Exogenous Metallothionein Potentiates the Insulin Response at Normal Glucose Concentrations in INS-1E Beta-Cells Without Disturbing Intracellular ZnT8 Expression.

As a consequence of the global epidemic of obesity, the incidence of type 2 diabetes (T2D) is increasing worldwide. T2D is characterized by hyperglycaemia, hyperinsulinaemia and a reduced insulin response in muscular and fatty tissue. Over time, an increased insulin demand leads to cellular fatigue and death of the insulin-producing β-cells. In response, the patients with T2D become insulin-dependent and subjected to the boundaries of lifelong insulin treatment. Preservation of β-cell insulin secretion and a sufficient β-cell mass is thus a corner stone in optimal T2D treatment. Physiologically, β-cell function and survival are closely related to zinc homeostasis. Zinc is essential for the primary functions of β-cells; insulin biosynthesis, insulin storage and insulin secretion and zinc are co-secreted with insulin in response to glucose stimulation. Hypozincaemia accompanied by hyperzincuria is often present in patients with diabetes 1. On the other hand, fluctuations in extracellular zinc levels can become toxic for β-cells if high concentrations arise locally during stress or increased insulin secretion 2. Regulation of cellular-free zinc homeostasis is orchestrated by two categories of zinc-carrying proteins; the zinc-buffering metallothioneins (MTs) and the zinc transporters also known as Zinc Transporters (ZnTs) and Zrt- and Irt-like proteins (ZIPs). The ZnTs are responsible for Zn2+ efflux from the cytoplasm to the extracellular matrix and into intracellular organelles like insulin-containing vesicles, whereas the ZIPs transport Zn2+ in the opposite direction 3. MTs tightly regulate the intracellular level of free zinc, and the levels of MTs are relatively high in the pancreas 3, 4, suggesting that MTs are involved in the normal function of the gland. New studies link dysregulation or dysfunction of zinc-transporting proteins with impaired insulin processing and impaired glucose metabolism 4, 6. Furthermore, polymorphisms in genes encoding for isoforms of MTs have been related to the development of type 2 diabetes and to the extent of diabetic complications 7. Transgenic mice, with β-cell-specific over-expression of MT-2 display, significantly reduced β-cell death, and the mice have a better preservation of insulin production when exposed to β-cell toxic Streptozotocin 8 . Moreover, a specific neuroprotective role has been postulated for extracellular MT 9. Here, we suggest that increasing MT levels by means of exogenous Zn7-MT-2A will be beneficial for β-cell function by contributing to an optimal zinc supply and regulation, testing this hypothesis in the glucose-sensitive, insulin-producing INS-1E β-cell culture. INS-1E (rat), originally kindly provided by Prof. C.B. Wollheim, Switzerland, was employed in this study. The INS-1E cells are cultured in a CO2 atmosphere in complete RPMI 1640 supplemented with 11 mM glucose, 10% (v/v) heat-inactivated foetal bovine serum, 50 μM β-mercaptoethanol, 2 mM l-Glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin as previously described 6. INS-1E cells were plated in 6-well plates (NUNC) in standard 11 mM glucose cell medium for minimum 24 hr prior to experiments. To ensure stable experimental conditions, 152 nM Zn7-Metallothionein-2A (Zn7-MT-2A; Bestenbalt LLC, Tallinn, Estonia) dissolved in 0.9% saline was supplied to the cell medium 6 hr before the experimental start. The cells were then exposed to RPMI 1640 with either 6 or 21 mM glucose and 152 nM Zn7-MT-2A for another 24 hr. To maintain the Zn7-MT-2A concentration, an additional 152 nM of Zn7-MT-2A was supplied every 6th hour until cell harvest. Control cells were not subjected to Zn7-MT-2A but exposed to similar changes of media during the experimental period. After the 24 hr of Zn7-MT-2A/glucose exposure, INS-1E cells were either harvested for RT-PCR or used for measurements of insulin content and insulin secretion. Samples and controls were performed in replicates of 5–6 for RT-PCR and in 3–4 for insulin measurement. After the 6 + 24-hr of Zn7-MT-2A exposure, the INS-1E cells were incubated for 2 hr in a Krebs-Ringer bicarbonate HEPES buffer (KRBH) at pH 7.4 containing 115 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.6 mM CaCl2, 1.2 mM KH2PO4, 20 mM HEPES, 5 mM NaHCO3, 0.1% (v/v) human serum albumin (HSA; Sigma, Brondby, Denmark) and supplemented with 6.6 mM or 21 mM glucose. The incubation medium was collected for analysis of insulin secretion, whereas the cells were collected in Earle's basal medium (Invitrogen, Taastrup, Denmark) by scarping with a rubber policeman followed by centrifugation. The pellet was split in two and resuspended in a buffer comprising 0.75% (v/v) glycine and 0.25% (v/v) bovine serum albumin with pH 8.8 for intracellular insulin determination, or in 0.1% M NaOH for protein determination. Total protein was determined using the BCA Protein Assay Reagent Kit from Pierce, USA (Bie & Berntsen A/S, Soborg, Denmark). Insulin concentration was determined using the ultra-sensitive Rat Insulin Elisa Kit from DRG Diagnostics (VWR, Soborg, Denmark). RNA was extracted using RNeasy Mini Kit Qiagen (VWR, Soborg, Denmark) and treated with DNase (VWR, Soborg, Denmark). Reverse transcription was carried out using 500 ng total RNA, ImProm-II™ Reverse Transcription System (Promega, Denmark) and oligo dT18 primers (TAC, Frederiksberg, Copenhagen, Denmark). Quantitative real-time PCR was performed in duplicate using IQ Sybr Green Supermix (Bio-Rad, Copenhagen, Denmark) in a MyiQ Two-Color Real-time PCR detection system (Bio-Rad). For all reactions, a melting curve was included. The results were analysed with iQ™5 Optical System Software, version 2.1. Starting quantities were calculated from a standard curve. Values were normalized to two stable genes as previously described 10. Data are presented as mean values with the standard error of the mean (S.E.M.). Unpaired t-test was used to determine statistical significance between two groups with p < 0.05 as the level of statistical significance. Zn7-MT-2A treatment induced the insulin secretion and increased the intracellular insulin content at 6.6 mM glucose (p = 0.0001 and p = 0.0325, respectively), whereas insulin secretion at 21 mM glucose was unaffected by Zn7-MT-2A supplementation (fig. 1A,B). As expected, the level of insulin secretion was increased by 21 mM glucose compared with 6.6 mM glucose in control cells while leaving the intracellular insulin content unaffected. Compared with normo-glucose levels (6.6 mM) exposure to a high glucose concentration (21 mM) is immediately reflected in the intracellular zinc homeostasis (fig. 1C–E). At high glucose concentrations, ZnT-3 was significantly up-regulated (p = 0.0089), whereas ZnT-5 (p = 0.0075) and ZnT-8 (p = 0.0061) were significantly down-regulated. Exposure to exogenous Zn7-MT-2A did not seem to alter the zinc homeostatic response to increased glucose. There was no significant down-regulation of the MT-1A expression in INS-1E cells at high glucose compared with normal glucose levels in the control cells; however, exposing INS-1E cells to exogenous Zn7-MT-2A in a high glucose environment led to a significant down-regulation of MT-1A transcription compared with a low glucose environment (p < 0.0001; fig. 1f). MT-3 gene expression was significantly up-regulated at 21 mM glucose in control cells (2.8-times, p = 0.0010) as well as in Zn7-MT-2A exposed cells (3.0-times, p < 0.0001) compared with 6.6 mM glucose (fig. 1g). The present study confirms that INS-1E is a physiologically functioning β-cell line displaying a glucose-sensitive insulin response as well as glucose-induced alteration in intracellular zinc homeostasis similar to previous report on the gene expression of ZnT-3, ZnT-5 and ZnT-8 5. Offering exogenous Zn7-MT-2A to this β-cell line, we observed direct effects on the insulin production and insulin response; That is, the presence of exogenous Zn7-MT-2A dramatically induces insulin secretion and increases the intracellular insulin content at 6.6 mM glucose. Increased insulin secretion is a cardinal feature of the sulfonylureas currently used in the treatment of T2D. Pathway analysis has previously indicated that several genes involved in increased activity of the secretory system such as SNARE 25 11 could be involved in the pharmacodynamics of sulfonylureas. The underlying mechanisms are, however, not known but could include fluctuations in intracellular-free zinc as such as known to be part of the beta-cell response to glucose stimulation 12. Much interest in the potential protective aspects of MTs towards T2D has focused on the antioxidant properties of these proteins. Recent research has on the other hand indicated that increased MT activity might not always be beneficial but in fact lead to an over-quenching of free oxygen radicals, which could lead to insulin resistance, for example through increased expression of insulin-signalling inhibitor PTP1B 13. So far, this phenomenon has only been described in adipocytes, highlighting the substantial regional differences in the role of MTs in cellular function. Zn7-MT-2A can carry up to seven zinc ions potentially available for cellular demands and could thus supply extra zinc in the current experiments. To elucidate this effect, we performed a pilot study showing no transcriptional effect of zinc in the concentration found during the experimental conditions (data not shown). The main source of zinc during these cell culturing experiments originates from the addition of 10% foetal calf serum that is 1/10 of the zinc concentration (14.0–18.0 μM) measured in plasma 14, 15, that is the zinc concentration in our experiment is somewhat lower than the in vivo situation. It is well known that the presence and function of the vesicular zinc transporters ZnT-3 and ZnT-8 are of importance for optimal insulin production in β-cells and the expression of ZnT-3, ZnT-5 and ZnT-8 respond to changes in glucose concentrations which we also describe in this present study 6. Our group has recently established 16 that changing the ambient zinc concentration affects the expression of the intracellular zinc transporters, ZnT-8 and ZnT-3, yet in our present experiments, addition of Zn7-MT-2A did not affect these zinc-sensitive intracellular zinc regulators. This indicates that exogenous MT does not act merely as a zinc supplement. Supporting this hypothesis, we found no gene induction of MT-1A upon Zn7-MT-2A addition. The MT-1A gene expression is known to be controlled by the metal response element-binding transcriptional factor (MTF)-1 17, and MT-1A expression is readily induced by zinc supplementation 7. It is possible that part of the effects of exogenous Zn7-MT-2A exposure is related to an internalization of the protein. Studies have shown that megalin, a protein belonging to the low density lipoprotein (LDL) receptor family, mediates endocytotic uptake of MT-2 and MT-1 in neuron cultures 18. Additionally, recent studies suggest that sorLA/LR11, another LDL receptor subtype, – known to be expressed in the pancreas – could be responsible for the uptake of MT-2A to collecting duct epithelial cells 19. As MT and ZnT regulate the intracellular zinc homeostasis in concert with the ZIP family, altered expression of these proteins regulating zinc influx to the cytoplasm might also contribute to the response of the beta-cells to exogenous Zn7-MT-2A. Up-regulation of key ZIPs, that is ZIP6, 7 and 8, and the resultant increase in intracellular-free zinc are part of the initial response of beta-cells towards high glucose 12. A higher baseline value of zinc, for example caused by high ambient zinc increasing the intracellular zinc level, can ameliorate this response 12. If Zn7-MT-2A is indeed internalized, the resulting increase in the zinc-buffering capacities of the cytoplasm might slightly reduce free cytoplasmatic zinc and hence potentiate the glucose-induced ZIP up-regulation in this way increasing insulin secretion. Although unaffected by extracellular MT supplements, we did on the other hand observe a down-regulation of MT-1A at high glucose concentration compared with low glucose concentration most likely caused by glucose-induced cAMP production known to have this effect on MT expression 12. Together with the reduced intracellular insulin content at the 21 mM glucose concentration, these findings could reflect that the INS-1E cells are at their maximum insulin secretion capacity when exposed to 21 mM glucose and might reflect cellular stress under this condition. This concurs with previous findings that high glucose levels increase β-cell stress and may induce apoptosis 6. Although not significantly improving insulin secretion at high glucose concentrations, an increased MT presence might still affect cellular survival due to the antioxidant scavenging properties of this protein. Interestingly, high glucose levels are reflected by the concomitant increase in MT-3 gene expression, whereas MT-3 is unaffected by the presence of extracellular Zn7-MT-2A. This result emphasizes that MT-3 and MT-1A react fundamentally different to stressors of β-cell function and therefore might play different roles in protecting β-cell function. In conclusion, this present study shows that exogenous, extracellular Zn7-MT-2A potentiates insulin production and secretions, revealing a possible therapeutic potential of MT administration for enhancing β-cell insulin processing capacity. The authors thank E. Cartsensen for her help with cell cultures and insulin measurements and K. Skjødt for her help with the Q-PCR technique. INS-1E cells were kindly provided by Professor Claes Wollheim and Pierre Maechler, Geneva, Switzerland. The authors declare that there are no conflicts of interest. This present study was funded by the A.P. Møller and Chastine Mc-Kinney Møller Foundation and the Desirée and Niels Ydes Foundation.