Single-Cell Transcriptomic Characterization And Energy Metabolism Mechanism Of Glucose Transporter In Pde6b Mouse Retina

Objectives:The retina has the highest relative energy consumption of any tissue,depending on a steady supply of glucose from the bloodstream.Glucose uptake is mediated by specific transporters whose regulation and expression are critical for the pathogenesis of many diseases,including diabetes and diabetic retinopathy.However,after performing single-cell RNA-sequencing(scRNA-seq)on the whole retina of C3H HeA Pde6b wild-type and rdl mice,we found that the abundance of GLUTs expression in mouse retinal tissue and the intensive energy expenditure of retina tissue did not seem to correspond too well,therefore,we decided to conduct further studies on GLUTs in the mouse retina.In this study,we made new discoveries in studying the expression of Glucose transporter-2(GLUT2)in mouse retinal cells,also made further progress in investigating the glucose transport mechanism and energy metabolism mechanism of photoreceptors.Methods:1.Perform scRNA-seq on whole retinal cells with C3H HeA Pde6b wild-type and rd1 mouse,and analyze the relevant results.2.Use immunofluorescence to show the GLUT2 expression in mouse neuroretina on mouse retinal cross-sections.3.Use organotypic retinal explants,cultivated under entirely controlled,serum free conditions to verify GLUT2 immunofluorescence results above.4.Animal and experimental groups:Postnatal day(P)5 C3H HeA Pde6brd1/rd1(rd1)and congenic C3H HeA Pde6b+/+wild-type(WT)mice of unspecified gender were used for in vitro organotypic retinal explants.On the premise that the bilateral retinas from the same animal do not enter the same group,the explants then randomized Divided into the following eight groups:control group,STZ low-concentration group,STZ medium-concentration group,STZ high-concentration group,hemichannel inhibitor intervention group,endocytosis blocker intervention group,STZ+hemichannel inhibitor intervention group,and STZ+endocytosis blocker intervention group.All explants were cultivated under entirely controlled,serum free conditions.5.Streptozotocin(STZ)exposure experiments:streptozotocin(STZ)is a cytotoxic drug similar in structure to glucose that transported exclusively by GLUT2,meclofenamic acid(MFA)is an inhibitor of connexin-formed hemi-channels,and Dynasore(DYN)is a blocker of clathrin-mediated endocytosis.According to different groups,the culture process and intervention of each experimental group are shown in following table:#126.Sampling:Explant culture was stopped at the 7th day(P11)by fixation and subsequent cryosectioning.7.Sample analysis:All samples were tested for Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)and poly-ADP-ribose(PAR)immunohistochemical staining,etc.Results:1.The scRNA-seq results showed that the expression of Slc2a1-Slc2a4 genes corresponding to GLUTs was not in line with expectations,especially GLUT2 was hardly expressed.2.GLUT2 expression was observed in horizontal cells of the mouse neuroretina,Staining on both retinal cross-sections and whole-mounts indicated that GLUT2 is often associated with inner retinal capillaries.3.Co-immunostaining of GLUT2 and Calbindin confirmed that horizontal cells were expressing GLUT2.4.In TUNEL assay,STZ did not measurably affect horizontal cells viability,while it ablated rod and cone photoreceptors in a concentration-dependent manner.5.Staining for PAR indicated that the detrimental effect of streptozotocin on photoreceptors may be associated with DNA-damage.6.The negative effect of STZ on the viability of rod photoreceptors was counteracted by co-administration of either MFA or DYN.7.Cone photoreceptors were not protected from STZ induced degeneration by neither MFA or DYN.Conclusions:1.The results of scRNA-seq in mouse retinal cells do not strictly correspond to protein expression;2.The expression of GLUT2 in horizontal cells may be related to the regulation of photoreceptor energy metabolism.3.There is a GLUT2-dependent glucose transport shuttle,from horizontal cells into photoreceptor synapses.4.Rod and cone photoreceptors have different glucose uptake mechanisms.