Department of Physiology, Michigan State University, East Lansing, MI, USA.
Department of Ophthalmology and Visual Sciences, School of Medicine, The University of Alabama at Birmingham, Birmingham, AL, USA.
Diabetologia. 2021 Jul;64(7):1674-1689. doi: 10.1007/s00125-021-05431-5. Epub 2021 Mar 26.
AIMS/HYPOTHESIS: Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage.
The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as liver X receptor alpha [LXRα]) acetylation was measured via western blot analysis.
IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression.
CONCLUSIONS/INTERPRETATION: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.
目的/假设：智人是在间歇性食物供应和两餐之间长时间禁食的条件下进化而来的。禁食期对于从膳食引起的氧化和代谢应激以及组织修复中恢复是很重要的。在当今社会，持续的高能量密度食物供应导致了慢性疾病的发病机制，包括糖尿病及其并发症，间歇性禁食（IF）和能量限制已被证明可以改善代谢健康。我们之前已经证明，IF 可预防 2 型糖尿病（db/db）小鼠模型中糖尿病性视网膜病变的发展；然而，禁食引起的健康益处和糖尿病患者禁食引起的风险的机制在很大程度上仍然未知。组蛋白去乙酰化酶 SIRT1 是一种营养感应酶，在糖尿病中下调。在这项研究中，通过 IF、模拟禁食的细胞培养条件（FMC）或使用 SRT1720 进行的药理学处理来评估 SIRT1 刺激对全身和视网膜代谢、全身和视网膜炎症以及血管和骨髓损伤的影响。
在 db/db 小鼠体内和牛视网膜内皮细胞或大鼠视网膜神经胶质/前体细胞 R28 血清饥饿 24 小时的体外模型中模拟 IF 的作用。通过定量 PCR（qPCR）分析 mRNA 表达。通过组蛋白去乙酰化酶活性测定法测量 SIRT1 活性。通过 Western blot 分析测量 NR1H3（也称为肝 X 受体 alpha [LXRα]）乙酰化。
与非禁食动物相比，IF 增加了小鼠肝脏和视网膜中的 Sirt1 mRNA 表达。IF 还使小鼠视网膜中的 SIRT1 活性增加了 8 倍，而 FMC 使视网膜内皮细胞中的 SIRT1 活性和表达增加与对照组相比。在神经元视网膜祖细胞（R28）中进行 FMC 处理后，Sirt1 的表达也增加了两倍。此外，FMC 导致 SIRT1 介导的 LXRα脱乙酰化，随后活性增加 2.4 倍，这可以通过编码 ATP 结合盒转运蛋白（Abca1 和 Abcg1）的基因的 mRNA 表达增加来衡量。当测试表达组成型乙酰化 LXRα 突变体的视网膜内皮细胞时，这些变化减少了。SIRT1/LXR/ABC 介导的胆固醇外排增加导致视网膜内皮细胞胆固醇水平降低。SRT1720 直接激活 SIRT1 导致 db/db 小鼠中糖尿病引起的炎症减少两倍，并改善了糖尿病引起的视觉功能障碍，这可以通过视网膜电图和光动反应来衡量。在骨髓中，预防了糖尿病引起的骨髓增生和减少了炎症细胞因子的表达。
结论/解释：总之，通过 IF 或通过药理学激活 SIRT1 信号转导代表了一种有效的治疗策略，它为与禁食方案相关的有利影响和预防糖尿病中小血管和骨髓功能障碍之间提供了机制联系。
