| BackgroundInsulin resistance-related diseases such as obesity and type 2 diabetes have become a global public health problem.Epidemiological investigation showed that the prevalence of diabetes in China has reached 10.4%,and the pre-diabetes state is as high as 35.7%.The relationship between environmental change and disease is receiving more and more academic attention,and the correlation between insulin resistance and the environment even exceeds its relationship with genetic susceptibility.Studies have found that biological clock disorders caused by light pollution,reduced sleep time and/or quality,and shift work can directly induce insulin resistance.And it is foreseeable that with the continuous advancement of China’s social industrialization,the national demand for 24h consumer services will continue to increase,and circadian rhythm changes will continue to prevail.However,its mechanism is unknown.Therefore,a targeted study of the impact of the biological clock on insulin sensitivity and the search for effective interventions will undoubtedly seek a new breakthrough for insulin resistance-related diseases.For a country like ours in a social transformation where biological clock disorders are widespread and insulin resistance-related diseases are spreading rapidly Is of great significance.In recent years,microRNAs(miRNAs)have received much attention in the role of affecting insulin sensitivity,and have been shown to be involved in many physiological processes such as regulating insulin production,secretion and regulating insulin target organ function.In addition,miRNAs present in exosomes can be used as signaling molecules to transmit signals to specific target organ cells.Exosome miR-155 derived from adipose tissue macrophages of obese mice can mediate the occurrence of insulin resistance,suggesting that exosome miRNAs can be used as therapeutic targets for insulin resistance-related diseases.It is worth noting that foreign studies have shown that expression of miR-140-5p,miR-185-5p,miR-328-p and miR-326-5p in the liver of mice in ZT14 group increased significantly compared with ZT2(Zeitgeber Time,the light-on time is recorded as ZT0),suggesting that miRNA expression has obvious rhythm.Based on this,we speculate that exosome miRNA may be a new mechanism and root cause of circadian clock disturbance mediated insulin resistance.In summary,we plan to use 24h continuous light to intervene in mice to build a mouse model of circadian clock disorder.Evaluate the activity patterns of mice throughout the day,the expression of skeletal muscle circadian core gene protein and mRNA and rhythm changes,so as to clarify the effect of continuous light on the integrity of the circadian clock system.At the same time,we further observed changes in skeletal muscle metabolism characteristics,lipid deposition and insulin sensitivity.In addition,mouse plasma exosomes were extracted for miRNA sequencing and bioinformatics analysis and preliminary verification,laying a foundation for further mechanism research and clinical development of intervention measures.Part Ⅰ Effects of constant light on skeletal muscle-fiber-type conversion and insulin sensitivity in miceObjectiveDisturbances in ambient light,decreased sleep time and/or quality,shift work,etc.,are closely related to insulin resistance.However,its specific mechanism has not yet been clarified.In this study,a 24-hour continuous light was used to successfully construct a circadian clock disorder model,to observe its effects on skeletal muscle metabolic characteristics and insulin sensitivity,and to explore its internal connections.MethodsIn this part,mice were intervened with different light,and randomly divided into the following two groups:normal light group(LD);24-hour continuous light group(LL).The activity patterns of mice were observed through CLAMS metabolic cages,and the changes of urine 6-MSL were detected by ELISA to determine the effect of continuous light on the integrity of the circadian clock system.The skeletal muscle is extracted every 4 hours,and the expression of skeletal muscle circadian core gene protein and mRNA and rhythm changes are observed.In addition,Western blot and Real-time PCR,tissue immunofluorescence and MRI were used to observe the changes of insulin sensitivity and skeletal muscle lipid deposition in each group of mice,and to evaluate the skeletal muscle lipid and glucose metabolism and changes of muscle fibers in each group of mice.Results1.Compared with LD group,the circadian rhythm characteristics of resting-activity mice in LL group were significantly degraded,and the cycle also changed,and the amount of daytime activity and nighttime activity increased and decreased respectively,and the difference was not statistically significant.Nighttime urine 6-SML level decreased,the difference was statistically significant.2.After 10 weeks of continuous light exposure,the expression levels and rhythms of Bmal1,Clock,Per2,Rev-erbα mRNA in the skeletal muscle core circadian clock genes of mice changed.3.After 10 weeks of continuous light exposure,the body weight of the mice in the LL group increased by 13%,and the serum free fatty acids,triglycerides,and cholesterol levels increased,the difference was statistically significant.4.The overall trend and area under the curve of the IPGTT and IPITT curves of mice under continuous light show that they are larger than those of the normal control group,the p-Akt/Akt level of skeletal muscle decreases and lipid deposition occurs.5.Immunofluorescence showed that the proportion of skeletal muscle slow-twitch fibers decreased and the proportion of fast-twitch fibers increased in the LL group.Real-time fluorescence quantitative PCR showed that the expression of slow-twitch fiber gene markers Myl2,Myh7 and TnnilmRNA in LL group decreased,while the expression of fast-twitch fiber gene markers Myh1mRNA increased.6.Compared with LD group mice,LL group mitochondrial biogenesis related genes PGC1α,Tfam,Cox2 and fatty acid oxidation key enzyme Fatp1,Cpt1b mRNA expression were significantly reduced.The expression levels of fatty acid synthase Fasn and glycolytic rate-limiting enzyme Pkfl mRNA were significantly increased compared with the control group.Conclusion1.Constant light damages the integrity of the circadian clock system in mice,and causes changes in the skeletal muscle circadian clock gene expression level and rhythm.2.Continuous light promotes ectopic lipid deposition and insulin resistance in skeletal muscle of mice.3.The skeletal muscle metabolic characteristics of mice under continuous light changes,muscle fiber type remodeling,slow-twitch fiber ratio decreases;skeletal muscle fatty acid oxidation function is impaired,fat synthesis and glycolysis function are enhanced.Part Ⅱ Effects of constant light on plasma Exo-miRNAs expression profiles in miceObjectiveCircadian disorders can mediate insulin resistance in mice,and Exo-miRNAs may play a key role in this process,but the mechanism is unknown.In this study,miRNA sequencing was performed on plasma exosomes in mice with circadian clock disorders.Exploring the changes of exosome miRNA expression profile under the background of circadian clock disorder and performing preliminary verification will lay the foundation for clarifying the specific mechanism of exosome miRNA in circadian clock disorder mediated insulin resistance.MethodsExosomes were extracted from the plasma of each group of mice.Exosomes specific proteins were detected by Western blot,morphological observation by fluoroscopy,and particle size distribution of exosomes by NTA method to verify whether the exosomes were successfully extracted.Exosome miRNA sequencing was performed,and GO analysis and KEGG Pathway analysis of differential miRNAs were performed by bioinformatics.Real-time PCR was used to verify the differentially expressed miRNAs.Results1.Western blot results showed that exosome-specific proteins CD9,CD63 and Alix were expressed in the LD and LL groups.In addition,under the transmission electron microscope,obvious sapodilla-like membranous vesicles with a diameter of 50～120 nm can be seen.NTA results suggest that the particle size distribution of the two groups is for 50～200 nm,and the average particle size is about 140 nm.2.Compared with LD group mice,there were 24 significant changes in plasma exosome miRNAs of LL group mice.Among them,there were 17 miRNAs that were significantly up-regulated and 7 that were significantly down-regulated.Further bi-cluster analysis of differentially expressed miRNAs revealed that the plasma exosomes secreted by the two groups of mice had good repeatability and obvious classification.3.The types of genetic biological processes targeted by differentially expressed miRNAs in LL-exosome are mainly enriched in biological processes,signal transduction and G protein coupled receptor signaling pathways.Most of the cell component types are enriched in membranes,membrane components and plasma membranes.The target genes in the molecular function category are mainly enriched in transferase activity,protein binding,and olfactory receptor activity.4.The KEGG analysis fed back a total of 71 related pathway information,of which the target genes were significantly enriched in the phosphatidylinositol signaling system,unsaturated fatty acid biosynthesis,FoxO signaling pathway,insulin signaling pathway,type 2 diabetes,pyruvate metabolism,AMPK and insulin resistance signaling pathways and other metabolic pathways.5.Continuous light up-regulates the expression of plasma exosomes and skeletal muscle miR-22-3p and miR-376a-3p,and down-regulates the expression of miR-223-3p;In addition,continuous light down-regulates the expression of plasma exosome miR-425-5p and upregulated the expression of skeletal muscle miR-425-5p.Conclusion1.Disturbance of circadian clock leads to changes in miRNAs expression profile of plasma exosomes in mice;2.miRNA-22,miR-223,miR-376 and miR-425 may play a key role in the regulation of insulin sensitivity by the biological clock. |
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