Analysis of the FAERS database

FAERS adverse event reports were obtained from the FDA website (http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Surveillance/AdverseDrugEffects/). Duplicated reports (among a total of 5,821,354 reports) from the first quarter of 2004 through the second quarter of 2014 were filtered by applying the FDA’s recommendation of adopting the most recent case number. Consequently, 4,547,841 remaining reports were analysed in this study. Arbitrary drug names, including trade names and abbreviations, were mapped into unified generic names via text mining. Adverse event risk was evaluated by calculating the reporting ORs with a 95% CI according to methods described in the literature34.

Briefly, individuals in the FAERS database were divided into the following four groups: (a) individuals who received the drug of interest (i.e., quetiapine or vitamin D) and exhibited DM-related adverse events; (b) individuals who received the drug of interest, but did not exhibit DM-related adverse events; (c) individuals who did not receive the drug of interest and exhibited DM-related adverse events; and (d) individuals who did not receive the drug of interest and did not exhibit DM-related adverse events. The OR with 95% CI was defined as follows:

where a, b, c and d refer to the number of individuals in each group, and log refers to the natural logarithm. If the lower limit of the 95% CI was >1, a significant association was assumed between use of the drug of interest (i.e., quetiapine) and the increased occurrence of DM-related adverse events. Contrarily, if the upper limit of the 95% CI was <1, a significant association was assumed between use of the drug of interest (i.e., vitamin D) and the decreased occurrence of DM-related adverse events.

The search terms for “DM” and “vitamin D” are described in Supplementary Tables 1 and 2, respectively. We regarded drug indication data as the patients’ primary diseases. To search for concomitant drugs associated with a decreased occurrence of quetiapine-induced, DM-related adverse events, the number of individuals treated with the concomitant drug of interest was restricted to a minimum of 1000, because ORs are prone to fluctuation when the sample size is small.

Combinatorial analysis of the GEO and KEGG PATHWAY databases

Microarray gene expression profiles were obtained from a previous study published in the GEO database (GEO Accession No. GSE 59923; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE59923). Expression data were analysed in the livers of rats orally treated with quetiapine (500 mg/kg, 5 days of exposure with daily dosing) or vehicle (carboxymethylcellulose). The analysis included genes participating in the insulin resistance pathway in skeletal muscle cells, and obtained from the KEGG PATHWAY database (Entry No. map04931; http://www.kegg.jp/dbget-bin/www_bget?pathway+map04931). For the expression analysis of each gene, log2-transformed microarray data were back-transformed to the original scale and presented relative to the vehicle-treated group.

Animals

All animal experiments were approved by the Kyoto University Animal Research Committee in accordance with the ethical guidelines of the Committee. All experiments were designed to minimise the use of animals and number of experiments. Male ICR mice (5–6 weeks of age) were purchased from Japan SLC (Shizuoka, Japan) and housed with a constant ambient temperature (24 ± 1 °C) and humidity (55% ± 10%) on a 12 h/12 h light/dark cycle. Food and water were freely available, except for during the fasting period before the glucose tolerance test.

Drugs and reagents

Quetiapine was purchased from Wako Pure Chemical Industries (Osaka, Japan). Methamphetamine was from Sumitomo Dainippon Pharma (Osaka, Japan). d-Glucose was from Nacalai Tesque (Kyoto, Japan). Insulin was from Biological Industries (Cromwell, CT, USA). 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG), LY294002, and dorsomorphin (compound C) were from Cayman Chemical (Ann Arbor, MI, USA). Calcitriol was from Toronto Research Chemicals (Ontario, Canada). Dulbecco’s modified Eagle’s medium (DMEM) and foetal bovine serum (FBS) were from Sigma-Aldrich (Saint-Louis, MO, USA). Horse serum (HS) was from Invitrogen Japan (Tokyo, Japan).

For in vivo study, quetiapine and methamphetamine were dissolved in distilled water plus 1% Tween 80 prior to use. d-Glucose was dissolved in distilled water before use. Quetiapine, methamphetamine and d-glucose were administered intraperitoneally at a volume of 10 ml/kg body weight.

Vitamin D 3 (cholecalciferol) was administered orally in the diet. MF diet (Oriental Yeast, Tokyo, Japan) containing 1370 IU vitamin D 3 /kg (8 IU vitamin D 3 /day based on daily consumption of 6 g chow/30 g body weight) served as the control diet. The cholecalciferol-supplemented diet consisted of a mixture of MF diet and 200,000 IU vitamin D 3 /kg (1200 IU/day).

Intraperitoneal glucose tolerance test

Mice were fed a control or cholecalciferol-supplemented diet for 1 week, followed by a 16 h fast before a 150 min intraperitoneal glucose tolerance test. Quetiapine (10 mg/kg) or vehicle (1% Tween 80) was administered intraperitoneally before the injection of d-glucose (3 g/kg). Blood samples were collected from the tail vein at the indicated time points, and blood glucose levels were measured using an Accu-Chek Blood Glucose Meter (Roche Diagnostics, Almere, the Netherlands). Blood insulin levels were measured using a Mouse Insulin Enzyme-Linked Immunosorbent Assay Kit (Morinaga Institute of Biological Science, Yokohama, Japan) and 5 μl blood samples collected at the indicated time points, as instructed by the manufacturer.

Real-time quantitative RT-PCR

Immediately after the glucose tolerance test, mice were sacrificed by cervical dislocation, and the right thigh muscles were collected. Total RNA was isolated using the ISOGEN Reagent (Nippon Gene, Tokyo, Japan), and the isolated RNA (0.5 μg) was reverse transcribed using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Real-time quantitative RT-PCR was performed using the StepOne Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) and the THUNDERBIRD SYBR qPCR Mix (Toyobo). Each PCR amplification consisted of heat activation for 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.

The oligonucleotide primers used for RT-PCR were as follows: 5′-GTA ACC CGT TGA ACC CCA TT-3′ and 5′-CCA TCC AAT CGG TAG TAG CG-3′ for the 18s ribosomal RNA gene (18s rRNA); 5′-CCC AGG CCA TCC CGA AAG-3′ and 5′-TCT CAA ATG GCC TGT GCT CC-3′ for Insr; 5′-TTA GGC AGC AAT GAG GGC AA-3′ and 5′-TCT TCA TTC TGC TGT GAT GTC CA-3′ for Irs1; 5′-GAC AGC GAA GCG ACG GC-3′ and 5′-GTC TGA TTT TAC TGC CAC GCT C-3′ for Pik3r1; 5′-CGC TTG CGG TCT GAT GTT TT-3′ and 5′-AAT ACC GCC TTT TCC AGC CA-3′ for Akt2; and 5′-TTA TTG CAG CGC CTG AGT CT-3′ and 5′-GGG TTC CCC ATC GTC AGA G-3′ for Slc2a4. The mRNA expression levels of each gene were normalised to that of 18s rRNA, which was measured in parallel in each sample and expressed relative to the vehicle-treated group.

Cell culture and differentiation

Mouse C2C12 myoblast cell line was kindly provided by Prof. H. Takeshima (Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto, Japan). The cells were cultured in 100-mm dishes in DMEM containing 10% heat-inactivated FBS at 37 °C with 5% CO 2 . One day after seeding cells in black 96-well plates (>70% confluence), the medium was switched to DMEM with 2% HS to differentiate cells into myotubes. The myotubes were used for experiments 3–5 days following differentiation.

Glucose uptake assay

Glucose uptake assay was performed by measuring the uptake of 2-NBDG, a fluorescent derivative of glucose. Differentiated myotubes were starved in a serum-free DMEM for 3 h before treatment with the drugs. After treatment, cells were stimulated with or without insulin (1 μM) dissolved in KRPH buffer (136 mM NaCl, 4.7 mM KCl, 1 mM MgSO 4 , 1 mM CaCl 2 , 5 mM KH 2 PO 4 , 20 mM HEPES) for 15 min followed by the addition of 2-NBDG (50 μM) for 20 min. After incubation, free 2-NBDG was washed out 3 times with KRPH buffer. The fluorescence retained in the cells was measured with a fluorescence microplate reader (FDSS/μCell; Hamamatsu Photonics, Shizuoka, Japan) at an excitation wavelength of 480 nm and an emission wavelength of 540 nm.

Statistical analysis

Statistical analyses of the FAERS and GEO databases were performed with R version 3.1.2 Software (R Foundation for Statistical Computing, Vienna, Austria). Data obtained from the animal experiments were analysed with GraphPad Prism 5 Software (GraphPad, San Diego, CA, USA). Differences between two groups were compared via an unpaired two-tailed t-test with Welch’s correction, while differences between more than two groups were compared via the Kruskal-Wallis test followed by Dunn’s post hoc test (Fig. 3) or the one-way ANOVA followed by Tukey’s post hoc test (Fig. 4). Time-course data were analysed by applying a two-way ANOVA for repeated measures, followed by the Bonferroni post hoc test. Differences were considered significant at p < 0.05.