【作者】 程杰；

【导师】 于晓； 孙金鹏；

【作者基本信息】 山东大学 ， 生理学， 2022， 博士

【摘要】 胰岛β细胞是体内唯一产生胰岛素的细胞,胰岛素约占胰岛β细胞总蛋白含量的10%。最近的研究表明,不仅功能完整的胰岛素,错误折叠的胰岛素经切割产生的胰岛素短肽也会在葡萄糖刺激下由胰岛β细胞中的囊泡释放到血液中。这些不同长度的胰岛素肽以及变性的胰岛素可以作为抗原触发胰岛自身免疫,这在功能上与1型糖尿病(Type 1 diabetesmellitus,T1D)的发病机制相关;同时2型糖尿病(Type2diabetesmellitus,T2D)由于胰岛素抵抗引起胰岛素需求增加,胰岛素肽水平也相应增加。值得注意的是,这些胰岛素肽片段与主要组织相容性复合体 2(major histocompatibility complex class Ⅱ,MHC-Ⅱ)分子的结合能力较差,具有微摩尔每升(μM)级别左右的亲和力,然而这些胰岛素肽在血浆中生理浓度却只有甚至不足纳摩尔每升(nM)级别。胰岛β细胞是否具有潜在的机制来感知这些低生理浓度的胰岛素肽片段并启动信号级联反应以调节胰岛稳态尚不清楚。此外,最近的证据表明,胰岛驻留型巨噬细胞的局部增殖在胰岛炎症和糖尿病发展中也起着关键作用。然而,胰岛对这一过程的调节在很大程度上未知。寻找胰岛分泌的胰岛素短肽的高亲和力受体(例如G蛋白偶联受体),并研究它们相应的功能,例如它们在胰岛素分泌中的作用以及胰岛β细胞与胰岛驻留巨噬细胞之间细胞通讯,将有助于阐明调节胰岛稳态的机制并对开发新的糖尿病治疗策略提供新的思路。哺乳动物通过嗅觉受体(Olfactory receptors,ORs)来感知气味信息。嗅觉受体是G蛋白偶联受体(G protein-coupled receptor,GPCR)的亚家族,并且是最大的哺乳动物基因家族之一。人体表达的826种GPCR中约有400种为嗅觉受体,主要表达在鼻粘膜上皮,负责将气味分子的化学信号转换为电信号,形成嗅觉感知。美国科学家Linda Buck和Richard Axel因为对嗅觉受体和嗅觉系统工作方式的发现而获得2004年诺贝尔生理学或医学奖。除了在气味感知中起主导作用外,近年来研究发现嗅觉受体在多种器官和组织中均有分布,并且在这些器官中发挥着不同的生理功能,例如介导精子趋化性、肌肉生成、癌细胞生长抑制、毛发生长、肝脂肪变性和脂肪组织脂肪分解等。然而,胰岛中嗅觉受体的基因表达谱和功能,以及对葡萄糖引起的胰岛素分泌与胰岛稳态的作用仍然未知。糖尿病相关的错误折叠的胰岛素切割产生的短肽以及胰岛素都可以由胰岛β细胞分泌。通过筛选胰岛中嗅觉受体的表达和定位,同时通过配体受体功能配对研究,我们将小鼠嗅觉受体Olfr109(对应人嗅觉受体OR12D3)确定为胰岛素短肽insB:9-23以及及变性胰岛素的受体。胰岛素肽insB:9-23通过激活Olfr109的Gi信号通路抑制胰岛素分泌;并通过激活β-arrestin1信号通路调控趋化因子2(C-C-motif-chemokineligand2,CCL2)的表达和分泌从而促进胰岛驻留巨噬细胞增殖,而且我们在β-arrestin-1-/-小鼠模型证明了这一点。在Pdx1-Cre+/-Olfr1 09fl/fl小鼠胰岛β细胞中特异性敲除Olfr109以及在Akita小鼠中敲除Olfr109显著改善了高脂喂养小鼠以及Akita小鼠的葡萄糖代谢和胰岛稳态。进一步的分子机制研究揭示了 insB:9-23和Olfr109之间的相互作用模式,同时开发了一种靶向Olfr109的特异性pepducin拮抗剂,能够显著改善肥胖小鼠和糖尿病模型小鼠的葡萄糖代谢。总的来说,我们发现了胰岛β细胞上嗅觉受体Olfr109可以作为质检蛋白,识别胰岛素肽及失活胰岛素,从而降低胰岛素分泌;在Akita小鼠或者长期高脂诱导的小鼠中,胰岛的代偿作用不能维持,过多的insB:9-23持续激活Olfr109会进一步降低葡萄糖引起的胰岛素分泌,并且促进胰岛驻留巨噬细胞的增殖,进而损害胰岛稳态和血糖稳态。更多还原

【Abstract】 Pancreatic islets are the only tissues in the body that produce insulin,and insulin accounts for about 10%of the total protein content of islet beta cells.Recent studies have shown that not only functionally intact insulin,but also short insulin peptides produced by cleavage of misfolded insulin are also released into the blood from vesicles in islet beta cells upon glucose stimulation.These insulin peptides of different lengths,as well as denatured insulin,can act as antigens to trigger islet autoimmunity,which is functionally related to the pathogenesis of type 1 diabetes(T1D);meanwhile,type 2 diabetes(T2D)increases insulin demand due to insulin resistance,and insulin Peptide levels also increased accordingly.Notably,the binding ability of these insulin peptide fragments to MHC II molecules is poor,with an affinity around micromolar(μM)level,but the physiological concentration of these insulin peptides in plasma is only even less than nanomolar(nM)level.Whether pancreatic beta cells have an underlying mechanism to sense these insulin fragments and initiate signaling cascades to regulate islet homeostasis is unclear.Furthermore,recent evidence suggests that local proliferation of islet-resident macrophages also plays a key role in islet inflammation and diabetes development.However,the mediation of this process by islets is largely unknown.Linking pancreatic islet secretions to their potential high-affinity receptors such as GPCRs and studying their corresponding functions,such as their role in insulin secretion and signaling between islet beta cells and islet-resident macrophages,will provide Help to elucidate the mechanism that regulates islet homeostasis and provide new ideas for the development of new anti-diabetic strategies.Mammals perceive odor information through olfactory receptors(ORs).Olfactory receptors are a subfamily of G protein-coupled receptors(GPCRs)and are one of the largest mammalian gene families.About 400 of the 826 GPCRs expressed in the human body are olfactory receptors,mainly expressed in the epithelium of the nasal mucosa,responsible for converting the chemical signals of odor molecules into electrical signals to form olfactory perception.American scientists Linda Buck and Richard Axel won the 2004 Nobel Prize in Physiology or Medicine for their discovery of olfactory receptors and how the olfactory system is organized.In addition to playing a leading role in odor perception,recent studies have found that olfactory receptors are distributed in a variety of organs and tissues,and play different physiological functions in these organs,such as mediating sperm chemotaxis,muscle production,Cancer cell growth inhibition,hair growth,liver steatosis and adipose tissue lipolysis,etc.However,the gene expression profile and function of olfactory receptors in pancreatic islets,as well as their effects on glucose-induced insulin and glucagon secretion,remaining unknown.Both diabetes-related insulin-cleaving peptides and insulin can be secreted by pancreatic beta cells.By screening the expression and localization of olfactory receptors(ORs)in pancreatic islets,we identified Olfr109(corresponds to human olfactory receptor OR12D3)as a receptor for insulin peptides and denatured insulin.The insulin peptide insB:9-23 reduces insulin secretion by activating the Olfr109 Gi signaling pathway,and promotes the proliferation of islet-resident macrophages through the β-cell-macrophage circuit and the β-arrestinl-mediated CCL2 pathway.The β-arrestin-1-/-mouse model demonstrates this.Islet-specific knockout of Olfr109 induced by Pdxl-Cre+/-Olfr109fl/fl and knockout of Olfr109 in Akita mice improved glucose metabolism and islet homeostasis in high-fat-fed mice as well as in Akita mice.In addition,we further determined the interaction mode between insB:9-23 and Olfr109,and further developed a specific pepducin antagonist targeting Olfr109,which can significantly improve glucose metabolism in obese mice and diabetic model mice.Overall,we found that the olfactory receptor Olfr109 on pancreatic β cells can act as a quality-checking protein to recognize insulin peptides early and inactivate insulin,thereby reducing insulin secretion.However,in long-term Akita or high-fat model mice,the compensatory effect of islets could not be maintained,and excessive insB:9-23 continued to activate Olfr109 to reduce insulin secretion and promote the proliferation of islet-resident macrophages to damage islets homeostasis and glucose homeostasis.更多还原