Biochemical Study And Possible Mechanism Of Olanzapine-induced Dyslipidemia

Background Schizophrenia causes severe mental distress to the patient.Although the second-generation anti-schizophrenic drugs such as olanzapine have a significant therapeutic effect on schizophrenia,they cause metabolic disorders(MS)in the body of the patient,causing serious adverse reactions such as insulin resistance(IR).Such adverse reactions such as dyslipidemia,glucose intolerance,inflammation,and IR have become essential factors in the low compliance of patients with clinical treatment of mental illness.This study found that Oxidized low-density lipoprotein(Ox-LDL)was significantly elevated in plasma levels during chronic olanzapine treatment.Fatty acid translocase(CD36)is a transporter for the uptake of Ox-LDL by adipose tissue.However,palmitoylation of CD36 as a necessary post-translational modification is the basis for its function as a fatty acid transporter.Therefore,the molecular mechanism of olanzapine-induced MS was explored from the perspective of olanzapine affecting CD36 palmitoylation modification.Objectives Based on long-term use of the anti-schizophrenic drug olanzapine may cause an abnormality in CD36 palmitoylation.To elucidate the cause of olanzapine-induced MS,explore the relationship between the significant accumulation of Ox-LDL caused by olanzapine and the degree of modification of CD36 palmitoylation.Palmitoyltransferase 4(Asp-His-His-Cys-4,DHHC4)is a catalytic enzyme for palmitoylation of CD36.This study further explores whether olanzapine affects DHHC4 activity and leads to the production of MS by the body.The study aimed to explore the possible mechanisms leading to MS from the perspective of olanzapine affecting palmitoylation of CD36.Methods 1.Animal experiments: The average body weight of 40 SPF female SD rats was randomly divided into olanzapine(OLZ)group and blank control(BL)group.The OLZ group was given 1.5 mg / kg olanzapine by gavage,and the BL group was given the same volume of sterile physiological saline once a day.Rats were weighed weekly.Fasting blood glucose levels and plasma insulin levels were measured every two weeks(2,4,6,and 8 weeks)until the end of the 8th week feeding experiment.In the 8th week,it was found that compared with the BL group,the body weight of the OLZ group increased significantly,and the homeostasis model assessment insulin resistance(HOMA-IR)increased significantly.The animals in the OLZ group produced MS and IR,indicating successful modeling.The successful model animals were subjected to an oral glucose tolerance test(OGTT),and the corresponding tissues and blood samples of the animals were collected.In this study,Western-blotting and real time Q-PCR methods were used to detect the protein and gene expression levels of related factors.The expression levels of TNF-?,IL-1?,IL-6,and Ox-LDL were measured by ELISA kit.2.Cytological experiments were carried out to explore the mechanism: the adipocyte cell line 3T3-L1 cells were induced,and the differentiation was induced successfully,and olanzapine was added for drug experiment observation.Expression analysis of CD36 in 3T3-L1 cells by Western-blot and Q-PCR was performed.The Ox-LDL standard was added to the 3T3-L1 cell culture medium,and the concentration of Ox-LDL in the 3T3-L1 cells was measured using an ELISA kit and analyzed.In the rescue experiment,293 T cells were used for transient DHHC4 transfection to construct an overexpression system.After the successful construction,a specific concentration of olanzapine was added to intervene,and western-blotting detected the degree of CD36 palmitoylation modification..Results 1.After 8 weeks of construction of the rat model,the body weight of the OLZ group was significantly increased by 20% compared with the BL group(P<0.05),and the fasting blood glucose of the OLZ group was significantly increased by 21%(P< 0.05).The plasma insulin level in the OLZ group was significantly increased by 65%(P<0.01),and the HOMA-IR in the OLZ group was significantly higher than that in the BL group by 95%(P<0.01).Besides,the area under the blood glucose concentration-time curve of the OGTT rats in the OLZ group was significantly higher than that in the BL group by 26%(P < 0.05).It indicates that long-term use of olanzapine leads to lipid metabolism and insulin resistance in rats.2.Compared with the BL group,the OLZ group had significantly increased TNF-?,IL-1?and IL-6 in the plasma and adipose tissue of the OLZ group(P<0.05).It was shown that olanzapine caused inflammation in rats.The level of Ox-LDL in the OLZ group was significantly improved by 79% compared with BL(P<0.01).The western blotting analysis showed that CD36 in the adipose tissue of the OLZ group was significantly increased by 591%(P<0.01).The plasma CD36 in the OLZ group was significantly increased by 98% compared with BL(P<0.01).Q-PCR detected the m RNA level of adipose tissue CD36.Compared with the BL group,the m RNA level of the OLZ group was significantly increased by 79%(P<0.01).Also,Western blotting analysis of the palmitoylation level of CD36 in adipose tissue showed that the level of CD36 palmitoylation in the adipose tissue of the OLZ group was significantly reduced by 81% compared with BL(P<0.01).The results indicate that olanzapine affects the level of CD36 palmitoylation modification and affects its uptake and absorption of Ox-LDL.3.Compared with the BL group,the long-term induction of olanzapine in rat adipose tissue also caused a significant decrease in the palmitoylation level of Glucose transporter type 4(GLUT4)protein by 91%(P<0.01).The palmitoylation level of rat sarcoma(Ras)protein was also significantly reduced by 70%(P<0.01).The above results indicate that olanzapine affects post-translational palmitoylation modification of these critical proteins.4.The results of 3T3-L1 adipocyte culture showed that the m RNA level of CD36 was significantly increased by 1351%(P<0.05)compared with the BL group,and the protein expression of CD36 in the OLZ group was also significantly increased.However,the level of CD36 palmitoylation in the membrane of 3T3-L1 adipocytes was significantly reduced by 84%(P<0.05),and the expression of CD36 in the membrane of OLZ group was significantly reduced by 54%(P<0.05).Moreover,the level of Ox-LDL absorbed by the OLZ group was also significantly reduced by 18%(P<0.05).In 293 T cells overexpressing DHHC4,compared to the BL group,DHHC4 significantly increased the level of CD36 palmitoylation on the cell membrane by 64%(P<0.05).Based on this overexpression system,olanzapine was added to the palmitoyl group of CD36.Significantly decreased by 77%(P<0.05)and significantly lower than 84%(P<0.05)of the BL group,indicating that olanzapine significantly inhibited DHHC4 activity,resulting in a significant decrease in CD36 palmitoylation levels.Conclusion After 8 weeks of long-term induction of olanzapine,rats in the OLZ group produced significant MS and IR,which were significantly increased in rat body weight,blood glucose,insulin,and HOMA-IR.A significant increase in inflammatory factors is an essential factor in IR.Abnormal levels of Ox-LDL in plasma are characteristic of metabolic disorders,and these markers are significantly elevated under the induction of olanzapine.The palmitoylation of CD36 is a necessary post-translational modification that directly affects the transport of fatty acids such as Ox-LDL.This study found that CD36 protein expression was significantly elevated in a rat model of chronic olanzapine-induced insulin resistance,whereas the level of palmitoylation of CD36 was significantly reduced,resulting in a significant increase in plasma Ox-LDL in the rat model.A significant increase in Ox-LDL is a significant marker of decreased levels of CD36 palmitic acid acylation.In 3T3-L1 adipocytes,olanzapine significantly increased the expression of CD36 and m RNA expression levels,but significantly inhibited the palmitoylation level of CD36,which significantly reduced the distribution of CD36 in the olanzapine group.It affects the uptake and absorption of Ox-LDL.To explore the mechanism by which olanzapine affects the palmitoylation of CD36,DHHC4 significantly increased the level of CD36 palmitoylation on the membranous membrane of 293 T cells in 293 T cells by constructing a DHHC4 overexpression system,however,under the intervention of olanzapine Overexpressed DHHC4 did not increase the level of palmitoylation of CD36,indicating that the activity of DHHC4 was significantly inhibited by olanzapine.This study investigated that olanzapine caused severe metabolic symptoms such as MS by affecting the palmitoylation of CD36,and may inhibit the level of CD36 palmitate acylation by inhibiting the activity of DHHC4.Exploring the mechanism of an intervention of olanzapine on DHHC4 activity will provide a feasible way to alleviate olanzapine-induced MS in the future.