The Impact Of Sidt2 Knockout On Insulin Secretion And Skeletal Muscle In Mouse Model

Part 1:The study for mechanism of impaired insulin secretion in Sidt2knockout mouseAims:To explore the expression and localization of Sidt2 in mouse pancreatic islets,and investigate the role of Sidt2 in glucose-induced insulin secretion in this study.Subjects and Methods:Sidt2 localization in?-cells was determined by qPCR,Western blotting and immunofluorescence assay.NAD?P?H responses to glucose in?-cells were analyzed by both fluorimetric assay and enzymatic recycling method.K_ATP and K_V2.1channel currents and cytosolic free Ca²⁺([Ca²⁺]_i)of?-cells were measured by whole cell patch-clamping with the later further confirmed by confocal microscopy with indicator Fura-2/AM.The pH within insulin secretory granules?ISGs?and lysosomes was meas-ured with indicator Lysosensor DND-189.Results:Sidt2 was expressed in mouse pancreatic?-cells and cell line INS-1 and co-localized with ISGs in islet?-cells.The islet?-cells in Sidt2^-/-mice had normal NAD?P?H responses,K_ATP and K_V2.1 currents,and membrane potentials,but exhibited a lower[Ca²⁺]_i peak with a longer lag time to glucose compared with those from normal mice.Under free extracellular calcium,[Ca²⁺]_i increase to glucose was low in WT?-cells while nearly absent in Sidt2^-/-?-cells.When 2-APB or ryanodine applied,the remaining[Ca²⁺]_i peak under stimulation of glucose in Sidt2^-/-?-cells was still significantly lower than in WT?-cells,but pretreatment with Bafilomycin A1 induced comparable[Ca²⁺]_i increase between these two groups indicating defective calcium traffic from the intracel-lular acidic organelle in Sidt2^-/-?-cells.There was no measurable difference in ISG pH in WT and Sidt2^-/-groups.After pretreatment with NAADP,the Ca²⁺response to high dose glucose was normal in Sidt2^-/-islet?-cells.The expression of CD38 increased under sti-mulation with glucose in WT and Sidt2^-/-islets,and there was no difference between these two groupsConclusions:These data indicate that impairment of insulin secretion in Sidt2^-/-mouse was primarily due to defects in the generation of NAADP signaling,further led to the de-fect of Ca²⁺releasing from acidic stores,and finally resulted in the impairment of[Ca²⁺]_i signaling.Our data support Sidt2 is a NAADP transporter in ISG membrane.Part 2: The impact of Sidt2 on skeletal muscle in skeletal muscle specific Sidt2 knockout mouseAims: To investigate the relationship of myopathy and glucose metabolism in Sidt2 knockout mouse,investigate the changes of gene expression patterns in the skeletal muscle-specific Sidt2 knockout mouse,analyze the difference in gene expression and find the possible pathways.Subjects and Methods: We developed the skeletal muscle-specific Sidt2 knockout mice by breeding the Sidt2 f lox/flox or Sidt2 f lox/-with mice carring the Cre recombinase gene driven by the muscle creatine kinase promoter.We assessed the impairment of myodynamia?Rotarod test?,observed the pathological changes of skeletal muscle?HE staining and electron microscope?,and estimate the glucose metabolism?fast blood glucose and glucose tolerance test?in the skeletal muscle-specific Sidt2 knockout mice.Furthermore,with the utilization of gene expression profile chip,we analyzed the difference in gene expression and explore the possible pathways in mouse model.Results: The skeletal muscle-specific Sidt2 knockout mice showed apparent pathological changes,and the impairment increased with the increase of age.There was no abnormality in glucose metabolism until six months.The gene expression chip showed 590 significantly differential genes?432 up-expression,158 down-expression?and 180 pathways?130 up-expression,53 down-expression?.The up-expression pathway included phagocytosis,lysosomes and the regulation of actin,and the down-expression included metabolic and calcium pathway.Conclusions: Sidt2 deletion led to primary myopathy.And the pathological changes had no effect on glucose metabolism until 6 months.The calcium pathway may be the main reason in the mechanism of myopathy.