初始水平或更低。實驗結束後萃取小鼠腦中下視丘、海馬迴、杏仁核的RNA進行RNA測序（RNA Sequencing; RNA-Seq）。利用生物路徑分析軟體（Ingenuity Pathway Analysis; IPA）和基因富集分析（Gene Set Enrichment Analysis; GSEA）進行分析後，發現Th17 activation pathway裡的RAR Related Orphan Receptor A（RORA）可能為miR-137的目標基因，且它可能會透過白细胞介素1β（Interleukin 1 beta; IL1B）去間接的活化食慾抑制基因前脑啡黑细胞促素皮促素（Pro-opiomelanocortin; POMC）。最後我們利用免疫熒光染色發現了在長期食用HFD的小鼠腦部下視丘中有Th17 cell增多的現象。 以上結果顯示，在活體中miR-137的減少影響了小鼠對高脂肪食物的食慾；而高脂肪飲食可能會透過免疫相關的路徑去調控位於下視丘的食慾相關神經元。

Obesity is caused by excess calories gained over calories expended, allowing energy to accumulate in fat cells as lipids. One method of reducing obesity is to control appetite so that the body consumes more than it gains. This study aimed to investigate the effects of a high-fat diet and genetic changes on appetite in mice. MicroRNA (miRNA) is a 22-nucleotide-long RNA molecule that has been shown to influence gene expression by binding to target genes.miR-137 in this study has a high level of expression in the brain. Previously, after being fed by HFD, mice with heterozygous (miR-137+/-) gained more body weight and ate more food than the control group (miR-137+/+), indicating that miR-137 is related to appetite. Due to the early death of knockout mice, miR-137+/- mice were primarily utilized in the experiment. According to the experimental design, adult mice were fed regular diet until they were six weeks old before being given HFD for three days. Heterozygous mice gained more weight and ate more food than the control group (miR-137+/+). They also had higher appetite-stimulating gene neuropeptide Y (NPY) levels. The association between miR-137 and appetite has been tentatively confirmed. On the other hand, we attempted to investigate the impact of high-fat diet on appetite. First, wild-type mice were divided into two groups and fed RD and HFD for six weeks. The group which ate HFD gained more weight and consumed less food, but there was no significant change in appetite-related genes. We hypothesized that a small amount of HFD could meet the daily energy demand of WT mice, so food intake was reduced, and thus the follow-up experiment time was reduced to three days. WT mice were then divided into four groups as follows: the first group was fed RD for three days, the second group was fed HFD for three days, the third group was fed HFD for 14 days, and the fourth group was fed HFD for three days followed by RD for three days. The mice's food consumption was recorded before and after the experiment. It was discovered that the food intake of mice increased significantly on the day of the HFD change and then decreased to the initial level or lowered the following day. Following the experiment, RNA was extracted from the hypothalamus, hippocampus, and amygdala for RNA sequencing. Using Ingenuity Pathway Analysis and Gene Set Enrichment Analysis, it was discovered that RAR Related Orphan Receptor A (RORA) in the Th17 activation pathway might be the target gene of miR-137. It may also indirectly activate the appetite suppressor gene Pro-opiomelanocortin (POMC) via Interleukin 1 beta (IL1B). Finally, we found an increase in Th17 cells in the brain's hypothalamus of mice consuming long-term HFD using immunofluorescence staining. These findings demonstrated that decreasing miR-137 in vivo affected mice's appetite for high-fat foods. On the other hand, high-fat diet may regulate appetite-related neurons in the hypothalamus via an immune-related pathway, which needs to be confirmed in future research.