The Regulatory Role And Mechanism Of Peripheral Blood Fructose On Intestinal Fructose Absorption

Aims:In recent years,numerous studies have shown that excess fructose intake may have an adverse effect on the glucose and lipid metabolism in human,and lead to many metabolic diseases such as obesity,diabetes,non-alcoholic fatty liver disease,hyperlipidemia and so on.The small intestine is the main site for the absorption of dietary fructose,and its absorption capacity is related to the expression level of fructose transporter Glut5 on the brush margin of the small intestine mucosa,which is regulated by the positive feedback of dietary fructose signal.Epidemiological data suggests that elevated fasting serum fructose level is an independent risk factor for type 2 diabetes and can significantly increase the risk of type 2 diabetes.The elevated peripheral blood fructose levels in humans are associated with excessive dietary fructose intake,increased endogenous synthesis,increased intestinal absorption,and impaired intestinal clearance.There are many studies related to peripheral blood fructose levels and metabolic diseases,however the regulatory mechanism between peripheral blood fructose and intestinal fructose absorption has not been clarified.This study aims to explore the regulatory role and possible mechanism between intestinal fructose absorption capacity and peripheral blood fructose level,and provides theoretical basis and therapeutic target for revealing the possible pathological mechanism of intestinal fructose absorption in metabolic diseases such as diabetes.Methods and results:In this study,the Cre/loxP system was used to construct the KHK liver-specific knockout mouse model（KHK-LO）,and we found that the mice could maintain glucose metabolism homeostasis under normal diet.However,given high fructose diet,KHK-LO mice developed obvious hyperfructosemia.The expression of Ch REBP and its downstream genes involving fructose metabolism and transporter in liver were detected by q PCR and Western Blot,immunofluorescence staining.Moreover,in order to observe the changes in the ability of liver to metabolize fructose,we used LC-MS technology to detect the content of fructose in portal vein（PV）and inferior vena cava（IVC）of mice.The results suggested that the metabolic ability of fructose in mice was impaired due to the lack of liver KHK,which reduced the clearance of fructose in portal venous blood and led to the increase of fructose content in inferior venous blood.In addition,the increase of fructose level in peripheral blood might also be regulated by absorptive capacity of intestine.By isotope labeling fructose absorption experiment,we found that the level of ¹³C-labeled fructose in portal venous blood increased significantly.This result combined with the up-regulation of fructose metabolism gene expression and the increase of Glut5 distributed in the brush margin of the small intestine mucosa,we presume that the deficiency of liver KHK can lead to the overactivation of the intestinal fructose metabolism pathway,thereby leading to the increased absorption of dietary fructose in the intestine.To further explore the regulatory role of peripheral blood fructose level on intestinal fructose sense and absorption,we introduced a hyperfructosemia model caused by non-dietary fructose.The wild-type C57BL/6J mice were subcutaneously injected with fructose water.The peripheral blood fructose level was detected to reflect the dynamic change of fructose clearance.The activation of Ch REBP and its downstream genes related to fructose metabolism and transport in small intestine were detected at the transcriptional and translational levels.The expression and localization of Glut5 on the brush edge of intestinal mucosa were observed by immunofluorescence staining.We found that increased fructose levels in peripheral blood can activate the intestinal fructose sensing pathway and participate in the regulation of intestinal fructose absorption.After maintaining a high concentration of peripheral blood fructose for a period of time,we observed that in mice simulating hyperfructosemia,the fructose levels in portal blood and inferior venous blood were significantly increased in the fructose absorption test,suggesting that the absorption capacity of dietary fructose in the intestine was enhanced.Thus,we proposed the core hypothesis:the promotion effect of peripheral blood fructose on intestinal fructose absorption depends on the activation of Ch REBP fructose sensing pathway in intestine.In order to explore the mechanism of fructose absorption induced by fructose from intestine,we established a KHK gut-specific knockout mouse model（KHK-GO）.Metabolic phenotype analysis found that the intestinal KHK knock out did not affect metabolism homeostasis of glucose in KHK-GO mice.After changing to the high fructose diet,the fed plasma fructose contents of control mice and KHK-GO mice were significantly higher than that of the normal diet group,but significant difference was not observed in the plasma fructose level,and the peripheral blood fructose of the two groups of mice fed with high fructose diet could be reduced to normal level after starvation.The next step was to verify whether intestinal KHK deficiency was involved in the regulation of intestinal absorption of dietary fructose.In the fructose absorption experiment of mice fed with general diet,we found that the intestinal fructose clearance ability was impaired after KHK deficient,and a large amount of fructose spilled into the portal vein through the intestinal epithelial cells,leading to a significant increase in the level of portal fructose in KHK-GO mice.However,in mice fed with high fructose diet,the levels of fructose in portal vein and inferior venous blood of the control group were significantly increased,but no significant changes were found in the KHK-GO group,suggesting that intestinal KHK-mediated fructose metabolism was necessary to promote intestinal fructose absorption.Combined with the changes in transcription levels of genes related to fructose metabolism and absorption in small intestine,we speculated that intestinal fructose absorption capacity was not only related to the activation state of Ch REBP,but also regulated by KHK-dependent fructose metabolism.Glut5 in small intestinal epithelial cells is a key transporter involved in intestinal fructose absorption and plays an important role in the regulation of intestinal fructose absorption by peripheral blood fructose.To clarify whether Glut5 was involved in the transport of fructose from peripheral blood to intestinal epithelial cells to activate the intestinal Ch REBP fructose sensing pathway,we used the Glut5 gut-specific knockout mouse model to conduct the experiment of subcutaneous injection of fructose water.The results showed that the Ch REBP fructose sensing pathway was activated after Glut5 deficiency,suggesting that the loss of intestinal Glut5 did not affect the activation of circulating fructose on intestinal Ch REBP and its target genesConclusion:In this study,we constructed a tissue-specific defect mouse model of key genes in fructose metabolism.Using LC-MS and biochemical analysis methods,we accurately measured the level of fructose in blood and the transcription and translation level of genes related to fructose metabolism and absorption in different tissues,describing the regulatory role of peripheral blood fructose on intestinal fructose absorption from the perspective of hepato-intestinal dialogue.In conclusion,a tissue-specific knockout mouse model of key genes,LC-MS and biochemical analysis methods in the process of fructose metabolism and transportation were constructed in this study.It accurately reflects the level of fructose and the transcription and translation level of genes related to fructose metabolism and absorption in different tissues.From the perspective of hepato-intestinal dialogue,the regulatory effect of peripheral blood fructose on intestinal fructose absorption and its mechanism are described,and we draw the following conclusions（1）The hyperfructosemia induced by dietary fructose in KHK-LO mice was caused by increased intestinal fructose absorption capacity and impaired liver fructose clearance capacity.（2）In addition to high blood glucose,hyperfructosemia can also promote intestinal absorption of fructose.（3）peripheral blood fructose can regulate intestinal fructose absorption capacity through the Ch REBP/Glut5 axis.（4）Intestinal KHK-mediated fructose metabolism is necessary to promote intestinal fructose absorption.（5）Intestinal intake of fructose from blood is independent of Glut5.Our study clarified the role of peripheral blood fructose in enhancing intestinal fructose absorption through the KHK/Ch REBP/Glut5 regulatory axis,illuminated the intestinal fructose absorption may adjust mechanism,enriched the forms of hepato-intestinal dialogue,showed the potential role of peripheral blood fructose in the regulation of metabolism,which is expected to provide theoretical basis and molecular targets for the precise diagnosis,treatment and prevention and control of metabolic syndrome and other chronic diseases.