Vitamin D supplementation is effective for olanzapine-induced dyslipidemia

Abstract

Olanzapine is an atypical antipsychotic drug that is clinically applied in patients with schizophrenia. It increases the risk of dyslipidemia, a disturbance of lipid metabolic homeostasis, usually characterized by increased low-density lipoprotein (LDL) cholesterol and triglycerides, and accompanied by decreased high-density lipoprotein (HDL) in the serum. In this study, analyzing the FDA Adverse Event Reporting System, JMDC insurance claims, and electronic medical records from Nihon University School of Medicine revealed that a co-treated drug, vitamin D, can reduce the incidence of olanzapine-induced dyslipidemia. In the following experimental validations of this hypothesis, short-term oral olanzapine administration in mice caused a simultaneous increase and decrease in the levels of LDL and HDL cholesterol, respectively, while the triglyceride level remained unaffected. Cholecalciferol supplementation attenuated these deteriorations in blood lipid profiles. RNA-seq analysis was conducted on three cell types that are closely related to maintaining cholesterol metabolic balance (hepatocytes, adipocytes, and C2C12) to verify the direct effects of olanzapine and the functional metabolites of cholecalciferol (calcifediol and calcitriol). Consequently, the expression of cholesterol-biosynthesis-related genes was reduced in calcifediol- and calcitriol-treated C2C12 cells, which was likely to be mediated by activating the vitamin D receptor that subsequently inhibited the cholesterol biosynthesis process via insulin-induced gene 2 regulation. This clinical big-data-based drug repurposing approach is effective in finding a novel treatment with high clinical predictability and a well-defined molecular mechanism.

Keywords: C2C12 cells; clinical big data; dyslipidemia; high-density lipoprotein; low-density lipoprotein; olanzapine; vitamin D.

FIGURE 1

Increased incidence of dyslipidemia with the prescription of drugs and confounding effects of concomitant drugs on the olanzapine-induced dyslipidemia in FDA Adverse Event Reporting System (FAERS) data. Volcano plots for visualizing the reporting odds ratio (ROR, on a log scale) and its statistical significance (absolute Z-score) are shown. Each circle indicates an individual drug, and the size of the circle reflects the number of patients taking the drug. (A) Strong and significant increases in the ROR for dyslipidemia were seen in patients using olanzapine. (B) Within the population taking olanzapine, confounding effects of concomitantly used drugs on the incidence of olanzapine-induced dyslipidemia were calculated thoroughly and plotted. Overall values are presented in (Supplementary Tables S2, S3).

FIGURE 2

Deteriorative effects of olanzapine on blood lipid profiles and body weight maintenance and the rescuing effects of vitamin D co-treatment from the JMDC insurance claims. The data of pre-prescription and post-prescription periods were collected from the nearest test data before or after the olanzapine prescription within 1 year. Background characteristics were matched between groups (n = 20) as shown in Table 2. (A–C) The annual blood test results from patients included in JMDC were extracted. (A) Serum concentrations of triglycerides, (B) low-density lipoprotein (LDL) cholesterol, and (C) high-density lipoprotein (HDL) cholesterol were expressed in units of mg/dL. (D,E) The annual body weights, heights, and waist circumferences included in the JMDC were extracted. (D) Body mass index (BMI) is calculated from body weights and heights and (E) waist circumferences were obtained by direct measurement. (F) the level of HbA1c expressed in units of percentage of blood. Data are shown as means ± SEM. *p < 0.05; a paired t-test of the pre-prescription period against the post-prescription period. ^# p < 0.05, ^## p < 0.01; an unpaired two-tailed t-test with Welch’s correction between the group with and without vitamin D supplementation.

FIGURE 3

Deteriorative effects of olanzapine on blood lipid profiles and the rescuing effects of vitamin D co-treatment from electronic health records of the clinical data warehouse of Nihon University School of Medicine. Blood test results were extracted every 3 months after the first prescription of olanzapine. (A) the triglycerides (n = 406 in olanzapine without vitamin D group; n = 23 in olanzapine with vitamin D group), (B) the LDL cholesterol levels (n = 154 in olanzapine without vitamin D group; n = 11 in olanzapine with vitamin D group), and (C) the HDL cholesterol levels (n = 258 in olanzapine without vitamin D group; n = 16 in olanzapine with vitamin D group) were expressed in units of mg/dL. Data are shown as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; a paired t-test of data on any months after the olanzapine prescription against the data on month 0 (baseline). ^# p < 0.05, ^## p < 0.01; an unpaired two-tailed t-test with Welch’s correction between the groups with and without vitamin D supplementation.

FIGURE 4

Olanzapine treatment caused dyslipidemia in a rodent model while vitamin D₃ supplements attenuated the worsening of the condition. (A) C57BL/6J mice were fed for 1 week with the medium-fat diet (containing 1.37 IU of cholecalciferol/g of chow) or cholecalciferol-supplemented medium-fat diet (containing 200 IU of cholecalciferol/g of chow). Each group of mice were divided into two groups and orally given 10 mg/kg/day of olanzapine or vehicle for 5 days; resulting 4 groups of mice (each n = 10) received normal chow and vehicle as control (C), normal chow and olanzapine (O), vitamin D₃-supplemented chow and vehicle (D), and vitamin D₃-supplemented chow and olanzapine (DO). Blood samples were collected from these mice 1 day after the last dose of olanzapine without fasting. (B) the triglycerides, (C) total cholesterol levels, (D) LDL cholesterol levels, and (E) HDL cholesterol levels were measured. Data are shown as means ± SEM. The multiple comparisons of olanzapine and cholecalciferol influences were compared by two-way analysis of variance (ANOVA) with post hoc Tukey’s multiple comparison test. *p < 0.05, **p < 0.01.

FIGURE 5

Volcano plots of gene expression in the cultured mice cells treated with vehicle, olanzapine, or olanzapine co-treated with calcifediol or calcitriol. The changing ranges were expressed as log₂-transformed fold-change (FC), and the significance was expressed as −log₁₀-transformed false discovery rate (FDR). (A) Demonstrations of the cholesterol biosynthesis-related gene changes under olanzapine treatment and calcifediol or calcitriol-olanzapine co-treatment. The genes significantly downregulated in the olanzapine-calcifediol co-treated C2C12 cells are tagged with blue dots, and the genes upregulated are tagged with red dots in all the data sets. (B) A schematic diagram illustrating the functions of downregulated genes (blue) and upregulated gene (red) in the process of cholesterol biosynthesis. Genes plotted with FC absolute value > 2 and FDR value > 0.05 were considered to be significantly changed by treatment.

FIGURE 6

Validation by quantitative RT-PCR results showing the expression of cholesterol biosynthesis-associated genes in the cultured C2C12 cell line. (A) 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr) and (B) Insulin-induced gene 2 (Insig2) were validated in the same treating condition as that in the RNA-seq experiments (n = 12). Results were normalized to ribosomal protein, large, P0 (Rplp0). (C) Hmgcr and (D) Insig2 were validated in calcifediol with gradient-changed concentrations increasing from 0 to 10 μM (n = 8–9). (E) Hmgcr and (F) Insig2 were validated in calcitriol with gradient-changed concentrations increasing from 0.01 μM to 1 μM. Data are shown as means ± SEM (n = 9). **p < 0.01, ****p < 0.0001. The comparisons of olanzapine and vitamin D influences were compared by two-way analysis of variance (ANOVA). The validation of the dose-dependent effects of calcifediol and calcitriol were compared by one-way ANOVA.

FIGURE 7

Validation by quantitative RT-PCR results showing calcitriol regulating the cholesterol biosynthesis-associated genes mediated by VDR in the C2C12 cell line. (A) The results of vitamin D receptor (VDR) protein levels in C2C12 cells, adipocytes, and hepatocytes, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (n = 3). (B) Hmgcr and (C) Insig2 were validated in ZK159222 with gradient-changed concentrations increasing from 0 to 3 μM (n = 6). Data are shown as means ± SEM. *p < 0.05, **p < 0.01. The validation of the dose-dependent effects of ZK150222 was compared by one-way analysis of variance (ANOVA).