Ith Emixustat (hydrochloride) decreased Akt and GSK3b phosphorylation and nuclear accumulation of Fyn. The expression of Nrf2 (A) and total and phosphorylated Akt (B) and GSK3b (C) were examined with Western blotting. The expression of Fyn in nuclei, cytosol, and total tissues were measured by Western blotting, for which the ratios of Fyn-N/Fyn-T (D) and Fyn-C/Fyn-T (E) were presented. Data are presented as mean 6 SD (n = 6 at least in each group). DM: diabetes; Fyn- N: Nuclear Fyn; Fyn- C: cytosolic Fyn; Fyn- T: Total tissue Fyn. * P,0.05 vs. control group; # P,0.05 vs. TPEN group; P,0.05 vs. DM group. doi:10.1371/journal.pone.0049257.gTPEN-induced hepatic cell death was rescued by supplementation of ZnTo investigate whether TPEN increased hepatic damage in the animal model is due to Zn deficiency, rather other TPEN’s direct toxicity, we have performed an in vitro study, in which HepG2 cells were exposed to TPEN at different doses (0.1?.0 mM) for 30 h to induce the cell death. 22948146 We found that 30-h exposure 
to TPEN at 0.5 and 1.0 mM induced a significant increase in apoptotic cell death, shown 15481974 by DNA fragmentation (Fig. 7A). We also found that when cells were exposed to 1.0 mM TPEN with and without Zn, the addition of Zn at 30?0 mM in the medium can completely rescue TPEN-induced apoptotic effect (Fig. 7B), suggesting that TPEN-induced apoptotic effect is due to its Zn chelation effect, instead of its direct toxic effect.DiscussionIn the present study, we have demonstrated the hepatic injury, including inflammatory response, lipid accumulation, and hepaticZn Deficiency Exacerbates Diabetic Liver InjuryFigure 6. Diabetes and TPEN treatment activated Akt negative regulators. Hepatic expressions of TRB3 (A), PTP1B (B), and phosphorylated PTEN (C) were examined by Western blotting. Data are presented as mean 6 SD (n = 6 at least in each group). DM: diabetes. * P,0.05 vs. control group; # P,0.05 vs. TPEN group; P,0.05 vs. DM group. doi:10.1371/journal.pone.0049257.gcell death along with the increased serum hepatic enzyme, in the type 1 diabetic animals. Diabetes-induced hepatic injury was Lixisenatide chemical information exacerbated by Zn deficiency induced by chronic treatment with TPEN. Nrf2 as an important transcription factor was found to be decreased in the liver of diabetic and Zn deficient groups, and further decreased in the liver of diabetic mice with Zn deficiency (Diabetes/TPEN). We also found that Zn deficiency exacerbated diabetic inhibition of Akt and GSK-3b phosphorylation along with an up-regulation of Akt negative regulators. The decreased phosphorylation of GSK-3b is accompanied with a significant increase in nuclear accumulation and decrease in cytosolic accumulation of Fyn. Therefore, we concluded that Zn deficiency significantly exacerbates diabetes-induced hepatic damage, which is likely because Zn deficiency exacerbates diabetic downregulation of Nrf2 expression and function by up-regulation of Akt negative regulators. Up-regulated Akt negative regulators down-regulate the phosphorylation of Akt and GSK-3b, leading to Fyn nuclear translocation that exports Nrf2 to cytosol where being degraded, as shown in Fig. 8. Although TPEN is a multi-heavy metal chelator, it has very high affinity for Zn and iron, and very low affinities for other metals such as calcium and magnesium [25]. Zn is the most abundant trace metal in the human body and has long been known to be an essential element for cell metabolism, antioxidation and survival [26,27]. Zn deficiency will lead to cell metabol.Ith decreased Akt and GSK3b phosphorylation and nuclear accumulation of Fyn. The expression of Nrf2 (A) and total and phosphorylated Akt (B) and GSK3b (C) were examined with Western blotting. The expression of Fyn in nuclei, cytosol, and total tissues were measured by Western blotting, for which the ratios of Fyn-N/Fyn-T (D) and Fyn-C/Fyn-T (E) were presented. Data are presented as mean 6 SD (n = 6 at least in each group). DM: diabetes; Fyn- N: Nuclear Fyn; Fyn- C: cytosolic Fyn; Fyn- T: Total tissue Fyn. * P,0.05 vs. control group; # P,0.05 vs. TPEN group; P,0.05 vs. DM group. doi:10.1371/journal.pone.0049257.gTPEN-induced hepatic cell death was rescued by supplementation of ZnTo investigate whether TPEN increased hepatic damage in the animal model is due to Zn deficiency, rather other TPEN’s direct toxicity, we have performed an in vitro study, in which HepG2 cells were exposed to TPEN at different doses (0.1?.0 mM) for 30 h to induce the cell death. 22948146 We found that 30-h exposure to TPEN at 0.5 and 1.0 mM induced a significant increase in apoptotic cell death, shown 15481974 by DNA fragmentation (Fig. 7A). We also found that when cells were exposed to 1.0 mM TPEN with and without Zn, 
the addition of Zn at 30?0 mM in the medium can completely rescue TPEN-induced apoptotic effect (Fig. 7B), suggesting that TPEN-induced apoptotic effect is due to its Zn chelation effect, instead of its direct toxic effect.DiscussionIn the present study, we have demonstrated the hepatic injury, including inflammatory response, lipid accumulation, and hepaticZn Deficiency Exacerbates Diabetic Liver InjuryFigure 6. Diabetes and TPEN treatment activated Akt negative regulators. Hepatic expressions of TRB3 (A), PTP1B (B), and phosphorylated PTEN (C) were examined by Western blotting. Data are presented as mean 6 SD (n = 6 at least in each group). DM: diabetes. * P,0.05 vs. control group; # P,0.05 vs. TPEN group; P,0.05 vs. DM group. doi:10.1371/journal.pone.0049257.gcell death along with the increased serum hepatic enzyme, in the type 1 diabetic animals. Diabetes-induced hepatic injury was exacerbated by Zn deficiency induced by chronic treatment with TPEN. Nrf2 as an important transcription factor was found to be decreased in the liver of diabetic and Zn deficient groups, and further decreased in the liver of diabetic mice with Zn deficiency (Diabetes/TPEN). We also found that Zn deficiency exacerbated diabetic inhibition of Akt and GSK-3b phosphorylation along with an up-regulation of Akt negative regulators. The decreased phosphorylation of GSK-3b is accompanied with a significant increase in nuclear accumulation and decrease in cytosolic accumulation of Fyn. Therefore, we concluded that Zn deficiency significantly exacerbates diabetes-induced hepatic damage, which is likely because Zn deficiency exacerbates diabetic downregulation of Nrf2 expression and function by up-regulation of Akt negative regulators. Up-regulated Akt negative regulators down-regulate the phosphorylation of Akt and GSK-3b, leading to Fyn nuclear translocation that exports Nrf2 to cytosol where being degraded, as shown in Fig. 8. Although TPEN is a multi-heavy metal chelator, it has very high affinity for Zn and iron, and very low affinities for other metals such as calcium and magnesium [25]. Zn is the most abundant trace metal in the human body and has long been known to be an essential element for cell metabolism, antioxidation and survival [26,27]. Zn deficiency will lead to cell metabol.
