Modulation of Zinc Transporter Expressions by Additional Zinc in C2C12 Cells Cultured in a High Glucose Environment and in the Presence of Insulin or Interleukin-6

Zn status has been related to various chronic diseases presenting oxidative stress and inflammation, such as type 2 diabetes. Zn supplementation has been suggested to be a potential coadjuvant in the management of this condition. Zn transporters constitute a key component in the maintenance of Zn homeostasis. Our aim was to evaluate the modulatory effect of additional Zn (10 or 100 µM; as a ZnSO4*7H20) on the mRNA relative expression of selected Zn transporters (ZnT1, ZnT5, ZnT7, ZIP6, ZIP7, ZIP10, ZIP14), in myoblast (C2C12) cells cultured in normal (10 mM) and high glucose (30 mM), and in the absence or presence of insulin (1 nM), and interleukin-6 (IL-6; 5 nM) for 24 h. The main findings of our study were that in high glucose conditions in absence of insulin or IL-6, additional Zn increased ZnT1 and ZIP6, and decreased ZnT5 and ZIP7 expressions. However, this situation is modified by insulin, where incremental Zn induced increased expressions of ZnT1, ZnT5, and all the ZIP transporters studied. In high glucose conditions and in the presence of IL-6, additional Zn caused increased expressions of ZnT7, ZIP7, and ZIP14, compared with results in the absence of IL-6. This study provides preliminary evidence for the differential expression of selected Zn transporters in C2C12 cells subjected to high glucose and incremental Zn, suggesting that important changes in intracellular Zn distribution take place in response to inflammatory and high-insulin environments. Further study is necessary to understand the implications of these findings.


Introduction
Since 1961, Zn has been recognized as an essential micronutrient in human nutrition. Zn deficiency is related to severe anemia, growth retardation, hypogonadism, and skin abnormalities [1]. Since then, Zn deficiency has been recognized as the main public health problem in low-, middle-, and high-income countries [2]. Zn plays key structural, catalytic, and gene expression roles. Zn is essential for the activity of more than 300 enzymes [3]. In the 1980s, the "Zn finger" motif was identified in the TFIIIA transcription factor of Xenopus [4]. Since then, more than 20 classes of "Zn fingers" have been described and are known to be functional motifs that interact with a variety of proteins, lipids, and nucleic acids [5]. In addition to the roles mentioned above, Zn as an ion can participate in signaling pathways [6].
In cells, Zn is distributed 50% in the cytoplasm, 30-40% in the nucleus and organelles, and 10% in membranes. Zn is mobilized at the cellular and organelle level by Zn transporters. Most of these Zn transporters are organ and organelle-specific. Their expression depends on the presence of Zn atoms and other factors [7]. Knowledge of the effects of modulators on Zn transporters is very limited. In humans, two major Zn transporter families have been identified: (1) Zrt-and Irt-like protein (ZIP) family (Slc39A); and (2) the ZnT family of Zn transporters (Slc30A) [8]. There are 14 ZIP proteins and 10 ZnT proteins that regulate Zn homeostasis [8][9][10]. In general, members of the ZIP family are Zn importers and those from the ZnT family are exporters. For instance, the uptake of Zn is regulated by Zn transporters of the ZIP family, located in membranes such as the plasma membrane, the endoplasmic reticulum, and the Golgi. ZIP family transporters import Zn from the extracellular environment or from organelles to increase the cytosolic Zn concentration. ZnT family transporters mediate the export of Zn from the cytosol into organelles or out of the cell. All ZnT transporters are located in the membranes of intracellular organelles except ZnT1, which is located in the plasma membrane [8]. The connection between the function of Zn transporters and physiological or pathological conditions has been a matter of interest. Thus, a mutation in the gene coding for ZIP4 was found to be responsible for acrodermatitis enteropathica. This transporter is the main Zn importer from the lumen to the enterocyte [11]. Another focus of attention has been the association between Zn status and type 2 diabetes (T2D). This issue has been reviewed by Ruz et al. [12]. The results on Zn status or Zn intake and T2D are inconsistent. In terms of Zn supplementation, two meta-analyses suggest some extent of beneficial effects on glycemic control biomarkers, although it must be noted that results have been mixed, mainly due to the marked differences in study designs and parameters used to assess the impact of interventions [13,14]. Although there are several potential mechanisms that could explain the association between Zn and glucose disorders, knowledge is still incomplete [12]. Zn is necessary for insulin synthesis and insulin action in peripheral tissues [3]. At the pancreatic beta cells, Zn is crucial for the synthesis, handling, and secretion of insulin. Zn transporter ZnT8 plays a crucial role [15]. This transporter is expressed almost exclusively in these pancreatic beta cells and it has been identified as a novel target autoantigen in patients with type 1 diabetes [16]. Deletion of ZnT8 impaired insulin secretion [17]. A single-nucleotide polymorphism of ZnT8 was associated with increased T2D risk [18].
Skeletal muscle plays a major role in the uptake of glucose stimulated by insulin in the post-prandial state. Under an insulin-resistant state, a condition that precedes T2D, there is a decrease in insulin signaling on IRS-1, the PI-3 kinase PI3K, and Akt, resulting in decreased translocation of GLUT4 with impaired glucose transport across the plasma membrane. As mentioned earlier, Zn also plays a role in the insulin signaling cascade. Several groups have identified that Zn is able to increase tyrosine phosphorylation of the IR-β sub-unit of the insulin receptor and enhance glucose transport in absence of insulin through the PI3K signal transduction pathway [19][20][21]. These results have shown that Zn might have insulin-mimetic properties on glucose and lipid metabolism as an inhibitor of protein tyrosine phosphatases as protein tyrosine phosphatase 1B (PTP1B) that is a negative regulator of insulin signaling [22]. Ionic Zn participates in the signaling cascades suggesting its intracellular distribution via the Zn transporters may play a pivotal role. In this study, we analyzed the modulatory effect of additional Zn on the expression of transporters located at the plasma membrane (ZIP6, ZIP10, ZIP14, and ZnT1), and early secretory pathway including endoplasmic reticulum and Golgi apparatus (ZnT5, ZnT7, and ZIP7) in myoblast (C2C12) cells cultured in normal and high glucose, and in the presence of insulin, and in an inflammatory environment by adding interleukin-6 (IL-6), a proinflammatory cytokine to the media.

Cell Culture
The C2C12 (ATCC® CRL-1772), an adherent myoblast cell line (Mus musculus), was used. This cell line differentiates rapidly in culture, forming contractile myotubes and producing characteristic muscle proteins. It was cultured with low or high glucose concentration (Merck, Chemical Co., Darmstadt, Germany) and with normal or high Zn (ZnSO 4 *7H 2 0; Merck, Chemical Co., Darmstadt, Germany) concentration, and in the absence or presence of insulin (Sigma-Aldrich, Chem Co, Germany) or interleukin-6 (IL-6; Sigma-Aldrich, Chem Co, Germany). The intracellular Zn levels and the expression of metallothionein-2 (MT2), a protein synthetized directly in relation to the availability of cell Zn availability, 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; Gibco, Thermo Fisher, MA, USA), 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher, MA, USA), and 10 IU/mL penicillin and streptomycin (Invitrogen, Thermo Fisher, MA, USA). Basal glucose and Zn content were 25 mM and 5 µM, respectively (control). Cells were maintained at 37 °C, 5% CO 2 , and 80% humidity. C2C12 cells were incubated for 24 h with Zn as ZnSO 4 *7H 2 0 10 µM compatible with a lower normal plasma Zn concentration or 100 µM (high concentration); and glucose 10 mM as a plasma concentration found in insulin resistance or 30 mM as concentration found in T2D. In addition, the cells were challenged with insulin (1 nM), a plasma concentration found in insulin resistance, or IL-6 (5 nM), a concentration that reflects an inflammatory state. The different treatments used are described in Table 1.

Intracellular Zn Concentration in C2C12 Cells
For total Zn quantification, after the incubation period, the C2C12 cells were trypsinized and then washed with phosphate-buffered saline (PBS, metal free) and centrifuged and the pellet suspended in 50 µL of PBS and separated into two aliquots. One of them was lysed with nitric acid (65%) (SupraPure, Merck, Chemical Co., Darmstadt, Germany) overnight at 60 °C. This sample was used to measure the total Zn concentration, using a flame atomic absorption spectrophotometer (PerkinElmer 2280, Boston USA). 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 [23].

qRT-PCR of Zn Transporter Genes
RNA from C2C12 cells was extracted using Trizol reagent according to product protocol (Invitrogen, USA). Extracted total RNA was treated with RNase-Free DNase Set (Qiagen, Düsseldorf, Germany) 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 (Gem Scientific Ltd, UK). Reverse transcription of RNA (1.5 µg) was done using an Affinity Scrip cDNA Synthesis Kit (Stratagene, Darmstadt, Germany). Real-time PCR was performed using Brilliant II SYBR Green QPCR Master Mix (Stratagene, Darmstadt, Germany) on a Step One (Applied Biosystems, USA). Beta-2-microglobulin (B2M) was used as a housekeeping gene.
The primers used are described in Table 2. The products of each set of primers were confirmed using agarose gel electrophoresis. The number of mRNA copies of target and housekeeping genes was 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 the absence of primer dimers. Agarose gel electrophoresis was performed to test amplicon specificity. Final results were reported according to the Pfaffl method [24], and they were expressed as fold change with respect to the control.

Statistical Analysis
The data were analyzed depending on the normality of the results. Parametric variables were presented as mean ± SD.

Total Zn Concentration and MT2 Relative Expression
Total Zn content in C2C12 cells exposed to different stimuli is shown in Fig. 1A. All Zn-treated cultures were significantly greater than the control, except the condition Gluc 30/Zn 10/Ins. Increasing Zn from 10 to 100 µM in the media was accompanied with greater intracellular Zn concentrations but only in 30 mM glucose. Regarding MT2 expressions (Fig. 1B), there was a general agreement with intracellular Zn concentration results, although it was noteworthy that expression values in 100 µM Zn conditions were of higher magnitude than observed in intracellular Zn content changes.

Relative Expression of Zn Transporters (ZnT1, ZnT5, and ZnT7) and ZIP Transporters (ZIP6, ZIP7, ZIP10, and ZIP14) in Normal and High Glucose and Zn Environments
The relative expression of Zn transporters ZnT1, ZnT5, and ZnT7 is shown in Fig. 2A. At 10 mM glucose, increasing the Zn concentration from 10 to 100 µM resulted in significant decreases in ZnT1 expression by 53%, and ZnT7 expression by 72%. In contrast, ZnT5 expression significantly increased by 4.5-fold. At 30 mM glucose, increasing the Zn concentration from 10 to 100 µM resulted in a significant increase in ZnT1 expression by 2.6-fold and a significant decrease in ZnT5 expression by 50%. No significant changes in the expression of ZnT7 were observed at 30 mM glucose. No significant changes in ZnT1 and ZnT5 expressions were observed in cells incubated with 10 µM Zn and 10 mM vs. 30 mM glucose, while the expression of ZnT7 was significantly reduced by 56% under these conditions. ZnT1 and ZnT7 in cells incubated with 100 µM Zn and 10 mM or 30 mM glucose exhibited significant increases in glucosedependent expression (4.1-fold and twofold, respectively). Conversely, ZnT5 expression was significantly decreased by 83% under the same conditions. while the expressions of ZIP6 and ZIP14 were significantly reduced by 45% and 57%, respectively; ZIP10 increased by threefold under these conditions. Cells incubated with 100 µM Zn and 10 mM or 30 mM glucose ZIP6 and ZIP10 exhibited significant increases in glucose-dependent expression (2.2-fold and 9.7-fold, respectively). No significant changes in ZIP7 and ZIP14 expression were observed in the same conditions.

Relative Expression of Zn Transporters (ZnT1, ZnT5, and ZnT7) and ZIP Transporters (ZIP6, ZIP7, ZIP10, and ZIP14) in a Normal and a High Glucose Environment: Effects of Additional Zn in the Presence of Insulin
The results of the relative expression of the ZnT (Fig. 3A) and ZIP (Fig. 3B) transporters studied under similar conditions as described above but adding insulin to the media are displayed in these figures. These experimental conditions were mainly aimed to observe, in the cell culture model, the effects resembling what it is observed in vivo in a high glucose and insulin resistance situation. At 10 mM glucose, increasing the Zn concentration from 10 to 100 µM resulted in a significant increase of ZIP10 expression by 1.6-fold. No significant changes in the expression of remaining ZnT and ZIP were studied at 10 mM glucose. It is noteworthy that in high glucose conditions (30 mM glucose), increasing Zn from 10 to 100 µM had a marked increasing effect on all but one of the Zn transporters studied. Moreover, in the case of ZnT1 and ZIP10, relative expression reached values well above 20-fold in contrast with the observations in the absence of insulin (see Fig. 2A   and ZIP10 expression by 8.7-fold and 7.8-fold were noted. In cells incubated with 100 µM Zn and 10 mM or 30 mM glucose, no significant changes were observed.

Discussion
The main findings of our study were that in high glucose conditions in the absence of insulin or IL-6, additional Zn increased ZnT1 and ZIP6 and decreased ZnT5 and ZIP7 expressions. However, this situation is modified by insulin, where incremental Zn induced increased expressions of ZnT1, ZnT5, and all the ZIP transporters studied. In high glucose conditions and in the presence of IL-6, additional Zn caused increased expressions of ZnT7, ZIP7, and ZIP14, compared with results in the absence of IL-6. These observations, although preliminary, are intended to contribute to understand events related to zinc supplementation in chronic conditions, such as insulin resistance and T2D. To insert these observations in a wider context, it is relevant to mention that Zn supplementation has been suggested to be a potential coadjuvant in the management of this condition as a result of its roles in oxidative stress, inflammation, and apoptosis [3]. Furthermore, zinc has been shown to be crucial for insulin synthesis and secretion at the pancreatic β cell [15] and has also been shown to have insulin-mimetic properties [22]. Several studies have shown an inverse relationship between Zn availability and cellular oxidative stress. Oteiza et al. [25] showed an increase in reactive oxygen species (ROS) production in 3T3 cells under low Zn concentrations. Ho et al. [26] demonstrated that ROS levels raised in C6 glioma cells from Zn-deficient rats; increased ROS production was related to an oxidative DNA damage in these cells. In addition, studies in lung fibroblasts, liver cells, neuroblastoma cells, and prostate epithelial cells showed increased levels of ROS and oxidative damage when grown in low Zn environments [27][28][29]. Inflammation is another major risk factor for chronic diseases and Zn has been pointed out to be essential for the normal function of the immune system in both innate and adaptive immunity responses. Zn deficiency results in reduced antibody production and cell-mediated immune response impairment. Also, Zn deficiency contributes to a chronic inflammatory state. These conditions are of particular interest in diseases such as T2D and cardiovascular diseases [30]. Zn regulates two major transcription factors that modulate inflammation: nuclear factor kappa-B (NF-κB) and hypoxia-inducible factor-1 alpha (HIF 1α), controlling the central inflammatory cascade [31]. Our group has recently published results on the antiapoptotic effects of additional zinc by modifying the expression of pro-and antiapoptotic genes in cultured myoblasts [32].
In order to make the effects of zinc in distinct tissues and cells possible, this element needs to be mobilized to the cell/organelle target. This is accomplished by Zn transporters. Although in general terms ZnT transporters are mainly exporters from the cytoplasm to organelles or to the extracellular space, and ZIP transporters are mainly importers to the cytoplasm, there are relevant particularities related to tissues and specific conditions [8]. In the context of T2D, ZnT8 has been the center of attention because its crucial role in the pancreatic beta cell has been demonstrated. Furthermore, its deletion causes major alterations in insulin synthesis and secretion [17]. This Zn transporter is almost exclusively expressed in these cells [7].
In glucose management, in addition to the pancreas and liver, skeletal muscle is highly relevant because it represents the largest mass of glucose utilization tissue and at the same time the major mass of body Zn. Knowledge on conditions present in insulin resistance and T2D, such as hyperglycemia, hyperinsulinemia, and inflammation, and how they relate to Zn is very limited. In order to explore such relationships, we used a myoblast cell model assessing the effect of additional Zn on selected Zn transporters cultured in normal and high glucose and in the absence or presence of insulin and a proinflammatory stimulus (IL-6).
Among the findings in our study, it was observed that in high glucose conditions and in the absence of insulin or IL-6, additional Zn increased expression of a Zn importer (ZIP6) and a Zn exporter (ZnT1) located at the membrane, and decreased expression of a Zn importer (ZIP7) and a Zn exporter (ZnT5) located at the early secretory pathway, suggesting additional Zn is able to be incorporated into the cells and inducing some adaptive changes attempting to set a new balance under a total cellular Zn increased condition. The effects of such changes on glucose uptake may be relevant. For instance, Ferdowsky and colleagues [33] reported that capsaicin and zinc were able to promote glucose uptake in C2C12 cells through a common calcium signaling pathway. Although the issue of glucose uptake was beyond the scope of the present study, it should be a matter of future investigations. This situation is marked modified in the presence of insulin, where incremental Zn induced increased expressions of ZnT1, ZnT5, and all four ZIP transporters studied. These expressions were of greater magnitude than those observed with high glucose alone.
It is well known that insulin increases glucose uptake into muscle cells by stimulating GLUT4 translocation to the cell membrane mediated by phosphorylation cascades. Zn also participates in insulin signaling cascades contributing to stimulate GLUT4 translocation [22]. Less known is the role of insulin inducing Ca +2 transients in cardiac and muscle cells. Actions of insulin on intracellular Ca2 + channel activation and their impact on GLUT4 traffic in muscle cells have been reviewed by Contreras-Ferrat et al. [34].
Calcium-conducting channels are also involved in the transport of Zn ions [35] which suggests this may be one of the mechanisms explaining the marked increased expression of all but one of the Zn transporter studied.
Compared with the high glucose alone condition, the presence of IL-6 in the media also modified some gene expressions when cultured in additional zinc media. Thus, IL-6 stimulated the expression of the Zn exporter ZnT7, and importers ZIP7 and ZIP14. Zn deficiency increases circulating IL-1β, IL-2, IL-6, and TNF-α while Zn supplementation induces the opposite effect in a dose-response manner by acting on NF-kB [36]. IL-6 is partially responsible for hypozincemia observed in inflammation by inducing expression of the Zn importer ZIP14 in the liver [37]. Further studies from the same group demonstrated that in mice following the administration of an inflammatory agent (lipopolysaccharide), the white adipose tissue and muscle had the highest ZIP14 expression, much higher than the liver, lung, and heart [38]. In our study, ZIP14 expression was moderately but significantly increased by additional Zn in both normal and high glucose in the presence of IL-6 but not in its absence. The meaning of these findings remains to be elucidated.
Information in the literature related to the issue under study is very limited. For instance, studies in skeletal muscle cell line C2C12 have shown that glucose metabolism is enhanced by ZIP7 via Akt phosphorylation, ZIP7 activates insulin receptor signaling by its binding to PTP1B [39]. Huang et al. [40] studied ZnT7 KO mice. They reported that a combination of decreased insulin secretion and increased insulin resistance may be responsible for the glucose intolerance observed in ZnT7 KO mice. The activity of the insulin signaling pathway was down-regulated in myocytes isolated from the femoral muscle of Znt7 KO mice. Besides the studies on ZnT7 and ZIP7 in muscle cells, knowledge on the potential roles of other Zn transporters in the glucose/ insulin homeostasis is scarce. For instance, Paskavitz et al. [41] studied the changes in Zn transporter expressions as the differentiation of C2C12 cell progresses. In terms of studies on insulin and the ZIP transporters studied here, there is an absence of reports in muscle cells. There are a few studies, however, in ZIP6 and ZIP7 in pancreatic β cells [42] and ZIP14 in pancreatic [43], pre-adipocytes [44], and hepatic cells [45]. Regarding ZnT transporters in muscle cells, no information was found with respect to ZnT1 and ZnT5. There is some evidence demonstrating that ZnT7 deficiency had adverse effects on fatty acid metabolism and insulin action in subcutaneous, but not epididymal fat in mice [46].
We consider these preliminary observations interesting, although we agree that implications are far from being fully understood as a result of the limited available information. On the other hand, these observations can serve as a point of departure for further studies.

Conclusions
This study provides preliminary evidence on the expression of selected ZnT and ZIP transporters in C2C12 cells subjected to high glucose and incremental Zn, emphasizing the relevance of inflammatory and high-insulin environments. The implications of these findings in T2D using Zn supplements deserve further study.

Competing interests
The authors declare no competing interests.

Ethics Approval and Consent to Participate
This article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors.