The Effect Of Liraglutide On The Apoptosis Of Human Nucleus Pulposus Cells Induced By High Glucose And On Glycemic Variability In Type 2 Diabetes

Glucagon-like peptide-1(GLP-1)is a key incretin hormone,secreted from enteroendocrine L cells,which enhances the secretion of insulin by islet beta cells and inhibits the secretion of glucagon by alpha cells in a glucose-dependent manner.In addition to the hypoglycemic effect,GLP-1 can also suppress the appetite and delay gastric emptying by affecting the vagal afferent fibers and hypothalamus,eventually resulting in weight loss.GLP-1 exerts its effect in vivo mostly through the GLP-1 receptor(GLP-1R).Under physiological conditions,GLP-1 in circulation is rapidly degraded by dipeptidyl peptidase IV(DPP-IV).Liraglutide,a synthetic GLP-1 receptor agonists(GLP-1RA),which has 97% homology to endogenous GLP-1 structure,can effectively resist degradation of DPP-IV and has a longer half-life,making it a powerful drug option for type 2 diabetes.Diabetes mellitus is one of the potential etiology of disc degeneration.The incidence of disc degeneration in diabetic patients is higher than that in non-diabetic patients.High glucose could cause negative effects on the disc cell's biology,such as inducing disc cell senescence and apoptosis.Because of the correlation between the two diseases,more and more attention has been paid to the effect of anti-hyperglycemia agents on disc degeneration.Apart from its beneficial effects on glycemic control,liraglutide could exert functions in a variety of tissues on the modulation of cell proliferation,differentiation,and apoptosis.However,little is known regarding the effects of liraglutide on nucleus pulposus cells(NPCs).In vitro studies,we mainly focused on the effects of liraglutide on apoptosis of human NPCs and explored the molecular mechanism of it,providing a new therapeutic strategy for diabetic patients with disc degeneration.Glycemic variability(GV)can be simply defined as the degree to which fluctuations of glucose between high(peaks)and low(nadir)levels exist.Generally,with the duration of type 2 diabetes,islet function declines progressively,insulin secretion decreases,and blood glucose fluctuation increases.Wide fluctuations in blood glucose levels can induce inflammation,oxidative stress and endothelial dysfunction,which can be linked to the development of macro-and microvascular complications of diabetes.Flash glucose monitoring(FGM)provides a novel method of continuously monitoring interstitial glucose levels for up to 14 days,which is beneficial to clinical medication guidance.In clinical studies,we treated poorly controlled type 2 diabetes mellitus patients with liraglutide in combination with continuous subcutaneous insulin infusion(CSII)for a short period of time.Ambulatory glucose profile was assessed using an FGM system for 14 days;the levels of serum oxidative stress and adipokines biomarkers were also measured.To evaluate the differences between the short-term combination of liraglutide and CSII and CSII alone in patients with type 2 diabetes mellitus.Part one Liraglutide inhibits high glucose-induced apoptosis of human nucleus pulposus cellsObjectives: To observe the effects of different concentrations of liraglutide on apoptosis of human NPCs and to confirm the expression of GLP-1R in human NPCs.Methods: The third-generation human NPCs were divided as follows:(1)Control group: cultured in NPC Medium;(2)high glucose group: cultured in 0.2M high glucose concentration;(3)various concentrations of liraglutide group: cultured in 0.2M high glucose medium containing various concentrations of liraglutide(10,100,or 1000 n M).Cell viability was measured by Ed U,cell apoptosis was assessed by flow cytometry and ELISA,caspase-3 activity was determined using a commercial caspase-3 activity kit,Western blot and immunofluorescence were used to detect the expression of GLP-1R in human NPCs after incubation for 48 h.Results:1.Western blot analysis and immunofluorescence showed that human NPCs expressed GLP-1R.2.Liraglutide(10,100,or 1000 n M)showed increased cell activity in a high glucose environment(P<0.05).The concentration of liraglutide up to 10 n M showed significant protective effection,and the maximal increase in proliferation activity was at 100 n M(P<0.05).3.The inhibition of liraglutide on high glucose-induced human NPCs apoptosis and caspase-3 activity was not in a dose-dependent manner(P<0.05).Liraglutide exerted the maximal anti-apoptotic effect at a concentration of 100 n M(P<0.05).Conclusions:1.GLP-1R is expressed in human NPCs.2.Liraglutide can inhibit the apoptosis of human NPCs induced by high glucose.Part two Liraglutide inhibits high glucose-induced human nucleus pulposus cells apoptosis through activation PI3K/Akt/mTOR and PI3K/Akt/GSK3? signaling pathwaysObjectives: To investigate the potential molecular mechanism of liraglutide against high glucose-induced apoptosis of human NPCs.Methods: The human NPCs were incubated with 100 n M liraglutide alone or combination with LY294002(inhibitor of PI3K),rapamycin(inhibitor of mTOR)and SB216763(inhibitor of GSK3?)in a high glucose culture for 48 h,respectively.ROS levels were detected by flow cytometric analysis.Cell apoptosis was evaluated by flow cytometry and cell death detection ELISA.Gene and protein expression levels were tested by quantitative real-time PCR(q RT-PCR)and Western blotting.Silencing GLP-1R by small interfering RNA(siRNA)transfection.The effect of silenced GLP-1R on the phosphorylation of Akt,the caspase-3 protein level and the apoptosis of NPCs were examined.Results:1.Liraglutide obviously decreased high glucose-induced ROS generation(P<0.05).The PI3 K inhibitor LY294002 partly reversed the effect of liraglutide in the high glucose environment(P<0.05).2.Liraglutide inhibited high glucose-induced apoptosis of human NPCs,promoted Bcl-2 gene and protein expression,and inhibited Bax and caspase-3 gene and protein expression(P<0.05).The PI3 K inhibitor LY294002 partly reversed the anti-apoptosis effect of liraglutide(P<0.05).3.100 n M liraglutide induced Akt,mTOR,and GSK3? phosphorylation and decreased caspase-3 levels(P<0.05).Meanwhile,these changes were blocked by PI3 K inhibitor LY294002(P<0.05).The phosphorylation of mTOR and GSK3? was almost completely inhibited by mTOR inhibitor rapamycin and GSK3? inhibitor SB216763,respectively(P<0.05).Treatment of human NPCs with rapamycin and SB216763 induced a significant increase in caspase-3 levels(P<0.05).4.Silencing GLP-1R with siRNA blocked the phosphorylation of Akt,increased the expression of caspase-3 after liraglutide treatment(P<0.05),and partly blocked the anti-apoptotic effect of liraglutide(P<0.05).Conclusions:1.Liraglutide can inhibit the apoptosis of human NPCs induced by high glucose through anti-oxidative stress and activation of P13K/Akt/ mTOR/caspase-3 and PI3K/Akt/GSK3?/caspase-3 signaling pathways.2.The anti-apoptotic effect of liraglutide on human NPCs in high glucose environment is realized by GLP-1 receptor dependent pathway Part three Effects of liraglutide combined with continuous subcutaneous insulin infusion on glycemic variability in type 2 diabetes mellitus: a study based on the flash glucose monitoring systemObjectives: Glycemic variability(GV)may be linked to the development of diabetic complications by inducing inflammation,oxidative stress,and endothelial dysfunction.Flash glucose monitoring(FGM)provides a novel method of continuously monitoring interstitial glucose levels for up to 14 days.In our study,FGM was used to evaluate the short-term combination of liraglutide and CSII versus CSII alone to reduce GV.Methods: This study randomly assigned poorly controlled type 2 diabetes mellitus patients treated with metformin and multiple daily injections of insulin(n=60)to either continuous subcutaneous insulin infusion(CSII)treatment or CSII in combination with liraglutide(CSII+Lira)treatment for 14 days during hospitalization.GV and time in range were assessed using an FGM system;weight,serum oxidative stress and adipokine levels were also evaluated at baseline and at the end of the study.Results: The weight and coefficient of variation were significantly reduced in the CSII+Lira group,while no significant change was observed in the CSII group.The changes differed significantly between the two groups in the mean blood glucose,mean amplitude of glycemic excursions,standard deviation,time in range(3.9-10.0mmol/L)and the hyperglycemia rate(>10mmol/L)(P<0.05).Compared with the CSII group,treatment with liraglutide increased the levels of serum adiponectin,heme oxygenase-1 and nuclear factor erythroid 2-related factor 2 and reduced the level of serum leptin.The difference was statistically significant(P<0.05).Conclusions: The addition of liraglutide to CSII therapy was superior to CSII alone in improving GV,time in range,weight and some cardiometabolic biomarkers,such as nuclear factor erythroid 2-related factor 2/heme oxygenase-1,leptin,and adiponectinin in type 2 diabetes.