| BackgroundType 2 diabetes mellitus(T2DM)is a chronic metabolic disease with a high incidence and difficult to cure,which seriously affects the function of multiple organs including the liver,bringing about significant social and economic burdens.In-depth exploration of the pathogenesis of T2DM and the development of corresponding intervention strategies have become a major medical challenge that urgently needs to be addressed in our country.The pathogenesis of T2DM involves various factors such as insulin resistance,glucose metabolic disorder,and abnormal lipid metabolism.Among them,the liver,as an important metabolic organ and a key target organ in the progression of diabetes,plays a crucial role in the regulation of glucose and lipid metabolism and T2DM metabolic disorders.Macrophages,as important components of the immune system,have functions in regulating inflammatory responses and clearing pathogens.Recent studies have shown that macrophages play an important role in the pathogenesis and progression of T2DM,including involvement in lipid metabolism and inflammation regulation through the release of extracellular vesicles(EVs).In T2DM patients,abnormal activation of macrophages and increased release of EVs may lead to hepatic steatosis and exacerbate the development of the disease,revealing their importance in metabolic disorders.Research has found that macrophage-derived EVs play a pathogenic role in hepatic steatosis in T2DM,but how to regulate the pathological release of EVs from macrophages to improve hepatic steatosis for the treatment of T2DM has not yet been studied.Mannose is a natural monosaccharide that physiologically exists in human blood at low levels.The concentration of free mannose in plasma is about 50-100 μM,which is only about 1/100 of the glucose content.Mannose enters cells through binding with corresponding receptors and undergoes two pathways,glycolysis or glycosylation,participating in inflammation regulation,immune response,and defense against bacteria,viruses,and other pathogens.It’s worth noting that mannose has a wide range of sources and can be supplemented through exogenous pathways such as intake of fruits,vegetables,and other food supplements.Mannose has broad application prospects in multiple fields and has shown good therapeutic efficacy in various diseases.Studies have found that plasma mannose concentration is elevated in the T2DM population and positively correlates with T2DM development.However,it is currently unclear whether this change is a concomitant metabolic regulatory response or a pathogenic cause of T2DM.Research has also reported that exogenous mannose(D-mannose)has a therapeutic effect on animal models of asthma,type 1 diabetes,hepatic steatosis,colitis,and other immune regulation and metabolic disorders.Given the beneficial therapeutic potential of mannose,exogenous mannose supplementation may still be beneficial for T2DM,but its efficacy requires further research.Therefore,exploring whether and how mannose regulates macrophage release of EVs and improves hepatic steatosis to treat T2DM is of significant theoretical and practical importance.This research direction is expected to provide a new perspective for us to deepen our understanding of the pathogenesis of T2DM and offer important references for developing more effective treatment strategies.AimThis study aims to use genetically obese leptin receptor-deficient db/db mice to investigate the correlation between hepatic glucose and lipid metabolic disorders caused by T2DM and the levels of mannose in the body.It seeks to elucidate the role and influencing factors of drinking water supplemented with mannose in treating T2DM,analyze the important function of mannose in regulating macrophage EVs release affecting liver metabolism for T2DM treatment,and finally delve into the mechanism by which mannose inhibits macrophage EVs and exerts therapeutic effects on T2DM.This research will provide experimental evidence for further understanding the mechanisms by which natural sugars participate in the pathogenesis of T2DM,reveal the role of mannose in regulating pathological cell-to-cell communication mediated by macrophage EVs,and establish a novel T2DM intervention strategy based on natural sugars influencing EVs release.Methods1.Animal model identification,T2DM glucose and lipid metabolism,and mannose levels analysisThe T2DM model was established using genetically obese db/db mice,and the following analyses were conducted to assess the modeling of T2DM:analysis of body weight,water intake,food consumption,and urine output;analysis of random blood glucose,fasting blood glucose,glycated hemoglobin(HbA1c),and serum levels of triglycerides(TG),cholesterol(TC),and free fatty acids(FFA)to evaluate T2DM glucose and lipid metabolic status;glucose tolerance test(GTT)and insulin tolerance test(ITT)to analyze key indicators of T2DM;high-performance liquid chromatography(HPLC)to measure mannose levels in mouse serum and liver,and extraction of liver tissue RNA to verify the expression levels of mannose synthesis and metabolic enzymes;administration of orally gavaged Cy5.5-labeled mannose(referred to as Cy5.5-mannose)to assess the exogenous biological distribution of mannose intake.2.Analysis of the therapeutic effect of exogenous mannose and its impact on microbiota and T cellsThe effects of adding 20%(w/v)mannose to drinking water on body weight gain,symptoms,random blood glucose levels,fasting blood glucose levels,HbAlc,and blood lipid levels in db/db mice were investigated,and the therapeutic effect of mannose on glucose tolerance and insulin sensitivity in db/db mice were examined.16S rDNA sequencing for analyzing the possible mechanism of mannose mediated by gut microbiota,flow cytometry(FCM)for detecting the proportion of peripheral blood T cells in each group,and histological staining with hematoxylin-eosin(H&E)for observing pathological changes in major organs of db/db mice.3.The effects of exogenous mannose on hepatic glucose and lipid metabolism and macrophage functionObservation of the gross appearance of isolated liver,analysis of liver weight/body weight ratio,histological analysis of liver tissues using H&E staining and Oil Red O(ORO)staining,assessment of hepatic lipid metabolism in db/db mice through liver lipid detection;analysis of hepatic glucose and lipid metabolism gene expression and insulin sensitivity in db/db mice using Western blot(WB)and quantitative real-time polymerase chain reaction(qRT-PCR);immunofluorescence(IF)analysis of the fate of mannose-targeted macrophage uptake;further analysis of the mechanism of action of mannose through network pharmacology and liver transcriptome sequencing;IF staining of macrophage activation/polarization markers tumor necrosis factor-alpha(TNF-α)and CD206 in the liver,enzyme-linked immunosorbent assay(ELISA)analysis of changes in macrophage polarization by detecting concentrations of TNF-α and interleukin-10(IL-10)in plasma;flow cytometry(FCM)analysis of extracellular vesicles(EVs)quantity and expression of macrophage marker F4/80.4.Experimental validation of the therapeutic efficacy and molecular mechanism of mannose through macrophage-derived EV release and replenishmentIdentification of macrophages and their EVs,studying the impact of mannose on the release of macrophage EVs under palmitate acid(PA)treatment,analyzing the relationship between abnormal hepatic glucose and lipid metabolism in db/db mice and the function of macrophage EVs,followed by validating the key role of macrophage EVs in the therapeutic effects of mannose through replenishment experiments.Tracking the in vivo fate and behavior of macrophage EVs labeled with PKH67 fluorescence,screening for mannose-regulated target genes through macrophage transcriptome sequencing,verifying the effects of mannose and PA co-treatment on target genes through WB and qRT-PCR experiments,further confirming the effects of regulating target genes through lentiviral transfection,and analyzing the possible mechanisms of mannose effects in macrophages treated with inhibitors of mannose metabolic pathways and their metabolites in a PA environment.Results1.The onset of T2DM in db/db mice is correlated with mannose levels and its metabolismIn the T2DM model of db/db mice,typical symptoms of diabetes such as obesity,increased water intake,food consumption,and urine output are observed,along with disrupted glucose and lipid metabolism,leading to abnormal glucose tolerance and insulin resistance.Compared to db/m mice,db/db mice show a significant increase in endogenous mannose levels in the serum and liver,with statistically significant differences(P<0.01).Additionally,mRNA expression of mannose synthesis enzymes and metabolic enzymes in the liver remains at parallel levels or is upregulated.Exogenous Cy5.5-mannose administered to db/db mice is rapidly absorbed through the intestines,accumulates in the liver and the kidney,and is related to biological utilization and excretion processes.2.Exogenous mannose improves T2DM in mice,and the therapeutic action is not related to intestinal microbiota and T cellsAdding mannose to drinking water does not significantly affect the body weight gain(P>0.05)or random blood glucose levels(P>0.05)of db/db mice,but can significantly reduce their fasting blood glucose levels(P<0.05)and restore the levels of HbAlc(P<0.01)and lipids(P<0.001)in the blood.In addition,oral mannose can improve the diabetic phenotype of db/db mice by reducing urine output,water intake,and food consumption.IPGTT and IPITT results confirm that mannose treatment improves glucose tolerance in db/db mice(P<0.01)and promotes insulin sensitivity(P<0.05).Furthermore,mannose does not affect the gut microbiota composition or peripheral blood T cell ratio of db/db mice,and has no significant effects on multiple organs,such as the heart,the lung,the kidney,and the spleen.3.Targeted inhibition of macrophage EV release by mannose alleviates hepatic steatosis and insulin resistanceTracking the fate of mannose in vivo reveals its enrichment in the liver.Further investigations show that mannose intake through drinking water improves the appearance of fatty liver and the liver weight/body weight ratio in mice(P<0.05).Histological examination confirms a reduction in fatty liver and decreased lipid droplet area(P<0.01)in db/db mice after mannose treatment.Additionally,mannose lowers the levels of TG(P<0.0001),TC(P<0.0001),FFA(P<0.001),and insulin resistance in the liver,restoring gene expression associated with hepatic glucose output and lipid metabolism.Tracking the cell targeting fate of mannose in vivo reveals its enrichment in liver macrophages.Network pharmacological methods and liver tissue transcriptome sequencing analysis suggest significant regulation of EVs transport by exogenous mannose.Combined analysis using IF and ELISA indicates that M1 macrophage polarization is inhibited by mannose(P<0.01),while M2 macrophage polarization is not significantly affected(P>0.05).Quantitative analysis of EVs derived from macrophages in mouse plasma reveals effective control of macrophage-derived EVs by exogenous mannose(P<0.0001).4.Mannose metabolism inhibits the expression of CD36 in macrophages to regulate EV releaseIn vitro simulation of the T2DM condition is performed using PA,and cultured macrophages are treated with mannose.It is found that mannose inhibits the release of EVs(P<0.0001)while the protein content of EVs is not affected(P>0.05).Furthermore,macrophage-derived EVs from db/db mice lead to metabolic disorders in hepatocytes and were primarily taken up by hepatocytes in vivo.Additionally,PA-induced pathological macrophage-derived EVs are replenished into db/db mice treated with mannose,which block the therapeutic effect of mannose on liver steatosis,resulting in significantly increased hepatic steatosis score(P<0.001).The CD36 gene regulated by mannose is screened out through transcriptome sequencing of macrophages,and WB and qRT-PCR detection techniques in vitro and in vivo jointly confirm the inhibitory effect of mannose on PA-induced CD36 expression(P<0.0001).The lentiviral overexpression of CD36 significantly attenuates the inhibitory effect of mannose treatment on macrophage-derived EV release(P<0.0001),confirming the crucial role of CD36 as a target for mannose-mediated regulation of macrophage-derived EV release.Moreover,macrophages treated with mannose metabolic pathway inhibitors and mannose metabolic cascade products in a PA environment reveal that mannose effectively inhibit the expression of CD36 and EV release,with statistically significant difference(P<0.0001).Conclusion1.Both endogenous and exogenous mannose exhibit metabolic process abnormalities in the pathological condition of T2DM,suggesting a certain correlation with the onset of T2DM in db/db mice.2.The safe improvement of T2DM symptoms in db/db mice by adding mannose to drinking water reveals a significant role of exogenous mannose in improving T2DM.3.Mannose targetedly inhibits macrophage-derived EV release to alleviate hepatic steatosis and insulin resistance,elucidating the important function of mannose in targeting macrophages to regulate liver metabolism.4.Mannose metabolism inhibits the expression of CD36 in macrophages,thereby controlling EV release,unraveling the key mechanism of mannose-controlled macrophage-derived EV release in treating T2DM.This study provides important reference for the in-depth exploring of the pathogenesis of T2DM,elucidating the macrophage communication and metabolic homeostasis maintenance mechanisms mediated by EVs,and establishing a natural sugar-based approach to T2DM by controlling the release of endogenous EVs. |
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