AIMS/HYPOTHESIS Progressive decline in functional beta cell mass is central to the development of type 2 diabetes. Elevated serum levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) are associated with beta cell failure in type 2 diabetes and eNAMPT immuno-neutralisation improves glucose tolerance in mouse models of diabetes. Despite this, the effects of eNAMPT on functional beta cell mass are poorly elucidated, with some studies having separately reported beta cell-protective effects of eNAMPT. eNAMPT exists in structurally and functionally distinct monomeric and dimeric forms. Dimerisation is essential for the NAD-biosynthetic capacity of NAMPT. Monomeric eNAMPT does not possess NAD-biosynthetic capacity and may exert distinct NAD-independent effects. This study aimed to fully characterise the structure-functional effects of eNAMPT on pancreatic beta cell functional mass and to relate these to beta cell failure in type 2 diabetes. METHODS CD-1 mice and serum from obese humans who were without diabetes, with impaired fasting glucose (IFG) or with type 2 diabetes (from the Body Fat, Surgery and Hormone [BodyFatS&H] study) or with or at risk of developing type 2 diabetes (from the VaSera trial) were used in this study. We generated recombinant wild-type and monomeric eNAMPT to explore the effects of eNAMPT on functional beta cell mass in isolated mouse and human islets. Beta cell function was determined by static and dynamic insulin secretion and intracellular calcium microfluorimetry. NAD-biosynthetic capacity of eNAMPT was assessed by colorimetric and fluorescent assays and by native mass spectrometry. Islet cell number was determined by immunohistochemical staining for insulin, glucagon and somatostatin, with islet apoptosis determined by caspase 3/7 activity. Markers of inflammation and beta cell identity were determined by quantitative reverse transcription PCR. Total, monomeric and dimeric eNAMPT and nicotinamide mononucleotide (NMN) were evaluated by ELISA, western blot and fluorometric assay using serum from non-diabetic, glucose intolerant and type 2 diabetic individuals. RESULTS eNAMPT exerts bimodal and concentration- and structure-functional-dependent effects on beta cell functional mass. At low physiological concentrations (~1 ng/ml), as seen in serum from humans without diabetes, eNAMPT enhances beta cell function through NAD-dependent mechanisms, consistent with eNAMPT being present as a dimer. However, as eNAMPT concentrations rise to ~5 ng/ml, as in type 2 diabetes, eNAMPT begins to adopt a monomeric form and mediates beta cell dysfunction, reduced beta cell identity and number, increased alpha cell number and increased apoptosis, through NAD-independent proinflammatory mechanisms. CONCLUSIONS/INTERPRETATION We have characterised a novel mechanism of beta cell dysfunction in type 2 diabetes. At low physiological levels, eNAMPT exists in dimer form and maintains beta cell function and identity through NAD-dependent mechanisms. However, as eNAMPT levels rise, as in type 2 diabetes, structure-functional changes occur resulting in marked elevation of monomeric eNAMPT, which induces a diabetic phenotype in pancreatic islets. Strategies to selectively target monomeric eNAMPT could represent promising therapeutic strategies for the treatment of type 2 diabetes.

中文翻译：

---

高浓度eNAMPT中的结构功能变化可介导2型糖尿病中的小鼠和人类β细胞功能障碍。

目的/假设功能性β细胞量的逐渐下降对于2型糖尿病的发展至关重要。血清中较高水平的细胞外烟酰胺磷酸核糖基转移酶（eNAMPT）与2型糖尿病的β细胞衰竭有关，eNAMPT免疫中和可改善糖尿病小鼠模型的葡萄糖耐量。尽管如此，有关eNAMPT对功能性β细胞质量的影响的了解仍很少，一些研究单独报告了eNAMPT对β细胞的保护作用。eNAMPT以结构和功能上不同的单体和二聚体形式存在。二聚化对于NAMPT的NAD生物合成能力至关重要。单体eNAMPT不具有NAD生物合成能力，并且可能发挥不同的NAD非依赖性作用。这项研究旨在全面表征eNAMPT对胰腺β细胞功能量的结构功能作用，并将其与2型糖尿病的β细胞衰竭相关。方法：来自未患有糖尿病，空腹血糖受损（IFG）或患有2型糖尿病（来自体脂，外科手术和激素[BodyFatS＆H]研究）或患有2型糖尿病或具有2型糖尿病风险的肥胖人类的CD-1小鼠和血清本研究使用了糖尿病（来自VaSera试验）。我们生成了重组野生型和单体eNAMPT，以探索eNAMPT对分离的小鼠和人类胰岛中功能性β细胞质量的影响。通过静态和动态胰岛素分泌和细胞内钙微荧光测定法确定β细胞功能。eNAMPT的NAD生物合成能力通过比色法和荧光法以及天然质谱法进行评估。胰岛细胞数通过胰岛素，胰高血糖素和生长抑素的免疫组织化学染色确定，胰岛凋亡通过caspase 3/7活性确定。通过定量逆转录PCR确定炎症标志物和β细胞身份。使用非糖尿病，糖耐量异常和2型糖尿病患者的血清，通过ELISA，western blot和荧光分析法评估了总的，单体的和二聚的eNAMPT和烟酰胺单核苷酸（NMN）。结果eNAMPT对β细胞功能质量发挥双峰作用以及浓度和结构功能依赖性。在低生理浓度（〜1 ng / ml）下，如未患糖尿病的人的血清中所见，eNAMPT通过NAD依赖性机制增强β细胞功能，这与eNAMPT作为二聚体存在是一致的。但是，随着eNAMPT浓度升高到约5 ng / ml（如在2型糖尿病中），eNAMPT开始采用单体形式并介导β细胞功能异常，β细胞同一性和数量减少，α细胞数量增加以及凋亡，通过NAD-独立的促炎机制。结论/解释我们已经表征了2型糖尿病中β细胞功能异常的新机制。在低生理水平下，eNAMPT以二聚体形式存在，并通过NAD依赖性机制维持β细胞功能和同一性。但是，随着eNAMPT水平的升高（如2型糖尿病），结构功能发生变化，导致单体eNAMPT明显升高，在胰腺胰岛中诱导糖尿病表型。选择性靶向单体eNAMPT的策略可能代表了治疗2型糖尿病的有前途的治疗策略。