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. 2016 Nov 11;291(46):23939-23951.
doi: 10.1074/jbc.M116.748632. Epub 2016 Oct 4.

Hepatic ZIP14-mediated Zinc Transport Contributes to Endosomal Insulin Receptor Trafficking and Glucose Metabolism

Tolunay Beker Aydemir  1 , Catalina Troche  1 , Min-Hyun Kim  1 , Robert J Cousins  2   3
Affiliations

Hepatic ZIP14-mediated Zinc Transport Contributes to Endosomal Insulin Receptor Trafficking and Glucose Metabolism

Tolunay Beker Aydemir et al. J Biol Chem. .

Abstract

Zinc influences signaling pathways through controlled targeted zinc transport. Zinc transporter Zip14 KO mice display a phenotype that includes impaired intestinal barrier function with low grade chronic inflammation, hyperinsulinemia, and increased body fat, which are signatures of diet-induced diabetes (type 2 diabetes) and obesity in humans. Hyperglycemia in type 2 diabetes and obesity is caused by insulin resistance. Insulin resistance results in inhibition of glucose uptake by liver and other peripheral tissues, principally adipose and muscle and with concurrently higher hepatic glucose production. Therefore, modulation of hepatic glucose metabolism is an important target for antidiabetic treatment approaches. We demonstrate that during glucose uptake, cell surface abundance of zinc transporter ZIP14 and mediated zinc transport increases. Zinc is distributed to multiple sites in hepatocytes through sequential translocation of ZIP14 from plasma membrane to early and late endosomes. Endosomes from Zip14 KO mice were zinc-deficient because activities of the zinc-dependent insulin-degrading proteases insulin-degrading enzyme and cathepsin D were impaired; hence insulin receptor activity increased. Transient increases in cytosolic zinc levels are concurrent with glucose uptake and suppression of glycogen synthesis. In contrast, Zip14 KO mice exhibited greater hepatic glycogen synthesis and impaired gluconeogenesis and glycolysis related to low cytosolic zinc levels. We can conclude that ZIP14-mediated zinc transport contributes to regulation of endosomal insulin receptor activity and glucose homeostasis in hepatocytes. Therefore, modulation of ZIP14 transport activity presents a new target for management of diabetes and other glucose-related disorders.

Keywords: Insulin degrading enzyme; gluconeogenesis; glycogen; glycolysis; insulin; mitochondria.

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Figures

FIGURE 1.
FIGURE 1.
Ablation of Zip14 prevents metabolic endotoxemia-induced hepatic insulin insensitivity. The mice were fed either chow diet or HFD for 6 weeks. A, serum endotoxin and insulin were measured by LAL reagents and ELISA, respectively. B, body weight. C, following OGTT, the area under the curve (AUC) was calculated. D, following ITT, the AUC was calculated. E, abundance of ZIP14 and IR pathway proteins were compared in WT and KO mice that were fasted or fed chow or HFD. F, genotypic differences in complex formation between IR and GLUT2. The results are means ± S.E. (n = 5–10 mice). IP, immunoprecipitation; ns, not significant; KOHF, HFD-fed KO mice; WTHF, HFD-fed WT mice.
FIGURE 2.
FIGURE 2.
Hepatic ZIP14 and IR activity during glucose uptake. Following glucose gavage, blood and liver tissue were collected. A, an array of IR pathway proteins was examined by Western blot. B, Glut2 protein and mRNA were measured by Western blot and qPCR, respectively. C, liver glucose were measured by glucose oxidase assay. D, serum insulin concentration was measured by ELISA. E, ZIP14 Western blot. F, liver zinc and Mt mRNA. The results are means ± S.E. (n = 410 mice). *, p < 0.05; #, p < 0.01; ¥, p < 0.001.
FIGURE 3.
FIGURE 3.
Subcellular ZIP14 localization and zinc redistribution during glucose uptake. After overnight serum starvation, HepG2 hepatocytes were treated with insulin (200 nm) and glucose (20 mm) and then harvested at the indicated points. Some HepG2 hepatocytes were transiently transfected with Zip14 siRNA, treated with insulin and glucose and harvested 3 h later. A, abundance of ZIP14 and IR pathway proteins in total cell lysate was examined by Western blot. Control and transfected cells were incubated with the non-metabolizable glucose analog 2NBDG along with insulin, and 2NBDG uptake was measured fluorometrically. Total zinc was measured by AAS. B, glucose uptake over time was determined by measuring both 2NBDG and total glucose. C, intracellular zinc concentration was assessed by AAS and labile zinc by fluorometry using FluoZin3-AM. D, ZIP14 abundance in PM and membrane rafts. E, ZIP14 abundance in cell surface. F, ZIP14 abundance in endosomes by Western blot and endosomal zinc concentrations were determined by fluorometry using FluoZin3-AM. The results are means ± S.E. (n = 3–5). *, p < 0.05; #, p < 0.01; ¥, p < 0.001. CSB, biotinylated cell surface proteins.
FIGURE 4.
FIGURE 4.
Zip14 ablation alters internalized IR activity. Subcellular fractions from liver of WT and KO mice were separated by sucrose gradient ultracentrifugation. A, at 0 and 90 min post-glucose gavage, the amount of ZIP14 and P-IR in early and late endosomes were analyzed by Western blotting (endosomes from n = 4 mice combined per lane). B, at steady state, the amount of ZIP14 and P-IR along with appropriate site markers were analyzed by Western blotting (n = 6 mice combined per lane). C, endosomal zinc concentrations were determined by FluoZin3-AM fluorescence (n = 6). D, cathepsin D activity in endosomes (n = 10). E, IDE activity in endosomes (n = 10). F, proposed model that describes the proposed role of zinc in endosomal IR regulation. The results are means ± S.E. Cyto, cytosol.
FIGURE 5.
FIGURE 5.
Zip14-mediated zinc transport influences hepatic glycogen. Glycogen was isolated from liver by ethanol precipitation, and following glycogen breakdown with amyloglucosidase, glucose was measured to calculate the glycogen content. A, glycogen concentration in HepG2 cells and an array of glycogen synthetic pathway proteins. B, glycogen concentration in HepG2 cells and glycogen synthetic pathway proteins after incubation with differing concentrations of zinc. C, liver glycogen and cytosolic zinc concentrations of WT and ZIP14 KO mice. D, glycogen synthetic pathway proteins in liver. E, liver glycogen concentration and an array of glycogen synthetic pathway proteins following a week of dietary zinc supplementation. F, proposed model for the influence of ZIP14-mediated transport of cytosolic zinc on glycogen synthesis. The results are means ± S.E. (n = 3–5 mice).
FIGURE 6.
FIGURE 6.
Impaired hepatic gluconeogenesis in Zip14 KO mice. A, 2 h after a zinc injection (i.p.), ITT, OGTT, and PTT were conducted. B, 2 h after a zinc injection, liver was collected, and mRNA and protein abundance of Glut2, Pepck, and G6Pase was analyzed. C–E, mice were fed either chow, ZnA, or ZnS. C and D, hepatic mRNA and protein abundance of Pepck (C) and G6Pase (D) were analyzed. E, PTT in ZnA and ZnS-fed mice and AUC of PTT. The results are means ± S.E. (n = 5–10 mice).
FIGURE 7.
FIGURE 7.
Zip14 and Zip8 are involved with ATP synthesis. Mitochondria were purified from HepG2 hepatocytes and mouse liver by Nycodenz gradient ultracentrifugation. A, ZIP14 Western blot from HepG2 lysates. Abundance of ZIP14 and ZIP8 in HepG2 cells during glucose uptake. B, total and mitochondrial zinc and ATP concentrations in HepG2 cells. C, percentage of change in total and mitochondrial zinc and ATP. D, left panel, ZIP14 and ZIP8 abundance in liver. Middle and right panels, percentage of change in hepatic total and mitochondrial zinc and ATP. E, liver pyruvate concentrations. F, proposed model for zinc integration of transporters ZIP14 and ZIP8 and glycolysis and mitochondrial ATP synthesis. The results are means ± S.E. (n = 5). *, p < 0.05; #, p < 0.01; ¥, p < 0.001. P Mit, pure mitochondria; C Mit, crude mitochondria; Mito, mitochondria.
FIGURE 8.
FIGURE 8.
Role of ZIP14 and zinc in hepatic glucose metabolism. Ins, insulin; Mit., mitochondria; Synt., synthesis.
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