RESUMEN
Zinc is an essential component of the insulin protein complex synthesized in ß cells. The intracellular compartmentalization and distribution of zinc are controlled by 24 transmembrane zinc transporters belonging to the ZnT or Zrt/Irt-like protein (ZIP) family. Downregulation of SLC39A14/ZIP14 has been reported in pancreatic islets of patients with type 2 diabetes (T2D) as well as mouse models of high-fat diet (HFD)- or db/db-induced obesity. Our previous studies observed mild hyperinsulinemia in mice with whole body knockout of Slc39a14 (Zip14 KO). Based on our current secondary data analysis from an integrative single-cell RNA-seq dataset of human whole pancreatic tissue, SLC39A14 (coding ZIP14) is the only other zinc transporter expressed abundantly in human ß cells besides well-known zinc transporter SLC30A8 (coding ZnT8). In the present work, using pancreatic ß cell-specific knockout of Slc39a14 (ß-Zip14 KO), we investigated the role of SLC39A14/ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses. Glucose-stimulated insulin secretion, zinc concentrations, and cellular localization of ZIP14 were assessed using in vivo, ex vivo, and in vitro assays using ß-Zip14 KO, isolated islets, and murine cell line MIN6. Metabolic evaluations were done on both chow- and HFD-fed mice using time-domain nuclear magnetic resonance and a comprehensive laboratory animal monitoring system. ZIP14 localizes on the endoplasmic reticulum regulating intracellular zinc trafficking in ß cells and serves as a negative regulator of glucose-stimulated insulin secretion. Deletion of Zip14 resulted in greater glucose-stimulated insulin secretion, increased energy expenditure, and shifted energy metabolism toward fatty acid utilization. HFD caused ß-Zip14 KO mice to develop greater islet hyperplasia, compensatory hyperinsulinemia, and mild insulin resistance and hyperglycemia. This study provided new insights into the contribution of metal transporter ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses.NEW & NOTEWORTHY Metal transporter SLC39A14/ZIP14 is downregulated in pancreatic islets of patients with T2D and mouse models of HFD- or db/db-induced obesity. However, the function of ZIP14-mediated intracellular zinc trafficking in ß cells is unknown. Our analyses revealed that SLC39A14 is the only Zn transporter expressed abundantly in human ß cells besides SLC30A8. Within the ß cells, ZIP14 is localized on the endoplasmic reticulum and serves as a negative regulator of insulin secretion, providing a potential therapeutic target for T2D.
Asunto(s)
Proteínas de Transporte de Catión , Diabetes Mellitus Tipo 2 , Hiperinsulinismo , Células Secretoras de Insulina , Humanos , Ratones , Animales , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Obesidad/genética , Obesidad/metabolismo , Zinc/metabolismo , Ratones NoqueadosRESUMEN
Objective: Zinc is an essential micronutrient that is critical for many physiological processes, including glucose metabolism, regulation of inflammation, and intestinal barrier function. Further, zinc dysregulation is associated with an increased risk of chronic inflammatory diseases such as type II diabetes, obesity, and inflammatory bowel disease. However, whether altered zinc status is a symptom or cause of disease onset remains unclear. Common symptoms of these three chronic diseases include the onset of increased intestinal permeability and zinc dyshomeostasis. The specific focus of this work is to investigate how dietary sources of intestinal permeability, such as high sucrose consumption, impact transporter-mediated zinc homeostasis and subsequent zinc-dependent physiology contributing to disease development. Method: We used in vivo subchronic sucrose treatment, ex vivo intestinal organoid culture, and in vitro cell systems. We analyze the alterations in zinc metabolism and intestinal permeability and metabolic outcomes. Results: We found that subchronic sucrose treatment resulted in systemic changes in steady-state zinc distribution and increased 65Zn transport (blood-to-intestine) along with greater ZIP14 expression at the basolateral membrane of the intestine. Further, sucrose treatment enhanced cell survival of intestinal epithelial cells, activation of the EGFR-AKT-STAT3 pathway, and intestinal permeability. Conclusion: Our work suggests that subchronic high sucrose consumption alters systemic and intestinal zinc homeostasis linking diet-induced changes in zinc homeostasis to the intestinal permeability and onset of precursors for chronic disease.
RESUMEN
Manganese exposure produces Parkinson's-like neurologic symptoms, suggesting a selective dysregulation of dopamine transmission. It is unknown, however, how manganese accumulates in dopaminergic brain regions or how it regulates the activity of dopamine neurons. Our in vivo studies in male C57BLJ mice suggest that manganese accumulates in dopamine neurons of the VTA and substantia nigra via nifedipine-sensitive Ca2+ channels. Manganese produces a Ca2+ channel-mediated current, which increases neurotransmitter release and rhythmic firing activity of dopamine neurons. These increases are prevented by blockade of Ca2+ channels and depend on downstream recruitment of Ca2+-activated potassium channels to the plasma membrane. These findings demonstrate the mechanism of manganese-induced dysfunction of dopamine neurons, and reveal a potential therapeutic target to attenuate manganese-induced impairment of dopamine transmission.SIGNIFICANCE STATEMENT Manganese is a trace element critical to many physiological processes. Overexposure to manganese is an environmental risk factor for neurologic disorders, such as a Parkinson's disease-like syndrome known as manganism. We found that manganese concentration-dependently increased the excitability of dopamine neurons, decreased the amplitude of action potentials, and narrowed action potential width. Blockade of Ca2+ channels prevented these effects as well as manganese accumulation in the mouse midbrain in vivo Our data provide a potential mechanism for manganese regulation of dopaminergic neurons.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Manganeso/metabolismo , Manganeso/toxicidad , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de ÓrganosRESUMEN
Skeletal muscle represents the largest pool of body zinc, however, little is known about muscle zinc homeostasis or muscle-specific zinc functions. Zip14 (Slc39a14) was the most highly expressed zinc transporter in skeletal muscle of mice in response to LPS-induced inflammation. We compared metabolic parameters of skeletal muscle from global Zip14 knockout (KO) and wild-type mice (WT). At basal steady state Zip14 KO mice exhibited a phenotype that included muscle wasting and metabolic endotoxemia. Microarray and qPCR analysis of gastrocnemius muscle RNA revealed that ablation of Zip14 produced increased muscle p-Mef2c, Hspb7 and miR-675-5p expression and increased p38 activation. ChIP assays showed enhanced binding of NF-[Formula: see text] to the Mef2c promoter. In contrast, LPS-induced systemic inflammation enhanced Zip14-dependent zinc uptake by muscle, increased expression of Atrogin1 and MuRF1 and markedly reduced MyoD. These signatures of muscle atrophy and cachexia were not influenced by Zip14 ablation, however. LPS-induced miR-675-3p and -5p expression was Zip14-dependent. Collectively, these results with an integrative model are consistent with a Zip14 function in skeletal muscle at steady state that supports myogenesis through suppression of metabolic endotoxemia and that Zip14 ablation coincides with sustained activity of phosphorylated components of signaling pathways including p-Mef2c, which causes Hspb7-dependent muscle wasting.
Asunto(s)
Proteínas de Transporte de Catión/deficiencia , Endotoxemia , Proteínas de Choque Térmico HSP27/metabolismo , MicroARNs/metabolismo , Músculo Esquelético/metabolismo , Síndrome Debilitante , Animales , Proteínas de Transporte de Catión/metabolismo , Endotoxemia/genética , Endotoxemia/metabolismo , Eliminación de Gen , Proteínas de Choque Térmico HSP27/genética , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones , Ratones Noqueados , MicroARNs/genética , Síndrome Debilitante/genética , Síndrome Debilitante/metabolismoRESUMEN
Hyperostosis Cranialis Interna (HCI) is a rare bone disorder characterized by progressive intracranial bone overgrowth at the skull. Here we identified by whole-exome sequencing a dominant mutation (L441R) in SLC39A14 (ZIP14). We show that L441R ZIP14 is no longer trafficked towards the plasma membrane and excessively accumulates intracellular zinc, resulting in hyper-activation of cAMP-CREB and NFAT signaling. Conditional knock-in mice overexpressing L438R Zip14 in osteoblasts have a severe skeletal phenotype marked by a drastic increase in cortical thickness due to an enhanced endosteal bone formation, resembling the underlying pathology in HCI patients. Remarkably, L438R Zip14 also generates an osteoporotic trabecular bone phenotype. The effects of osteoblastic overexpression of L438R Zip14 therefore mimic the disparate actions of estrogen on cortical and trabecular bone through osteoblasts. Collectively, we reveal ZIP14 as a novel regulator of bone homeostasis, and that manipulating ZIP14 might be a therapeutic strategy for bone diseases.
Asunto(s)
Proteínas de Transporte de Catión/genética , Homeostasis/genética , Hiperostosis/genética , Mutación , Osteosclerosis/genética , Base del Cráneo/anomalías , Animales , Línea Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Hiperostosis/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/citología , Osteoblastos/metabolismo , Osteosclerosis/metabolismo , Transducción de Señal/genética , Base del Cráneo/metabolismo , Zinc/metabolismoRESUMEN
Mutations in human ZIP14 have been linked to symptoms of the early onset of Parkinsonism and Dystonia. This phenotype is likely related to excess manganese accumulation in the CNS. The metal transporter ZIP14 (SLC39A14) is viewed primarily as a zinc transporter that is inducible via proinflammatory stimuli. In vitro evidence shows that ZIP14 can also transport manganese. To examine a role for ZIP14 in manganese homeostasis, we used Zip14 knock-out (KO) male and female mice to conduct comparative metabolic, imaging, and functional studies. Manganese accumulation was fourfold to fivefold higher in brains of Zip14 KO mice compared with young adult wild-type mice. There was less accumulation of subcutaneously administered 54Mn in the liver, gallbladder, and gastrointestinal tract of the KO mice, suggesting that manganese elimination is impaired with Zip14 ablation. Impaired elimination creates the opportunity for atypical manganese accumulation in tissues, including the brain. The intensity of MR images from brains of the Zip14 KO mice is indicative of major manganese accumulation. In agreement with excessive manganese accumulation was the impaired motor function observed in the Zip14 KO mice. These results also demonstrate that ZIP14 is not essential for manganese uptake by the brain. Nevertheless, the upregulation of signatures of brain injury observed in the Zip14 KO mice demonstrates that normal ZIP14 function is an essential factor required to prevent manganese-linked neurodegeneration.SIGNIFICANCE STATEMENT Manganese is an essential micronutrient. When acquired in excess, manganese accumulates in tissues of the CNS and is associated with neurodegenerative disease, particularly Parkinson-like syndrome and dystonia. Some members of the ZIP metal transporter family transport manganese. Using mutant mice deficient in the ZIP14 metal transporter, we have discovered that ZIP14 is essential for manganese elimination via the gastrointestinal tract, and a lack of ZIP14 results in manganese accumulation in critical tissues such as the brain, as measured by MRI, and produces signatures of brain injury and impaired motor function. Humans with altered ZIP14 function would lack this gatekeeper function of ZIP14 and therefore would be prone to manganese-related neurological diseases.
Asunto(s)
Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Intoxicación por Manganeso/genética , Intoxicación por Manganeso/metabolismo , Manganeso/metabolismo , Actividad Motora/genética , Animales , Química Encefálica/genética , Femenino , Motilidad Gastrointestinal/genética , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distribución Tisular , Zinc/metabolismo , Zinc/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Endosomas/metabolismo , Glucosa/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Receptor de Insulina/metabolismo , Zinc/metabolismo , Animales , Proteínas de Transporte de Catión/genética , Endosomas/genética , Glucosa/genética , Glucógeno/biosíntesis , Glucógeno/genética , Ratones , Ratones Noqueados , Transporte de Proteínas/fisiología , Receptor de Insulina/genéticaRESUMEN
Inflammation and zinc dyshomeostasis are two common hallmarks of aging. A major zinc transporter ZIP14 (slc39a14) is upregulated by proinflammatory stimuli, e.g. interleukin-6. We have evaluated the influence of age on the Zip14 KO phenotype using wild-type (WT) and Zip14 knockout (KO) mice. Aging produced a major increase in serum IL-6 concentrations that was dramatically augmented in the Zip14 KO mice. In keeping with enhanced serum IL-6 concentrations, aging produced tissue-specific increases in zinc concentration of skeletal muscle and white adipose tissue. Metabolic endotoxemia produced by Zip14 ablation is maintained in aged KO mice. Muscle non-heme iron (NHI) was increased in aged WT mice but not in aged Zip14 KO mice demonstrating NHI uptake by muscle is ZIP14-dependent and increases with age. NF-κB and STAT3 activation was greater in aged mice, but was tissue specific and inversely related to tissue zinc. Micro-CT analysis revealed that Zip14 KO mice had markedly reduced trabecular bone that was greatly amplified with aging. These results demonstrate that the inflammation-responsive zinc transporter ZIP14 has phenotypic effects that are amplified with aging.
Asunto(s)
Envejecimiento/metabolismo , Proteínas de Transporte de Catión/genética , Zinc/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Hueso Esponjoso/metabolismo , Eliminación de Gen , Inflamación/metabolismo , Interleucina-6/sangre , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
To evaluate the effect of diet on metabolic control and zinc metabolism in patients with type 2 diabetes mellitus (T2DM). One-week balanced diet was provided to 10 Brazilians patients with T2DM. Nutritional assessment, laboratorial parameters and expression of zinc transporter and inflammatory genes in peripheral blood mononuclear cells (PBMC) were performed. Healthy non-diabetic subjects of the same demographic were recruited to provide baseline data. Diabetic patients had higher body mass index and greater fasting plasma glucose, plasma tumor necrosis factor α (TNFα) and plasma interleukin 6 (IL6) levels compared with healthy subjects. In addition, the expression of transporters 4 (ZnT4) mRNA was lower and IL6 mRNA was higher in PBMC of these diabetic patients than in healthy subject. One week after a balanced diet was provided, fasting plasma glucose decreased significantly as did TNFα, IL6 and Metallothionein 1 (MT1) mRNAs. No change was observed in zinc transporter expression in PBMC after the dietary intervention. A healthy eating pattern maintained for one week was able to improve metabolic control of diabetic patients by lowering fasting plasma glucose. This metabolic control may be related to down-regulation of zinc-related transcripts from PBMCs, as TNFα, IL6 and MT1 mRNA.
RESUMEN
Zinc is a signaling molecule in numerous metabolic pathways, the coordination of which occurs through activity of zinc transporters. The expression of zinc transporter Zip14 (Slc39a14), a zinc importer of the solute carrier 39 family, is stimulated under proinflammatory conditions. Adipose tissue upregulates Zip14 during lipopolysaccharide-induced endotoxemia. A null mutation of Zip14 (KO) revealed that phenotypic changes in adipose include increased cytokine production, increased plasma leptin, hypertrophied adipocytes, and dampened insulin signaling. Adipose tissue from KO mice had increased levels of preadipocyte markers, lower expression of the differentiation marker (PPARγ), and activation of NF-κB and STAT3 pathways. Our overall hypothesis was that ZIP14 would play a role in adipocyte differentiation and inflammatory obesity. Global Zip14 KO causes systemic endotoxemia. The observed metabolic changes in adipose metabolism were reversed when oral antibiotics were administrated, indicating that circulating levels of endotoxin were in part responsible for the adipose phenotype. To evaluate a mechanism, 3T3-L1 cells were differentiated into adipocytes and treated with siRNA to knock down Zip14. These cells had an impaired ability to mobilize zinc, which caused dysregulation of inflammatory pathways (JAK2/STAT3 and NF-κB). The Zip14 deletion may limit the availability of intracellular zinc, yielding the unique phenotype of inflammation coupled with hypertrophy. Taken together, these results suggest that aberrant zinc distribution observed with Zip14 ablation impacts adipose cytokine production and metabolism, ultimately increasing fat deposition when exposed to endotoxin. To our knowledge, this is the first investigation into the mechanistic role of ZIP14 in adipose tissue regulation and metabolism.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo Blanco/metabolismo , Proteínas de Transporte de Catión/genética , Citocinas/metabolismo , Endotoxemia/metabolismo , Células 3T3-L1 , Adipocitos/patología , Tejido Adiposo , Tejido Adiposo Blanco/patología , Adiposidad , Animales , Western Blotting , Proteínas de Transporte de Catión/metabolismo , Diferenciación Celular , Técnicas de Silenciamiento del Gen , Hipertrofia , Inflamación , Janus Quinasa 2/metabolismo , Leptina/metabolismo , Lipopolisacáridos , Ratones , Ratones Noqueados , Microscopía Confocal , FN-kappa B/metabolismo , PPAR gamma/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de SeñalRESUMEN
Zinc transporters have been characterized to further understand the absorption and metabolism of dietary zinc. Our goal was to characterize zinc transporter Slc39a11 (ZIP11) expression and its subcellular localization within cells of the murine gastrointestinal tract of mice and to determine if dietary zinc regulates ZIP11. The greatest ZIP11 expression was in the stomach, cecum, and colon. Both Zip11 mRNA and ZIP11 protein were shown to be downregulated during dietary zinc restriction (<1 mg Zn/kg) in the murine stomach tissue but were unaffected in the colon. Acute repletion with zinc did not restore Zip11 mRNA levels in the stomach. Immunohistochemistry (IHC) revealed high ZIP11 levels in the lower regions of gastric glands and parietal cells of the stomach. IHC analysis of the colon showed a marked ZIP11 abundance within the cytoplasm of the colonic epithelial cells. IHC also showed an increase in ZIP11 expression in the colon during zinc restriction. There is a robust abundance of ZIP11 in the nuclei of cells of both stomach and colon. Our experiments suggest that when dietary zinc intake is compromised, the colon may increase zinc transporter expression to improve the efficiency for absorption via increased expression of specific zinc transporters, including ZIP11 and also zinc transporter Slc39a4. In conclusion, ZIP11 is highly expressed within the murine stomach and colon and appears to be partially regulated by dietary zinc intake within these tissues. ZIP11 may play a specialized role in zinc homeostasis within these tissues, helping to maintain mucosal integrity and function.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Núcleo Celular/efectos de los fármacos , Colon/metabolismo , Mucosa Gástrica/metabolismo , Zinc/farmacología , Animales , Secuencia de Bases , Proteínas de Transporte de Catión/genética , Cartilla de ADN , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Zinc/administración & dosificaciónRESUMEN
ZIP14 (slc39A14) is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia) of acute inflammation. ZIP14 can transport Zn(2+) and non-transferrin-bound Fe(2+) in vitro. Using a Zip14(-/-) mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using ZIP14(-/-) mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, Zip14(-/-) mice absorbed more iron. The Zip14 KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of Zip14-/- mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The Zip14(-/-) phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The Zip14 KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are altered in the Zip14(-/-) mice and their phenotype shows defects in glucose homeostasis.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Endotoxemia/metabolismo , Glucosa/metabolismo , Inmunidad Innata/fisiología , Hierro/metabolismo , Hígado/metabolismo , Zinc/metabolismo , Proteínas de Transporte de Catión/genética , Endotoxemia/genética , Femenino , Humanos , Inmunidad Innata/genética , MasculinoRESUMEN
BACKGROUND & AIMS: Zinc homeostasis in cells is maintained through tight regulation of zinc influx, efflux, and distribution to intracellular organelles by zinc transporters. The Zrt-Irt-like protein (ZIP) transporters facilitate zinc influx to the cytosol. Expression of the ZIP family member Zip14 can be induced by inflammatory cytokines, which also initiate liver regeneration. Hepatocyte proliferation is required for liver regeneration. Zinc regulates cell proliferation, tissue growth, and many mitogenic signaling pathways; we investigated its role in hepatocytes. METHODS: Wild-type and Zip14(-/-) mice that underwent partial hepatectomy (70% of liver removed) were used as models of liver regeneration. We also analyzed AML12 hepatocytes that overexpressed Zip14. Proliferation was assessed with proliferating cell nuclear antigen, CD1, and Ki67 markers and along with assays of zinc content was related to protein tyrosine phosphatase 1B (PTP1B) and extracellular signal-regulated kinase 1/2 signaling. RESULTS: Zip14 was up-regulated and hepatic zinc content increased during liver regeneration. Increased hepatic zinc inhibited activity of the phosphatase PTP1B and increased phosphorylation of c-Met, which promoted hepatocyte proliferation. AML12 cells that overexpressed Zip14 increased in zinc content and proliferation; PTP1B was inhibited and phosphorylation of c-Met increased. The increases in hepatic levels of zinc and hepatocyte proliferation that occurred following partial hepatectomy were not observed in Zip14(-/-) mice. CONCLUSIONS: The transporter Zip14 mediates hepatic uptake of zinc during liver regeneration and for hepatocyte proliferation. These findings indicate that zinc transporter activity regulates liver tissue growth by sequestering zinc. Reagents that regulate ZIP14 activity might be developed as therapeutics to promote liver regeneration in patients with chronic liver disease.
Asunto(s)
Proteínas de Transporte de Catión/farmacología , Proliferación Celular/efectos de los fármacos , Hepatocitos/metabolismo , Regeneración Hepática/fisiología , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Línea Celular , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Regulación hacia Arriba , Zinc/metabolismo , Zinc/farmacologíaRESUMEN
An effective measure to assess zinc status of humans has remained elusive, in contrast to iron, where a number of indicators of metabolism/function are available. Using monocytes, T lymphocytes, and granulocytes isolated by magnetic sorting and dried blood spots (DBS) derived from 50 mul of peripheral blood, we evaluated the response of metallothionein (MT), zinc transporter, and cytokine genes to a modest (15 mg of Zn per day) dietary zinc supplement in human subjects. Transcript abundance was measured by quantitative real-time RT-PCR (QRT-PCR). Zinc supplementation increased MT mRNA abundance by up to 2-fold in RNA from leukocyte subsets, and 4-fold in RNA from DBS. Transcript levels for the zinc transporter genes ZnT1 and Zip3 were increased and decreased, respectively, by zinc supplementation. Expression of the ZnT and Zip genes among leukocyte subsets differ by up to 270-fold. Monocytes and granulocytes from supplemented subjects were activated by LPS, whereas T lymphocytes were activated by mimicking antigen presentation. With zinc consumption, TNF-alpha and IL-1beta expression was greater in activated monocytes and granulocytes, and IFN-gamma mRNA levels were higher in activated T lymphocytes. These studies show that QRT-PCR is a tool to reliably measure transcript abundance for nutritionally responsive genes in human subjects, and that a small sample of whole dried blood, when appropriately collected, can be used as the source of total RNA for QRT-PCR analysis. The results obtained also show that zinc supplementation of human subjects programs specific leukocytic subsets to show enhanced cytokine expression upon activation by stimulators of immunity.
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Proteínas de Transporte de Catión/genética , Citocinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Metalotioneína/genética , Zinc/farmacología , Adulto , Suplementos Dietéticos , Humanos , Masculino , ARN Mensajero/genética , Transcripción Genética/genética , Zinc/administración & dosificaciónRESUMEN
Infection and inflammation produce systemic responses that include hypozincemia and hypoferremia. The latter involves regulation of the iron transporter ferroportin 1 by hepcidin. The mechanism of reduced plasma zinc is not known. Transcripts of the two zinc transporter gene families (ZnT and Zip) were screened for regulation in mouse liver after turpentine-induced inflammation and LPS administration. Zip14 mRNA was the transporter transcript most up-regulated by inflammation and LPS. IL-6 knockout (IL-6(-/-)) mice did not exhibit either hypozincemia or the induction of Zip14 with turpentine inflammation. However, in IL-6(-/-) mice, LPS produced a milder hypozincemic response but no Zip14 induction. Northern analysis showed Zip14 up-regulation was specific for the liver, with one major transcript. Immunohistochemistry, using an antibody to an extracellular Zip14 epitope, showed both LPS and turpentine increased abundance of Zip14 at the plasma membrane of hepatocytes. IL-6 produced increased expression of Zip14 in primary hepatocytes cultures and localization of the protein to the plasma membrane. Transfection of mZip14 cDNA into human embryonic kidney cells increased zinc uptake as measured by both a fluorescent probe for free Zn(2+) and (65)Zn accumulation, as well as by metallothionein mRNA induction, all indicating that Zip14 functions as a zinc importer. Zip14 was localized in plasma membrane of the transfected cells. These in vivo and in vitro experiments demonstrate that Zip14 expression is up-regulated through IL-6, and that this zinc transporter most likely plays a major role in the mechanism responsible for hypozincemia that accompanies the acute-phase response to inflammation and infection.