Long-term intermittent fasting restores function of the gut–liver axis and prevents development of diabetic retinopathy in db/db mice. Mus musculus

Constant availability of food can contribute to the pathogenesis of metabolic syndrome and type 2 diabetes. Short term intermittent fasting (IF) can reset the central, light-entrained (suprachiastmatic nucleus) clock and also the peripheral, food-entrained (liver) clock to restore metabolic homeostasis in T2D. We asked if long term IF could prevent development of diabetic retinopathy (DR) in a type 2 diabetes model, the db/db mouse. After 7 months, IF corrected diabetes-induced increases in triglycerides, cholesteryl esters and diglycerides. IF protocol in db/db mice also prevented development of DR. In addition, host frequency and time of food intake affected the gut microbiome composition. IF led to decreased levels of Clostridiales and Akkermansia muciniphila in db/db mice and these changes in flora were accompanied by increased gut mucin, goblet cell number and villus length. Increased levels of Firmicutes in db/db mice on IF supported improved bile acid metabolism. To confirm that the restoration of bile acid function could contribute to the beneficial effects induced by IF on DR, the dual FXR/TGR-5 agonist INT-767 was administered to a second diabetes model, DBA2J mice injected with streptozotocin (STZ) and placed on Western diet (WD). In this model, INT-767 prevented development of DR. These findings support the concept that long-term IF mediates multiple beneficial effect by restoring the gut-liver axis homeostasis. Overall design: Liver samples from 3 db/db mice per group were collected at zeitgeber (ZT) time ZT9 (daytime) and ZT21 (nighttime) from both the diabetic ad-lib and IF groups and were used for microarray analysis. Total RNA was extracted using miRNeasy Mini kit (Qiagen, Valencia, CA) and standard procedures were used for hybridization, staining and scanning GeneChip® Mouse Gene 2.0 ST Arrays (Affymetrix, Santa Clara, CA). CEL files were analyzed with Expression Console software (Affymetrix) and the statistical analysis was performed with Transcriptome Analysis Console (Affymetrix) and in R (version 2.15.3). A linear model with covariates of long-term IF effect, ZT time effect and feeding/fasting effect was built and the goodness of fit between the full model and null model was tested by likelihood ratio test. False discovery rate (FDR) correction was conducted to adjust for multiple testing. In this study, the genes with FDR less than 0.3 was selected for further post hoc analysis. Only genes with p values for the comparasion between long term IF treatment and ad-lib less than 0.05 and absolute fold-change larger than 1.5 were selected as statistically significantly expressed genes.

持续的食物供给可促进代谢综合征与2型糖尿病的发病进程。短期间歇性禁食（intermittent fasting, IF）可重置受光调控的中枢生物钟（视交叉上核，suprachiasmatic nucleus）以及受食物调控的外周生物钟（肝脏），从而恢复2型糖尿病（T2D）模型中的代谢稳态。本研究旨在探讨长期间歇性禁食是否可在2型糖尿病模型db/db小鼠中预防糖尿病视网膜病变（diabetic retinopathy, DR）的发生。

干预7个月后，间歇性禁食可纠正糖尿病诱导的甘油三酯、胆固醇酯及甘油二酯水平升高。db/db小鼠的间歇性禁食方案同样可阻止糖尿病视网膜病变的进展。此外，宿主的进食频率与进食时间会影响肠道菌群的组成结构。在db/db小鼠中，间歇性禁食可降低梭菌目（Clostridiales）与嗜黏蛋白阿克曼氏菌（Akkermansia muciniphila）的丰度，同时伴随肠道黏蛋白、杯状细胞数量及肠绒毛长度的增加。接受间歇性禁食的db/db小鼠中厚壁菌门（Firmicutes）丰度升高，这一变化有助于改善胆汁酸代谢。

为验证胆汁酸功能恢复是否有助于介导间歇性禁食对糖尿病视网膜病变的有益作用，本研究向第二种糖尿病模型——经链脲佐菌素（streptozotocin, STZ）注射并饲喂西式饮食（Western diet, WD）的DBA/2J小鼠——给予双重FXR/TGR5激动剂INT-767。在该模型中，INT-767可阻止糖尿病视网膜病变的发生。上述研究结果支持以下结论：长期间歇性禁食可通过恢复肠-肝轴稳态发挥多重有益作用。

实验整体设计：分别在节律时间（zeitgeber, ZT）ZT9（日间）与ZT21（夜间），从糖尿病自由进食组与间歇性禁食组的每一组3只db/db小鼠中采集肝脏样本，用于芯片分析。使用miRNeasy Mini试剂盒（Qiagen, 美国加利福尼亚州巴伦西亚）提取总RNA，并按照标准流程进行杂交、染色及GeneChip®小鼠基因2.0 ST芯片（Affymetrix, 美国加利福尼亚州圣克拉拉）的扫描。使用Expression Console软件（Affymetrix）分析CEL文件，并通过Transcriptome Analysis Console（Affymetrix）及R语言（版本2.15.3）完成统计分析。构建包含长期间歇性禁食效应、节律时间效应及进食/禁食效应作为协变量的线性模型，并通过似然比检验比较全模型与零模型的拟合优度。采用错误发现率（false discovery rate, FDR）校正以处理多重检验问题。本研究选取错误发现率小于0.3的基因进行后续事后分析。仅选取长期间歇性禁食处理与自由进食组比较的P值小于0.05且绝对倍数变化大于1.5的基因，作为具有统计学显著性的差异表达基因。