【作者】 李梅；

【导师】 周中凯；

【作者基本信息】 天津科技大学 ， 食品科学与工程， 2021， 博士

【摘要】 代谢综合征正在成为21世纪的主要医疗保健问题之一,并且与易患胰岛素抵抗的代谢器官的病理紊乱以及2型糖尿病（T2D）和心血管疾病有关。T2D及其并发症是世界上最受关注的内分泌代谢疾病。它们的特点是脂质、葡萄糖代谢和胰岛素功效紊乱,最终导致全身稳态紊乱。肠道微生物群可能通过发酵难以消化的膳食成分产生短链脂肪酸（SCFAs）来影响代谢表型。本研究首先研究了不同SCFAs对体外肝脂肪变性模型脂质代谢的影响,其次,从SCFAs的生产特性出发,制备了不同取代度的酰化淀粉,并对同一取代度下不同SCFAs酰化淀粉的精细结构、消化特性、发酵特性及其对T2D的改善作用进行了研究。（1）研究SCFAs对游离脂肪酸（FFA）诱导的大鼠肝细胞（BRL 3A）脂肪变性细胞模型氧化应激、脂质积累和信号转导通路的影响。结果表明,SCFAs干预在增加超氧化物歧化酶（SOD）酶活水平的同时降低了丙二醛（MDA）、活性氧（ROS）和细胞甘油三酯（TG）的水平,且具有浓度依赖性。此外,SCFAs通过激活AMPK和PPAR信号通路抑制脂质合成并促进脂质氧化。更重要的是,与单独的乙酸盐或丙酸盐相比,SCFA的混合物(M_{Na Ac}:M_{Na Pr}:M_{Na Bu}=3:1:1)或单独的丁酸盐显著增强了对脂质积累的抑制作用,并且与其他组相比,SCFAs和Na Bu处理组之间观察到类似的差异表达基因谱,表明SCFAs（特别是丁酸盐）的存在对于调节脂质代谢具有重要作用。（2）研究不同SCFAs酰化对淀粉精细结构、消化和发酵特性的影响。核磁共振（NMR）光谱证实了酰基质子的存在,傅立叶变换红外（FTIR）光谱在1730 cm^-1处羰基C=O的伸缩振动进一步证实了酰化的发生。X射线衍射仪（XRD）结果表明,酰化破坏了淀粉的内部结构,为淀粉分子的重排提供了机会,从而在淀粉颗粒内部形成了更有序的聚集结构,这些多尺度结构的变化增加了抗性淀粉（RS）的形成并降低了消化过程中葡萄糖的产生。此外,酰化过程中引入的SCFAs可以在发酵过程中被肠道菌群有效释放,特异性增加了相应酰化SCFA的产量。（3）研究天然RS（高直链玉米淀粉,HAMS）和短链脂肪酸酰化淀粉（SCFAs-RS）对T2D大鼠肝功能、脂质成分和氧化应激的干预作用。酰化淀粉干预后,糖尿病大鼠器官肿大逆转,肝血脂显著降低,口服葡萄糖耐受不良得到改善。与HAMS相比,SCFAs-RS治疗可显著改善糖尿病大鼠的高血糖、高胰岛素血症、高脂血症和氧化应激。对于机制研究,我们发现SCFAs-RS通过增加胰岛素受体底物（IRS）、Akt、葡萄糖转运蛋白2（Glut2）的表达和降低核因子κB（NFκB）的表达对T2D大鼠的胰岛素抵抗表现出显著的保护作用。根据目前研究结果得出结论,SCFAs-RS通过激活NFκB和IRS-1/AKT/GLUT2通路具有潜在的抗糖尿病作用。（4）研究天然RS（HAMS）或SCFA-RS干预后肠道微生物群变化与T2D指数之间的相关性。结果表明,与HAMS相比,摄入SCFA-RS对T2D指标的改善作用更大,包括体重减轻、空腹血糖（FBG）降低、血清胰岛素水平减少和改善氨基酸代谢紊乱,证明了SCFAs和RS在肠道中双重功能的重要性。丁酸弧菌属（Butyricimonas）的丰度与血清FBG和糖化血清蛋白水平呈负相关性,10种细菌与HOMA-IR呈正显著性,这表明某些关键微生物群与胰腺功能有关。胰岛素敏感性的变化也可能与氨基酸代谢紊乱的改善有关。与天然HAMS相比,所有酰化淀粉显著增加了SCFAs产生菌的生长,并且这种变化与血清和粪便样品中相应的SCFAs浓度高度一致。此外,丙酰化淀粉（HAMSP）促进双歧杆菌（Bifidobacterium）丰度,而乙酰化淀粉（HAMSA）和丁酰化淀粉（HAMSB）有利于粪球菌属（Coprococcus）、丁酸单胞菌（Butyricimonas）和布劳特氏菌属（Blautia）富集,这可能表明它们的干预途径不同。总之,目前的研究证明,酰化淀粉干预在改善体重减轻、血糖控制和脂质代谢方面有效改善高脂饮食协同链脲佐菌素诱导的T2D,保护脏器组织免受氧化应激损伤。酰化淀粉的降血糖作用与抑制炎症反应和氧化应激,以及调节失衡的肠道菌群有关。我们的研究将促进对酰化淀粉在减轻血糖、炎症、氧化应激和代谢紊乱方面功效的理解,并为开发基于酰化淀粉的代谢紊乱治疗策略提供见解。更多还原

【Abstract】 Metabolic syndrome is becoming one of the major health-care problems of the twenty-first century and is associated with a wide spectrum of pathological disturbances in metabolic organs predisposed towards insulin resistance,as well as type 2 diabetes（T2D）and cardiovascular disease.T2D and its complications are the most concerned endocrine and metabolic diseases in the world.They are characterized by disorders of lipid,glucose metabolism and insulin efficacy,which ultimately lead to disorders of systemic homeostasis.The gut microbiota might affect the metabolic phenotype by fermenting indigestible dietary components and thereby producing short-chain fatty acids（SCFAs）.This study first studied the effects of different SCFAs on lipid metabolism in an in vitro liver steatosis model.Secondly,starting from the production characteristics of SCFAs,acylated starches with different degrees of substitution were prepared.The multi-scale structure,digestion characteristics,fermentation characteristics of acylated starches with different SCFAs under the same degree of substitution were studied,and their effects on T2D were studied.（1）The current study applied free fatty acid（FFA）-induced cell model of hepatic steatosis in an in vitro model of cultured rat hepatocytes（BRL 3A cells）,and measured the effects of SCFAs on oxidative stress,lipid accumulation and signal transduction pathways.The results indicated that a certain concentration of SCFAs increased the level of superoxide dismutase（SOD）and decreased the levels of malondialdehyde（MDA）,reactive oxygen species（ROS）,and cellular triglycerides（TG）,and these effects were characterized with concentration dependent.In addition,SCFAs inhibit the expression of lipid synthesis genes and up-regulate the expression of lipid oxidation-related genes by activating AMPK and PPAR signaling pathways.More importantly,either a mixture of SCFAs(M_{Na Ac}:M_{Na Pr}:M_{Na Bu}=3:1:1)or butyrate alone enhanced their corresponding inhibition of lipid accumulation compared to acetate or propionate,individually,and a higher in similarly differentially expressed genes profile was observed between SCFAs and Na Bu treatment groups compared to others,indicating the importance of the existence of butyrate for the regulation of lipids metabolism.（2）The effect of acylation with various short-chain fatty acids on starch fine structure,digestion and gut microbiota fermentation property was investigated.The presence of acyl protons was convinced by Nuclear magnetic resonance（NMR）spectra followed by the further support from Fourier transform infrared（FTIR）spectra with a carbonyl C=O vibration at 1730 cm^–1 into the acylated starch molecules.X-ray diffractometer（XRD）studies revealed that the acylation destroyed the internal structure for providing a chance of rearrangement of starch molecules,suggesting more ordered aggregation structures was formed inside the starch granules.Kinetics of in-vitro hydrolytic enzymatic model and Pearson correlation coefficients further confirmed the association between multi-scale structural order and digestion characters.Acyl groups introduced by acetylated,propionylated and butyrylated starch could be effectively released by the intestinal flora during the fermentation,specifically increasing their corresponding SCFAs production,respectively.（3）This study investigated the interventional effect of resistant starch（RS）,and acylated starch（SCFAs plus resistant starch,SCFA-RS）on liver function,lipid composition and oxidative stress in T2D rats.After acylated starch intervention,organ enlargement was reversed,liver lipids were significantly decreased,and oral glucose intolerance was improved in diabetic rats.Treatment with acylated starches（SCFAs-RS）significantly ameliorated hyperglycemia,hyperinsulinemia,hyperlipidemia,and oxidative stress in diabetic rats compared with HAMS.For the mechanism study,we have found that SCFAs-RS exhibited a significant protective effect on insulin resistance（IR）in T2DM rats through increasing expressions of insulin receptor substrate（IRS）,Akt,glucose transporters 2（Glut2）and decreasing the expressions of nuclear factor kappa-B（NFκB）.According to our present findings,we could conclude that SCFAs-RS possessed the potential anti-diabetic effects through activating NFκB and IRS-1/AKT/GLUT2 pathways.（4）This study investigated the correlations between the gut microbiota and type 2diabetes（T2D）indexes using either native resistant starch（from high amylose maize starch,HAMS）or acylated starch（SCFAs plus resistant starch,SCFA-RS）.The current results showed that,compared to HAMS,consumption of SCFA-RS achieved a greater impact on the improvement of T2D indexes in term of body weight loss,fasting blood glucose（FBG）,serum insulin level,and amino acid metabolism,indicating the importance of the dual function from both SCFAs and RS in the gut.The abundance of Butyricimonas was negatively significant with the level of serum FBG and glycated serum protein,and 10bacteria were positively significant with the HOMA-IR,which may indicate the association of some key microbiota with the pancreatic function.Insulin sensitivity may be related to the improvement of amino acids metabolism as well.All the acylated starch significantly enhanced the growth of SCFAs-producing bacteria compared to its native HAMS,and this change was highly consistent with their corresponding SCFAs concentration both in serum and fecal samples.The propionylated HAMS promoted the abundance of Bifidobacterium,while acetylated and butylated HAMS benefited the enrichment of Coprococcus,Butyricimonas and Blautia,which may indicate their different intervention pathway.In summary,the current study proves that acylated starch intervention is effective at improving HFD-STZ-induced T2D in term of the improved weight loss,glycemic control,and lipid metabolism,protected the tissue against oxidative stress injury.The hypoglycemic effects of acylated starch are related to the inhibition of the inflammatory response and oxidative stress,and the regulation of the imbalanced intestinal microbiota.Our research would promote the understanding of the efficacy of acylated starch in alleviating blood glucose,inflammation,oxidative stress,and metabolic disorders,and provide insights for the development of acylated starch-based treatment strategies for metabolic disorders.更多还原