| Part? The sphingolipids metabolism in spingomyelin synthase2-knock outmiceObjective: To observe the total SMS activity,SMS2 m RNA expression and sphingolipids metabolism in the islets of spingomyelin synthase2-knock out(SMS2-KO)mice.Methods:Genotype identifications were conducted in SMS2-KO mice and wide type mice before experiments.Islets from wild type mice and SMS2-KO mice were also isolated.Thin-layer chromatography(TLC)was used to measure total SMS activity in islets.RNA was extracted from these islets and gene expressiones of sphingolipid metabolism related enzymes(SMS1,SMS2,GCS,SMPD1,SMPD2,SMPD3,SMPD4)were analyzed.Liquid chromatography-tandem MS(LC-MS/MS)was used to measure sphingomyelin,ceramide,sphingosine-1-phosphate and dihydroceramide in the serum and islets.Results:(1)SMS2-KO mice showed specific 354 bp bands.WT mice showed specific 252 bp bands.SMS2 deficiencydiminished SMS activity in islets by about 49%(P<0.05).(2)As indicated in the Table 2,SMS2 KO mice showed a significant decreased in SM levels(P<0.001)and significantly increased ceramide levels in islets(P<0.001).SM-C16 and SM-C24:1 was the predominant SM species.The levels of SM species containing acyl chains(SM-C16,C18,C18:1,C24:1)in the WT mice were significantly higher than SMS2-KO mice.The predominant Cer species in both the WT mice and SMS2-KO mice were Cer-C16 and Cer-24:1.Moreover,the levels of Cer species containing acyl chains(Cer-C16,C18,C20,C22,C24,C24:1)in the WT mice were significantly lower than SMS2-KO mice.There were no significant changes in S1 P and DHCer levels between the two groups.The SM and Cer levels showed a similar viariation trend.(3)As expected,islets from SMS2-KO mice scacely have SMS2 m RNA expression(P<0.001).SMS2 deficiency does not influence SMS1 m RNA levels in islets.We also measured sphingomyelin phosphodiesterase(SMPD)and glucosylceramide syntheses(GCS)m RNA levels in islets,and did not find any significant changes between WT and SMS2-KO mice(P>0.05),indicating these enzymes might not be rate limiting ones determing the concentration of ceramide and sphingomyelin levels.Conclusion:(1)SMS2 deficiency significantly decreased SMS activity and reduced SMS2 m RNA levels in islets.Moreover,SMS2 deficiency does not influence SMS1 m RNA levels.(2)SMS2 deficiency significantly decreased SM levels and significantly increased ceramide levels in the plasma and the islets.SMS2 deficiency has no significant influence on S1 P and DHCer levels.Part II Glucose metabolism and pancreatic ? cell function in Sphingomyelin Syntheses 2-knock outmiceObjective: To investigate SMS2 deficiency on glucose metabolism and pancreatic ? cell function,and to explore its possible mechanism.Methods: SMS2-KO and wild typemice were fed on a normal diet since four weeks to nine weeks old of age.Daily food intake and body weight were kept a record of to observe body weight and metabolism changes.Fasting blood glucose was measured.Intraperitoneal injection of glucose tolerance test(IPGTT)and intraperitoneal injection of insulin tolerance test(IPITT)were done at nine weeks old of age to assess the glucose tolerance and insulin sensitivity.Serum insulin levels at fasting and after glucose injection(30min and 60min)were measured.Mice were sacrificed and islets of the two groups were collected to extracted insulin content in acid ethanol extraction liquid.The islets were also used to conduct glucose-stimulated insulin secretion(GSIS)experiments.Total cellular ATP concentration in isolated islets was determined following a 1h incubation period in 3.3 m M or 16.7 m M glucose.Pancreas biopsies were done andislet size,density and morphological changes were observed,with the aim to explore the reason for insulin secretion decrease in SMS2-KO mice.Furthermore,electron microscopy was applied to observe pancreatic ? cell cytolasmic docked insulin granules vesicular,and potential structure changes in mitochondria and rough endoplasmic reticulum.Results:(1)On a chow diet,no differences were found between WT and SMS2-KO mice in body weight and food consumption.(2)IPGTT showed that SMS2-KO mice had significantly lowerblood glucose levels at 30,60 and 120 min compared with WT mice(P<0.05),indicating SMS2-KO mice performed better in their ability to remove glucose from the circulation.(3)IPITT showed that SMS2 deficiency increased insulin sensitivity.There was no significant difference in the fasting plasma insulin level between SMS2-KO mice and WT mice.However,in SMS2-KO mice,the plasma insulin level after injection of glucose(30min and 60min)tended to be lower than WT mice,but not reaching significant difference.(4)Compared with WT mice,SMS2-KO mice showed decreased glucose stimulating insulin secretion while there was no difference in the islet insulin content between the two groups.(6)There was no significant difference in the total cellular ATP concentration in isolated islets incubation period,neither in 3.3 m M nor in 16.7 m M glucose.(7)We observed no difference in islet size,density or morphology changes.(8)Islets electron microscopy showed no difference in pancreatic ? cell cytolasmic docked insulin granules vesicular,mitochondria and rough endoplasmic reticulum morphalogy between the two group.Conclusions: SMS2 deficiency increased insulin sensitivity.SMS2 deficiency decreased theglucose stimulated insulin secretion,but the increased insulin sensitivity could compensate insulin hyposecretion to maintain normal glucose metabolism... |
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