DMEM was purchased from Gibco, Thermo Fisher, USA. Fetal bovine serum (FBS), antibiotic/antimycotic mix, TRIzol LS Reagent, and Turbo DNase-free kit were acquired from Invitrogen, USA. Culture bottles and other reagents were purchased from Sigma-Aldrich, Chem Co, Germany. High Capacity cDNA Reverse Transcription Kit; Fast SYBR® Green Master Mix were purchase from Applied Biosystems, USA.
The cell line C2C12 (ATCC® CRL-1772) was used. It was cultured with low or high glucose concentration and with normal or high Zn concentration, and in the absence or presence of insulin or Interleukin 6 (IL 6). The intracellular Zn concentration and the expression of metallothionein-2 (MT2) and selected Zn transporters (ZnT1, ZnT5, ZnT7, ZIP6, ZIP7, ZIP10, and ZIP14) were determined. C2C12 cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM), 10% fetal bovine se-rum (FBS) and 10 IU/mL penicillin and streptomycin. Basal glucose and Zn content were 25 mM and 5 µM, respectively (control). Cells were maintained at standard temperature and CO2.
C2C12 cells were incubated for 24 hours with: Zn (ZnSO4) 10 or 100 µM and/or glucose 10 or 30 mM, in addition, the cells were challenged with insulin 1 nM or IL 6 5 nM for 24 h. The different treatments were: Gluc 10/Zn 10= glucose 10 mM, Zn 10 μM; Gluc 10/Zn 100= glucose 10 mM, Zn 100 μM; Gluc 30/Zn 10= glucose 30 mM, Zn 10 μM; Gluc 30/Zn 100= glucose 30 mM, Zn 100 μM; Gluc 10/Zn 10/Ins= glucose 10 mM, Zn 10 μM, insulin 1 nM; Gluc 10/Zn 100/Ins= glucose 10 mM, Zn 100 μM, insulin 1nM; Gluc 30/Zn 10/Ins= glucose 30 mM, Zn 10 μM, insulin 1 nM; Gluc 30/Zn 100/Ins= glucose 30 mM, Zn 100 μM, insulin 1 nM; Gluc 10/Zn 10/IL6= glucose 10 mM, Zn 10 μM, interleukin 6 5 nM; Gluc 10/Zn 100/IL6= glucose 10 mM, Zn 100 μM, interleukin 6 5 nM; Gluc 30/Zn 10/IL6= glucose 30 mM, Zn 10 μM, interleukin 6 5 nM; Gluc 30/Zn 100/IL6= glucose 30 mM, Zn 100 μM, interleukin 6 5 nM.
The intracellular Zn concentration and mRNA relative abundance of ZnT1, ZnT5, ZnT7, ZIP6, ZIP7, ZIP10, ZIP14, and MT2 were determined by qRT-PCR. The experiments were made in triplicate and repeated at least three times.
Intracellular Zn concentration in C2C12 cells
For total Zn quantification, C2C12 cells were separated into two aliquots. One of them was lysed with nitric acid (65%) (SupraPure, Merck, Chemical Co., Darmstadt, Germany) overnight at 60 ºC. This sample was used to measure the total Zn concentration, using an atomic absorption spectrophotometer (Perkin-Elmer 2280). A 3-point standard curve was used, with Zn standard solution (CertiPur 1.19806, Merck, Chemical Co., Darmstadt, Germany). Total metal content was expressed as µg metal/µg protein per sample. The second aliquot was used to determine total protein concentration .
qRT-PCR of Zn transporter genes
RNA from C2C12 cells was extracted using Trizol reagent according to product protocol (Invitrogen). Extracted total RNA was treated with RNase-Free DNase Set (Qiagen) according to product instructions. Total RNA concentration was measured by absorption at 260 nm. RNA purity and concentration were checked by determining the OD ratio at 260/280 nm using a Biowave II Spectrophotometer. Reverse transcription of RNA (1.5 µg) was done using an Affinity Scrip cDNA Synthesis Kit (Stratagene). Real Time PCR was performed using Brilliant II SYBR Green QPCR Master Mix (Stratagene) on a Step One, Applied Biosystems (USA). Beta-2-microglobulin (B2M) was used as housekeeping gene.
The primers used were: (1) B2M: CCGCCTCACATTGAAATCCA and CTGCAGGCGTATGTATCAGT (NM_009735.3); (2) ZnT1: CACGACTACCCATT-GCTCAAGGA and CGTCAACGTCTCGAAGCTCTTTCA (NM_009579.3); (3) ZnT5: TGCTCTTTGACTGCTCGGCTTT and CTCTATTCGGCCATACCCATAGGA (NM_ 022-885.2); (4) ZnT7: TGCAAAGAACTCCTCCCTCGTTG and TCCTTGTAACTG TTGCACCCTCTG (NC_000069.7); (5) ZIP6: ACTGCCGGCTTGTTCATGTATGTC and ACTGCCGGCTTGTTCATGTATGTC (NC_000084.6); (6) ZIP7: TCTGGCCATTGG TGCTTCCTTT and AGACTGGACCAGGATGGCAAAA (NC_000083.6); (7) MT2A: TCGGAAGCCTCTTTGCAGAT and CGCCTGCAAATGCAAACAATG (NC_000074.6); (8) Zip10: ATAGCCTGGATGGTGATCATGGGT and TATGGATGTGCTGATGCCT CCAGT (NC_0000 67.6); (9) ZIP14: TCCATTGGAACGCTGCTCTC and AAACAC CATGCAGACTTGGAG (NM_ 001135152.1);
The products of each set of primers were confirmed using agarose gel electrophoresis. The number of mRNA copies of target and housekeeping genes were calculated according to the standard curve method. PCR amplification efficiency of each primer pair was calculated from the slope of the standard curve. Melting curve analysis was constructed to verify the presence of gene-specific amplification and for absence of primer dimers.
The results are expressed as median and interquartile range. Gene expression results are expressed as fold change related to Beta-2-microglobulin (B2M) used as housekeeping gene. Statistical analyses were performed with Graph-Pad Prism 6.0 software (San Diego, CA, USA). The data were analyzed depending on the normality of the results. Comparisons were conducted according to One-way ANOVA (Tukey´s post-hoc test) or Kruskal-Wallis test (Dunn´s post-hoc test) as corresponds. Statistical significance was considered when p< 0.05.