The Effect Of Glucosamine On The Metabolism Of Apolipoprotein M And Its Mechanism

Apolipoprotein M, a novel apolipoprotein discovered by Ning Xu and Dahlb(a|¨)ck, is mainly located in High-density lipoprotein(HDL) in the blood. By influencing preβ-HDL formation and cholesterol efflux, apoM is thought to be an important regulative factor in HDL metabolism and affects on the initiation and developmet of atherosclerosis. The expression and secretion of apoM are regulated by many physiological factors. ApoM is associated with coronary heart disease(CHD), diabetes and other diseases with dyslipidemia. The serum apoM concentration is significantly reduced in diabetic patients. Single nucleotide polymorphism (SNP) T-778C in the proximal promoter region of apoM gene was associated with the levels of plasma cholesterol and fasting plasma gluocose and also conferred the risk in the development of type 2 diabetes among Han Chinese. ApoM is reduced in diabetic rats and exogenous insulin administrations partially reverses the abnormal apoM expression. Insulin, insulin-like growth factor I (IGF-I), and IGF-I potential peptide (IGF-IPP) significantly inhibits apoM expression, in a dose- and a time-dependent manner, in the HepG2 cells. The machanism of this discrepancy of experiment result in vivo and in vitro is still unknown. Hyperglycemia down-regulates apoM expression in vivo and in vitro, but its mechanism need further investigation. Hyperglycemia stimulates the hexosamine pathway and leads to the increase of the endogenous glucosamine and metabolites of glycosylation. So we suggest the hexosamine biosynthesis pathway,a branch of the glycolytic pathway,be responsible for the suppression of apoM in hyperglycemia condition. Glucosamine is usually used to mimic the hexosamine pathway, so we investigate the influence of glucosamine on the expression of apoM.Glucosamine is an amino monosaccharide that is an essential component of mucopolysaccharides and chitin. Gluocosamine is a prominent component of the hexosamnie pathway and has many physiological function. Usual therapeutic doses of glucosamine administration show no adverse effects on glucose homeostasis. But the increased flux of metabolites through the hexosamine biosynthesis pathway impairs glucose transport and insulin action mediated by altered gene expression, and then changed the metabolism of glucose and lipoproteins. The hexosamine pathway induced by glucosamine is associated by insulin resistance. But other studies found glucosamine may attenuated the vessel injury induced by hyperglycemia and regulate the expression of lipoprotein genes. Glucosamine impairs hepatic apolipoprotein B100 (apoB100) assembly and secretion by inducing endoplasmic reticulum (ER) stress and enhancing co-translational degradation via proteasomal degradation and post-translational degradation via nonproteasomal pathways. Glucosamnie induces apolipoprotein A1 mRNA and protein levels by prolonging its mRNA half-life. The influence of glucosamine on apoM is unknown and the research about its relationship would take new sight into the pathophysiological procedure of the atherosclerosis and would help apoM to be an attractive target for the treatment of diabetes, obesity, hyperinsulinemia and dyslipidemia.Part one The effect of glucosamine on the expression of apoM in HepG2 cellsObjective: To explore the influence of glucosamine on the expression of apoM and apoA1 in vitro.Methods: The effect of glucosamine on apoM and apoA1mRNA expression in the human hepatocyte cell line, HepG2,is measured by Real-time PCR,after 24 hours of treatment with various concentrations of glucosamine(0, 1mmol/L, 5mmol/L).Results: The apoM and apoA1 mRNA levels in human HepG2 cells were significantly different among the groups.The apoM mRNA levels were increased 60%(P<0.05)compared to the controls at 1 mmol/L glucosamine group. While at 5 mmol/L glucosamine group, apoM mRNA were increased 47.7%(P>0.05).The apoA1 mRNA levels at 1 mmol/L and 5 mmol/L glucosamine groups were increased 1.03 fold (P>0.05) and 2.74 fold(P<0.05), respectively.Conclusion: The expression of apoM and apoA1 is up-regulated by glucosamine in HepG2 cells within a certain range of concentrations. Too high or too low concentration of glucosamine has no significant influence on the expression of apoM and apoA1.Part two The effect of glucosamine on apoM expression and secretion in healthy SD ratsObjective: To explore the influence of exogenous glucosamine on the expression and secretion of apoM in vivo.Methods: Healthy rat models were infused with glucosamine(1mmol/L, 10mmol/L) through tail veins for 6 hours continuously, at the speed of 2.5ml per hour by microinfusion pumps. The controls were infused with saline in the same way . The concentration of apoM in the serum was assayed by western-blot and the mRNA levels of apoM in the liver were measured by Real-Time PCR. The serum glucose, insulin, HDL, LDL, cholesterol, triglyceride and lipoprotein a were measured by standard chemistry methods at the same time.Results: After 6-hour continuous infusion through tail veins, the hepatic apoM mRNA levels among the groups differed significantly(H=7.344,P=0.0254).The apoM mRNA levels in 1mM glucosamine group were increased 9.6%(P>0.05) and 10mM glucosamine group were increased 3.40 fold(P<0.05)compared to the controls. While the serum apoM levels increased slightly and without statistical significance(H=0.98,P=0.6126). At the same time, HDL in 10mM glucosamine group were increased 20.3%( P<0.05)compared to the saline group. There were no significant change in the levels of LDL, triglyceride and lipoprotein a among groups.Conclusion: The hepatic expression of apoM was up-regulated by glucosamine in healthy rats and the serum apoM levels increased slightly. A short time high levels of glucosamine had no significant influence on the expression of serum triglyceride, cholesterol, LDL and apolipoprotein a. But HDL were increased significantly suggesting glucosamine may enhance the serum levels of HDL and the trend be similar to apoM.Part three The mechanism of glucosamine in regulating apoM expression and secretion.Objective: To explore the role of hexosamine pathway in regulating the expression and secretion of apoM by using GFAT inhibitor, azaserine. And together with insulin or azaserine, the mechanism of regulative role of glucosamine was investigated.Methods:①A was control group. B was added with 5 umol/Lazaserine. C was 5umol/L azaserine together with 10mmol/L glucosamine. After 24-hour incubation, the HepG2 cells were collected and apoM mRNA was measured by RT-PCR.②A was control group. B was added with 5 mmol/L glucosamine and C was 5mmol/L glucosamine together with 1ug/ml insulin. The apoM and apoA1 mRNAs in HepG2 cells were measured by RT-PCR.③A was control group. B was added with 20umol/L azaserine. C was 1ug/ml insulin together with 20umol/L azaserine and D was 5mmol/L glucosamine together with 1ug/ml insulin and 20umol/L azaserine. The apoM and apoA1 mRNAs in HepG2 cells were measured by RT-PCR.Results:①The apoM mRNA was decreased 35.4%(P<0.05)in azaserine group,and partially reversed by exogenous glucosamine.②Compared to the control group, the apoM mRNA was increased 51.1% (P<0.01) in 5mM glucosamine group and increased 78.2% (P<0.01) in 5mM glucosamine + 1ug/ml insulin group.③The apoM mRNA was increased 120% (P<0.01) in glucosamine + insulin + azaserine group compared to the azaserine group, and was increased 68.9% (P<0.05) compared to the azaserine + insulin group. The apoA1 mRNA was increased 3.6 fold (P<0.01) in glucosamine + insulin + azaserine group compared to the azaserine group, and was increased 1.85 fold (P<0.01) compared to the azaserine + insulin group.Conclusion: The expression of apoM was regulated by glucosamine through hexosamine pathway and both exogenous and endogenous glucosamine were involved in the regulation of apoM. Insulin enhanced the up-regulative effect of glucosamine on the expression of apoM, but insulin itself wasn’t the main regulative factor. The hexosamine pathway was not responsible for the down-regulation of apoM in hyperglycemia condition.