A serotonin 5-HT2B agonist slightly increased insulin secretion, while a 5-HT2C antagonist slightly decreased it. it. Other agonists and antagonists for serotonin receptors did not impact insulin secretion. A histamine H1 agonist increased insulin secretion, whereas an H1 antagonist and H2 agonist suppressed it. Our results suggest that dopamine (D2, D3 and D4), serotonin (5-HT2B and 5-HT2C), and histamine (H1 and H2) receptors, which are expressed on pancreatic -cells, directly modulate insulin secretion from pancreatic -cells. Thus, olanzapine may induce hyperglycemia in LY-3177833 clinical settings by suppressing insulin secretion from pancreatic -cells through inhibition of dopamine D3, serotonin 5-HT2B and 5-HT2C, and histamine H1 receptors. and animal studies, these findings shed new light around the mechanisms underlying olanzapine-induced hyperglycemia. Materials and Methods Chemicals Olanzapine and haloperidol were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Dopamine hydrochloride and bromocriptine were purchased from Sigma-Aldrich (St Louis, MO, USA). 7-Hydroxy PIPAT, ABT724, TCB2, BW723C86, Ro60C0175, WAY181187, 2-PEA, NGB2904, sonepiprazole, MDL11939, SB204741, SB399885, trans-triprolidine, amthamine, and tiotidine were from Tocris Bioscience (Bristol, England, UK). SB242084 Rabbit polyclonal to PAK1 was obtained from Toronto Research Chemicals (Ontario, Canada). All other chemicals used were of the highest purity available. Cell culture HIT-T15 cells were obtained from Sumitomo Dainippon Pharma (Osaka, Japan). Cells were cultured in Hams F12K medium (Sigma-Aldrich) made up of 10% fetal bovine serum, 100 models/mL penicillin G, 100?g/mL streptomycin, and 10?mM glucose which corresponds to the physiological blood concentrations in human in an atmosphere of 5% CO2/95% air flow at 37?C. Cells were subcultured once a week using 0.25% EDTA and 0.038% trypsin. New medium was replaced every 2 days. Cells were used between passages 80 and 100. RT-PCR analysis Total RNA was extracted from HIT-T15 cells using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions. Next, total RNA was utilized for reverse transcription to synthesize cDNA using a ReverTra Ace qPCR RT kit (Qiagen). PCR was performed with an iCycler (Bio-Rad Laboratories, Hercules, CA, USA) using KOD-Plus-DNA polymerase (Toyobo, Osaka, Japan). Conditions for PCR were as follows: initial denaturation at 94?C for 2?min; denaturation at 94?C for 30?sec; annealing at optimal temperatures for dopamine, serotonin, and histamine receptors for 30?sec; and extension at 68?C for 1?min (35 cycles). Primers, annealing temperatures, and product sizes for each receptor are summarized in Table?2. To examine expression of mRNA for dopamine D3 and D4 receptors, and all serotonin receptors, we performed two-step PCR with nested primers due to their lower expression in HIT-T15 cells. Nested primers for each receptor are summarized in Table?2. Conditions for the second round of PCR were the same as those for the first round. PCR products were electrophoresed with a 2% agarose gel and visualized under ultraviolet light with ethidium bromide. Table 1 Effects of chemicals on HIT-T15 cell viability.
olanzapine101.2??9.1bromocriptine108.2??13.1haloperidol105.1??4.77-hydroxy PIPAT94.9??4.1NGB2904106.4??3.3ABT724104.7??3.3sonepiprazole99.7??8.1TCB2101.0??9.1MDL11939121.4??23.9BW723C8694.7??7.9SB204741103.2??10.9Ro60C0175103.2??16.0SB24208499.8??5.0WAY18118795.6??12.5SB39985113.4??8.62-pyridylethylamine105.2??7.2trans-triprolidine102.7??3.4amthamine89.1??21.2tiotidine93.5??17.2 Open in a separate window Table 2 Primer sequences, annealing temperatures, and product sizes.
dopamine D2forward: 5-TCGCCATTTGTCTGGGTCCTG-365261reverse: 5-TGCCCTTTGAGGGGGGTCTTC-3dopamine D3 (1st PCR)forward: 5-GTCTGGAATTTCAGCCGCATTTGCTGTGA -362119reverse: 5-ATGACCACTGCTGTGTACCTGTCTATGCTG-3(2nd PCR)forward: 5-CAGCCGCATTTGCTGTGATG-36294reverse: 5-GTACCTGTCTATGCTGATGGCA-3dopamine D4 (1st PCR)forward: 5-GTCCGCTCATGCTACTGCT-360344reverse: 5-GACTCTCATTGCCTTGCGCTC-3(2nd PCR)forward: 5-GCTACTGCTTTACTGGGCCAC-360329reverse: 5-TCATTGCCTTGCGCTCCCTT-3serotonin 5-HT2A (1st PCR)forward: 5-CTGGTCATCATGGCAGTGTCCCTAGAGAA-367291reverse: 5-GGTTCTGGAGTTGAAGCGGCTATGGTGGA-3(2nd PCR)forward: 5-TGATGTCACTTGCCATAGCTG-355105reverse: 5-AGAGCTTGCTGGGCAAAG-3serotonin 5-HT2B (1st PCR)forward: 5-ATGCCGATTGCCCTCTTGAC-367185reverse: 5-CGGGAGTTGCACTGATTGG-3(2nd PCR)forward: 5-GCCGATTGCCCTCTTGACA-362182reverse: 5-GGGAGTTGCACTGATTGGC-3serotonin 5-HT2C (1st PCR)forward: 5-GGGTCCTTCGTGGCATTCTTCATCCCG-365273reverse: 5-CTTTTCGTTGTTGATAGCTTGCATGGTGCC-3(2nd PCR)forward: 5-GTGGCATTCTTCATCCCGTTG-362254reverse: 5-TTGATAGCTTGCATGGTGCT-3serotonin 5-HT6 (1st PCR)forward: 5-ATGCTGAACGCGCTGTATGG-360140reverse: 5-GAGAGGATGAGCAGGTAGCG-3(2nd PCR)forward: 5-GTATGGGCGCTGGGTGCTA-360112reverse: 5-GTAGCGGTCCAGGCTGATG-3histamine H1forward: 5-ACTTGAACCGAGAGCGGAAG-360178reverse: 5-GGGTTCAGCGTGGAGTTGAT-3histamine H2 (1st PCR)forward: 5-CCAGCTCCTGTGACTCCAGA-360353reverse: 5-GGGTTTGGGAAGGTCTGATG-3(2nd PCR)forward: LY-3177833 5-GATCCCTTGCACAAACCCAAC-36097reverse: 5-TCCTGGTCTGTAGTGTGCGT-3 Open in a separate windows Insulin secretion assay Insulin secretion assays were performed according to previous reports29,30. Briefly, HIT-T15 cells were seeded at a density of 1 1.0??105 cells/well in 24-well plates and cultured for 72?h after seeding. Next, cells were pre-incubated with new medium made up of 1% dimethylsulfoxide (DMSO) for LY-3177833 30?min at 37?C. After pre-incubation, cells were incubated with new medium for 1?h at 37?C. To examine the effects of olanzapine or agonists/antagonists for each receptor on insulin secretion, each compound was added to the medium at numerous concentrations during incubation. Compounds tested are shown in Table?3. After incubation, the concentration of insulin released into the medium was determined using a rat Insulin ELISA kit (Morinaga Institute of Biological Science, Yokohama, Japan) according to LY-3177833 our previously reported method31,32. Next, residual cells were washed with phosphate-buffered saline (pH 7.4), and lysed with 0.3?M NaOH. Concentrations of total protein were determined by Lowry method with bovine serum albumin as the standard. Amounts of insulin secretion were normalized to the total protein content of each well. XTT assay HIT-T15 cells were seeded at a density of 1 1.5??104 cells/well in 96-well plates and cultured for 24?h. Next, cells were replaced with serum-free Hams F12K medium made up of optimal concentrations of olanzapine, an agonist or antagonist of each receptor, or 1% LY-3177833 DMSO (control). Cells were incubated for 1?h at 37?C. After washing, 200?L of Hanks Balanced Salt Answer containing 225?M XTT and.