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標題: | 克弗爾分離菌株Lactobacillus kefiranofaciens M1 及 Lactobacillus mali APS1 對於飲食誘導肥胖影響之研究 The study of Lactobacillus kefiranofaciens M1 and Lactobacillus mali APS1 on diet-induced obesity |
作者: | Yu-Chun Lin 林幼君 |
指導教授: | 陳明汝(Ming-Ju Chen) |
關鍵字: | 肥胖,克弗爾,Lactobacillus kefiranofaciens M1,Lactobacillus mali APS1,腸道菌相,代謝物組, Obesity,Kefir,Lactobacillus kefiranofaciens M1,Lactobacillus mali APS1,Gut microbiome,Metabolome, |
出版年 : | 2019 |
學位: | 博士 |
摘要: | 肥胖與過重的流行率在過去10年內於全球快速地增加,特別是飲食所引起的菌相失衡(dysbiosis),會導致肥胖及相關代謝併發症。然而,利用益生菌株作為改善菌相平衡與調節免疫作用,是改善飲食肥胖的可行策略之一。過去本研究室篩選鑑定之克弗爾分離乳酸菌株 Lactobacillus (L.) kefiranofaciens M1(以下簡稱M1)及 L. mali APS1(以下簡稱APS1),已證實具有改善發炎及促進免疫平衡等益處,因此我們假設此二菌株,具有改善菌相失衡引起之肥胖與代謝病症之潛力,故本研究聚焦以高脂飲食建立肥胖小鼠動物模式,評估 M1 及 APS1 乳酸菌對於飲食引起肥胖之主因,包括慢性發炎反應、腸道菌相組成及能量代謝平衡等三個層面之影響與機制進行探討。為了測試這個假設,試驗將小鼠區分為4個組別包括對照組(normal diet, ND + saline)、高脂飼糧處理組(high fat diet, HFD + saline)、HFD M1處理組(HFD + 5x108 CFU/mL M1)及 HFD APS1 處理組(HFD + 5x108 CFU/mL APS1)進行試驗。首先,我們發現在同樣具有免疫平衡作用的受試菌株,對於代謝之調控卻產生相反結果。其中 APS1 處理組之小鼠,在高脂飲食的攝取下,可有效延緩體重與體脂肪增加,與正常飲食對照組之小鼠無顯著差異。APS1 處理亦維持周邊組織對於胰島素作用之敏感性,有效改善胰島素阻抗與可能誘發之第二型糖尿病等代謝疾病。反之,M1 處理組之小鼠在體重上卻有相反之表現。
為探討相關原因,我們深入了解此二菌株對於脂肪生成作用,以及發炎反應與代謝調控。在影響脂肪分化的作用上,於高脂飲食誘導肥胖小鼠脂肪組織內發現,APS1處理可影響脂肪分化相關轉錄因子之表現,包括抑制過氧化物酶體增殖物啟動受體γ(peroxisome proliferator activated receptor gamma, PPAR-γ)表現,使下游脂肪生成功能基因如脂肪酸合成酶複合體(fatty acid synthase, FAS)及脂肪酸結合蛋白(fatty acid binding protein-4 , FABP-4)等表現量下降。然而,M1 處理則觀察到相反的結果,M1 不僅活化上述路徑之表現,在 3T3-L1 前身脂肪細胞分化之過程,亦觀察到細胞中出現較多的油滴累積,與動物試驗之結果相吻合。此外,我們在肥胖小鼠及 M1 處理小鼠之脂肪,觀察到高量的巨噬細胞表面抗原標記之基因表現(F4/80、CD68及CD11c),亦經由 F4/80 與 CD11c 染色觀察到肝臟組織中巨噬細胞的浸潤現象。進一步從血液循環中的蛋白質表現中發現,M1 雖與 APS1 處理同樣可降低循環中介白素-6(Interleukin-6, IL-6)及腫瘤壞死因子-α(tumor necrosis factor-α, TNFα)等前發炎反應細胞激素之生成,然而卻無法如同APS1處理,可誘發抗發炎因子如 介白素-10 (Interleukin-10, IL-10) 及 介白素-4 (Interleukin-4, IL-4) 上升,因此無法使脂肪組織內,促發炎型 M1 巨噬細胞(classically activated macrophage, M1)藉由 IL-10 活化,成為抗發炎 M2 巨噬細胞(alternatively activated macrophage, M2),進而產生胰島素作用出現胰島素阻抗(insulin resistance)症狀。 為了解上述現象是否為受試菌株直接調節腸道菌群,或是經由改變腸道微生物代謝物而引起,我們觀察 M1 與 APS1 介入對於腸道菌群組成及血液中代謝物組成之變化。試驗結果發現,APS1 降低厚壁菌門和擬桿菌門比例(Firmicutes / Bacteroidetes ratio, F/B ratio),亦增加Bacteroides與 Adlercreutzia菌種之表現,二菌種分別與與調節糞便中短鏈脂肪酸(short-chain fatty acids, SCFA)及能量代謝有關,共同影響食慾相關的荷爾蒙表現及能量代謝。然而M1處理組則增加 F/B 比例,亦增加肥胖相關之菌屬相對豐度如乳酸球菌屬(Lactococcus)、瘤胃球菌屬 (Ruminococcus)及 Dorea菌屬。APS1 及 M1 處理不僅改變腸道微生物相對豐度之表現,同時影響不同菌屬之間的交互作用;上述現象不僅與體增重累積呈現相關性,且不同處理之群組所產生之代謝物質具有明顯之區分,證實 M1 與 APS1 處理亦改變了宿主的代謝輪廓。在 M1 處理組中,S24_7 及 Erysipelotrichaceae 菌科與體重呈現負相關,亦與能量代謝必須之酯化肉鹼(esterified-L-carnitine)呈現正相關之表現。另外,Lachnospiraceae 及 Streptococcaceae 菌科高量的豐度表現,亦與慢性發炎指標及造成胰島素阻抗之胺基酸代謝物呈現正相關。 本研究證明 M1 與 APS1 對於飲食誘導肥胖具有不同調控之結果,後續為應用 APS1 可改善肥胖與胰島素阻抗等代謝症狀之特性,作為非傳統食品保健應用,我們根據Organization for Economic Cooperation and Development(OECD)規範針對 APS1 之安全性進行基因突變性、致畸性及餵食毒性等毒性測試等分析。試驗結果發現 APS1 無論是在沙門氏菌突變回復、體外哺乳類細胞染色體異常分析及囓齒類週邊血液微核試驗,皆無觀測到毒性反應; 此外,經口連續餵食懷孕雌鼠 10 日,並不會引發懷孕雌鼠毒性反應及影響胚胎發育; 而每日經口連續餵食大鼠 90 天,APS1 菌株之無毒性顯示劑量(no-observed-adverse-effect level; NOAEL) 均為大於 1.67 g/kg/day,且此劑量為人體建議攝取量之 100 倍。 綜觀上述,本研究成果證明 M1 及 APS1 菌株對於飲食誘導肥胖具有不同之調節影響。透過調節菌相組成及代謝物影響宿主生理代謝功能之完整研究,本研究提供了乳酸菌可作為改善飲食誘導肥胖之策略選項。然而乳酸菌之機能特性需定義至菌株,因此我們期望 APS1 菌株可發展為一種可應用於人類食品與動物保健之代謝調節補充食品。 An alarming increase in the prevalence of overweight and obesity worldwide has occurred in the past decade. Dietary changes are central drivers of obesity-associated microbiome. Probiotics generally recognized as safe for human use, have beneficial effects on improvement of obesity through manipulating gut microbiota composition. In our previous works, Lactobacillus mali APS1 (APS1) and L. kefiranofaciens M1 (M1), isolated and identified from sugary kefir grains and milk kefir, respectively, demonstrated the multiple health benefits, such as anti-inflammation, anti-allergy and anti-colitis activities. Since the features of anti-inflammation and intestinal protection of APS1 and M1 are positively related to anti-obesity, we hypothesized that APS1 and M1 could ameliorate the metabolic syndromes through modification of metabolites and reduction of inflammation by alternation of intestinal microbiota in the present study. To test the hypothesis, mice were treated with normal diet (ND) + saline, high fat diet (HFD) + saline, HFD + 5x108 CFU/mL M1 and HFD + 5x108 CFU/mL APS1 for 10 weeks. Surprisingly, these two strains exhibited completely opposite results in anti-obesity. APS1 intervention significantly reduced body weight gain, body composition, liver weight and size of adipocytes. APS1 also impaired glucose–insulin homeostasis to prevent insulin resistance. While, we unexpectedly found that M1 showed higher body weight gain and body fat than their HFD counterparts. To further investigate the mechanisms underlying these differences for both strains, we first identified the roles of M1 and APS1 on adipogenesis. Results indicated that administration of APS1 significantly reduced mRNA levels of adipogenic transcription factors (peroxisome proliferator activated receptor alpha (PPAR-γ) and de-novo lipogenesis pathway markers fatty acid synthase (FAS) and fatty acid binding protein 4 (FABP-4) in HFD mouse model. Whereas, up-regulation in the expression of adipogenic transcriptional cascade was observed in M1 group. Secondly, we examined inflammation status in serum and in adipose tissue. Results showed that mice fed with APS1 and M1 could significantly reduce the levels of TNF-α and Interleukin-6 (IL-6) when comparing with HFD group. Only APS1 group upregulated anti-inflammatory cytokines, Interleukin-10 (IL-10). Additionally, a higher level of classically activated macrophage (M1 ∅) expression in adipose tissue was observed in M1 group. Next, we studied how M1 and APS1 alternated adipogenesis pathway and inflammation status by examining fecal microbiota and serum metabolites. We found APS1 normalized the Firmicutes/Bacteroidetes (F/B) ratio and increased the proportion of genus Bacteroides, Adlercreutzia as well as increased a significant level of fecal SCFAs resulting to modulating appetite related hormone and energy expenditure. However, M1 remained in the proportion of a decrease in Bacteroidetes and an increase in Firmicutes caused by HFD, whereas increased the proportion of genus Lactococcus, [Ruminococcus] and Dorea. Moreover, APS1 and M1 not only changed bacterial composition, but also influenced on bacterial interactions, showing the correlation with body weight gain via modulating metabolomic relationships. We demonstrated the families Christensenellaceae and S24_7 negatively correlated with body weight gain in HFDM1 through increases in octanoyl-L-carnitine (OLC) and propionyl-L-carnitine (PLC) and OLC, the essential substrates for energy expenditure. Additionally, higher relative abundance of Lachnospiraceae, Streptococcaceae positively correlated with chronic inflammation and insulin resistance. For potential food application, safety assessment of APS1 on genotoxicity, mutagenicity, developmental toxicity and oral toxicology was performed according to the Organization for Economic Co-operation and Development (OECD) Guidelines. The results showed that APS1 did not exhibit mutagenic and genotoxic effects at any dose in our experiments. The data support the no-observed-adverse-effect level (NOAEL) for maternal treatment-related reproductive toxicity, fetal development and 90-day repeated dose oral exposure for the APS1 was 1,670 mg/kg b.w./day. An adult consumer weighting 60 kg would ingest 100.2 g of APS1 per day would be around 100-fold greater than the recommended dosage, which provides a basis for food product and dietary supplements use. Overall, the finding of this study proves that M1 and APS1exbited different modulation on diet-induced obesity. Our study provides an example of how probiotics could serve as a potential therapy for obesity by modulating physiological function through alternating microbiota. APS1 could be a novel potential probiotic supplement to ameliorate features of obesity in both human and animals in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21759 |
DOI: | 10.6342/NTU201900517 |
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顯示於系所單位: | 動物科學技術學系 |
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