Zn²⁺ is required for the activity of many mitochondrial proteins, which regulate mitochondrial dynamics, apoptosis and mitophagy. However, it is not understood how the proper mitochondrial Zn²⁺ level is achieved to maintain mitochondrial homeostasis. Using Caenorhabditis elegans, we reveal here that a pair of mitochondrion-localized transporters controls the mitochondrial level of Zn²⁺. We demonstrate that SLC-30A9/ZnT9 is a mitochondrial Zn²⁺ exporter. Loss of SLC-30A9 leads to mitochondrial Zn²⁺ accumulation, which damages mitochondria, impairs animal development and shortens the life span. We further identify SLC-25A25/SCaMC-2 as an important regulator of mitochondrial Zn²⁺ import. Loss of SLC-25A25 suppresses the abnormal mitochondrial Zn²⁺ accumulation and defective mitochondrial structure and functions caused by loss of SLC-30A9. Moreover, we reveal that the endoplasmic reticulum contains the Zn²⁺ pool from which mitochondrial Zn²⁺ is imported. These findings establish the molecular basis for controlling the correct mitochondrial Zn²⁺ levels for normal mitochondrial structure and functions.
Animals ; Caenorhabditis elegans/metabolism* ; Cation Transport Proteins/genetics* ; Homeostasis ; Mitochondria/metabolism* ; Zinc/metabolism*

Platinum drugs are widely used in the treatment of various solid tumors, but their resistance to platinum is the most significant obstacle to successful treatment. Copper transporter 1 (CTR1) is the specific transporter for copper, and it mainly locates at the plasma membrane and plays a role in pumping copper into the cell. CTR1 is also the major platinum influx transporter and plays a key role in platinum resistance. The expression, polymorphism, and degradation of CTR1 affect platinum resistance in tumors. Therefore, CTR1 may be a potential predictive biomarker of platinum resistance and a therapeutic target for overcoming platinum resistance.
Antineoplastic Agents ; therapeutic use ; Cation Transport Proteins ; genetics ; metabolism ; Cisplatin ; therapeutic use ; Copper ; Copper Transporter 1 ; Drug Resistance, Neoplasm ; genetics ; Platinum ; therapeutic use ; Research ; trends

OBJECTIVETo investigate the mutations of SLC22A5 gene in patients with systemic primary carnitine deficiency (CDSP).

METHODSHigh liquid chromatography tandem mass spectrometry (HPLC/MS/MS) was applied to screen congenital genetic metabolic disease and eight patients with CDSP were diagnosed among 77 511 samples. The SLC22A5 gene mutation was detected using massarray technology and sanger sequencing. Using SIFT and PolyPhen-2 to predict the function of protein for novel variations.

RESULTSTotal detection rate of gene mutation is 100% in the eight patients with CDSP. Seven patients had compound heterozygous mutations and one patient had homozygous mutations. Six different mutations were identified, including one nonsense mutation [c.760C>T(p.R254X)] and five missense mutations[c.51C>G(p.F17L), c.250T>A(p.Y84N), c.1195C>T(p.R399W), c.1196G>A(p.R399Q), c.1400C>G(p.S467C)]. The c.250T>A(p.Y84N) was a novel variation, the novel variation was predicted to have affected protein structure and function. The c.760C>T (p.R254X)was the most frequently seen mutation, which was followed by the c.1400C>G(p.S467C).

CONCLUSIONThis study confirmed the diagnosis of eight patients with CDSP on the gene level. Six mutations were found in the SLC22A5 gene, including one novel mutation which expanded the mutational spectrum of the SLC22A5 gene.

Adult ; Amino Acid Sequence ; Base Sequence ; Cardiomyopathies ; diagnosis ; genetics ; metabolism ; Carnitine ; deficiency ; genetics ; metabolism ; DNA Mutational Analysis ; methods ; Female ; Gene Frequency ; Genotype ; Humans ; Hyperammonemia ; diagnosis ; genetics ; metabolism ; Infant, Newborn ; Male ; Muscular Diseases ; diagnosis ; genetics ; metabolism ; Mutation ; Organic Cation Transport Proteins ; genetics ; metabolism ; Reproducibility of Results ; Sensitivity and Specificity ; Sequence Homology, Amino Acid ; Solute Carrier Family 22 Member 5 ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Objective: To investigate the expressions of solute carrier family 22 member 14 (SLC22A14) and sperm-associated antigen 6 (SPAG6) in the sperm of idiopathic asthenospermia men. METHODS: We collected semen samples from 50 idiopathic asthenozoospermia patients and another 50 normal sperm donors, purified the sperm by discontinuous density centrifugation on Percoll gradients, and then determined the mRNA and protein expressions of SLC22A14 and SPAG6 by RT-PCR and Western blot, respectively. RESULTS: Compared with the normal controls, the idiopathic asthenozoospermia patients showed significantly decreased mRNA expressions of SLC22A14 (0.77 ± 0.08 vs 0.53 ± 0.10, P<0.01) and SPAG6 (0.78 ± 0.09 vs0.52 ± 0.10 , P<0.01) and protein expressions of SLC22A14 (0.80 ± 0.09 vs 0.55 ± 0.10 , P<0.01) and SPAG6 (0.78 ± 0.09 vs 0.56 ± 0.09, P<0.01). CONCLUSIONS T The expressions of SLC22A14 and SPAG6 are reduced in the sperm of the patients with idiopathic asthenospermia, which may be one of the important causes of asthenospermia.
Asthenozoospermia ; metabolism ; Blotting, Western ; Ejaculation ; Humans ; Male ; Microtubule Proteins ; genetics ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Proteomics ; RNA, Messenger ; metabolism ; Sperm Motility ; Spermatozoa ; metabolism

PURPOSE: Diabetic nephropathy (DN) is a prevalent chronic microvascular complication of diabetes mellitus involving disturbances in electrolytes and the acid-base balance caused by a disorder of glucose metabolism. NHE1 is a Na+/H+ exchanger responsible for keeping intracellular pH (pHi) balance and cell growth. Our study aimed to investigate roles of NHE1 in high glucose (HG)-induced apoptosis in renal tubular epithelial cells. MATERIALS AND METHODS: Renal epithelial tubular cell line HK-2 was cultured in medium containing 5 mM or 30 mM glucose. Then, cell apoptosis, oxidative stress, NHE1 expression, and pHi were evaluated. NHE1 siRNA and inhibitor were used to evaluate its role in cell apoptosis. RESULTS: HG significantly increased cell apoptosis and the production of reactive oxygen species (ROS) and 8-OHdG (p<0.05). Meanwhile, we found that HG induced the expression of NHE1 and increased the pHi from 7.0 to 7.6 after 48 h of incubation. However, inhibiting NHE1 using its specific siRNA or antagonist DMA markedly reduced cell apoptosis stimulated by HG. In addition, suppressing cellular oxidative stress using antioxidants, such as glutathione and N-acetyl cysteine, significantly reduced the production of ROS, accompanied by a decrease in NHE1. We also found that activated cyclic GMP-Dependent Protein Kinase Type I (PKG) signaling promoted the production of ROS, which contributed to the regulation of NHE1 functions. CONCLUSION: Our study indicated that HG activates PKG signaling and elevates the production of ROS, which was responsible for the induction of NHE1 expression and dysfunction, as well as subsequent cell apoptosis, in renal tubular epithelial cells.
Antioxidants/metabolism ; Apoptosis/*drug effects ; Cation Transport Proteins/*metabolism ; Cell Cycle/drug effects ; Cell Line ; Dose-Response Relationship, Drug ; Epithelial Cells/*cytology/drug effects/*metabolism ; Glucose/*pharmacology ; Glutathione/metabolism ; Humans ; Kidney Tubules/*cytology ; Oxidative Stress/*drug effects ; Reactive Oxygen Species/metabolism ; Signal Transduction/drug effects ; Sodium-Hydrogen Antiporter/*metabolism

OBJECTIVETo explore the correlation between Pi and Shen by observing the relationship between the metabolism of aristolochic acid (AA) and mRNA and protein expression levels of organic anion transporting polypeptide (oatp) superfamily member 2a1 and 2 b1 (oatp2al and oatp2bl) in renal, small intestinal, and large intestinal tissues of Pi deficiency syndrome (PDS) model rats.

METHODSTotally 46 Sprague-Dawley (SD) rats were randomly divided into four groups, i.e., the blank group (n = 12), the PDS group (n = 22), the AA-I group (n = 6), and the PDS AA-I group (n = 6). PDS model was established by subcutaneously injecting Reserpine at the daily dose of 5 mg/kg for 16 successive days. Carotid intubation was performed in 6 rats selected from the blank group and the PDS group. Pharmacokinetics of AA-I were detected at 5, 15, 30, 45, and 60 min after gastrogavage of AA-I. AA-I concentrations in renal, small intestinal, and large intestinal tissues of 10 rats selected from the PDS group were determined. Normal saline was administered to 6 rats selected from the PDS group and the blank group by gastrogavage. Renal, small intestinal, and large intestinal tissues were collected in the AA-I group and the PDS AA-I group at 60 min after gastrogavage of AA-I. mRNA and protein expression levels of oatp2a1 and oatp2b1 in each tissue were detected using real-time polymerase chain reaction (RT- PCR) and Western blot.

RESULTSCompared with the blank group, plasma concentrations of in vivo AA-I were obviously higher in the PDS group at 15, 30, 45, and 60 min after gastrogavage of AA-I with statistical difference (P < 0.05). Plasma concentrations of AA-I were obviously decreased at 60 min after gastrogavage of AA-I; AA-I concentrations in renal and large intestinal tissues were elevated; AA-I concentrations in small intestinal tissues were obviously reduced in the PDS group. There was no statistical difference in mRNA expression levels of oatp2a1 and oatp2b1 in the aforesaid three tissues of rats between the blank group and the PDS group. Compared with the blank group, mRNA expression levels of oatp2a1 and oatp2b1 decreased in small intestinal tissues of the AA-I group, and the mRNA expression level of oatp2a1 in large intestinal tissues significantly decreased (P < 0.05, P < 0.01). Compared with the PDS group, mRNA expression levels of oatp2a1 and oatp2b1 increased in renal tissues of the PDS AA-I group (P < 0.05); mRNA expression levels of oatp2b1 increased in large intestinal tissues of the PDS AA-I group (P < 0.05).

CONCLUSIONSThe difference in AA-I metabolism might be associated with changed expression levels of oatp2a1 and oatp2b1 in renal, small intestinal, and large intestinal tissues under Pi deficiency induced loss of transportation. Shen and Dachang played important roles in substance metabolism under Pi deficiency state, which proved Pi-Shen correlated in Chinese medical theories.

Animals ; Anions ; Aristolochic Acids ; metabolism ; Drugs, Chinese Herbal ; Kidney ; Medicine, Chinese Traditional ; Organic Cation Transport Proteins ; metabolism ; Peptides ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the potential involvement of DMT1 (IRE) protein in the brain vascular system in vivo during Pb exposure.

METHODSThree groups of male Sprague-Dawley rats were exposed to Pb in drinking water, among which two groups were concurrently administered by oral gavage once every other day as the low and high Fe treatment group, respectively, for 6 weeks. At the same time, the group only supplied with high Fe was also set as a reference. The animals were decapitated, then brain capillary-rich fraction was isolate from cerebral cortex. Western blot method was used to identify protein expression, and RT-PCR to detect the change of the mRNA.

RESULTSPb exposure significantly increased Pb concentrations in cerebral cortex. Low Fe dose significantly reduced the cortex Pb levels, However, high Fe dose increased the cortex Pb levels. Interestingly, changes of DMT1 (IRE) protein in brain capillary-rich fraction were highly related to the Pb level, but those of DMT1 (IRE) mRNA were not significantly different. Moreover, the consistent changes in the levels of p-ERK1/2 or IRP1 with the changes in the levels of DMT1 (IRE).

CONCLUSIONThese results suggest that Pb is transported into the brain through DMT1 (IRE), and the ERK MAPK pathway is involved in DMT1 (IRE)-mediated transport regulation in brain vascular system in vivo.

Animals ; Blood-Brain Barrier ; drug effects ; metabolism ; Cation Transport Proteins ; drug effects ; genetics ; physiology ; Cerebral Cortex ; drug effects ; metabolism ; Dietary Supplements ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Gene Expression Regulation ; drug effects ; Iron ; administration & dosage ; metabolism ; Lead ; administration & dosage ; pharmacokinetics ; MAP Kinase Signaling System ; physiology ; Male ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley

To establish single- and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindIII and Kpn I restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)- hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 µg · mL(-1), all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 µmol · L(-1) cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.
Animals ; Biological Transport ; Cimetidine ; pharmacology ; DNA, Complementary ; Dogs ; Drug Interactions ; Humans ; Madin Darby Canine Kidney Cells ; Metformin ; pharmacology ; Organic Cation Transport Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo evaluate the utility of zinc transporter-8 (ZnT8) in the improvement of type 1 diabetes mellitus (T1DM) diagnosis and prediction, and to explore whether ZnT8 is a potential therapeutic target in T1DM.

DATA SOURCESA search was conducted within the medical database PubMed for relevant articles published from 2001 to 2015. The search terms are as follows: "ZnT8," "type 1 diabetes," "latent autoimmune diabetes in adults," "type 2 diabetes," "islet autoantibodies," "zinc supplement," "T cells," "β cell," "immune therapy." We also searched the reference lists of selected articles.

STUDY SELECTIONEnglish-language original articles and critical reviews concerning ZnT8 and the clinical applications of islet autoantibodies in diabetes were reviewed.

RESULTSThe basic function of ZnT8 is maintaining intracellular zinc homeostasis, which modulates the process of insulin biosynthesis, storage, and secretion. Autoantibodies against ZnT8 (ZnT8A) and ZnT8-specific T cells are the reliable biomarkers for the identification, stratification, and characterization of T1DM. Additionally, the results from the animal models and clinical trials have shown that ZnT8 is a diabetogenic antigen, suggesting the possibility of ZnT8-specific immunotherapy as an alternative for T1DM therapy.

CONCLUSIONSZnT8 is a novel islet autoantigen with a widely potential for clinical applications in T1DM. However, before the large-scale clinical applications, there are still many problems to be solved.

Animals ; Autoantibodies ; immunology ; Autoantigens ; immunology ; Cation Transport Proteins ; immunology ; metabolism ; Diabetes Mellitus, Type 1 ; immunology ; metabolism ; Humans

OBJECTIVETo study the changes in the expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FP1) in the substantia nigra (SN) of rats with manganese-induced parkinsonism.

METHODSEighty Sprague-Dawley rats were randomly divided into four groups. Rats in the control group were injected intraperitoneally with saline solution. Rats in the low-dose, medium-dose, and high-dose groups were injected intraperitoneally with 5, 15, and 20 mg/kg MnC12 solution, respectively, for 16 weeks. Three behavioral tests were performed at the 16th week. The concentration of Mn2+ in the SN was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES), and the positive expression of tyrosine hydroxylase (TH) was measured by immunohistochemical staining to determine whether rats with manganese-induced parkinsonism were successfully produced. The expression of DMT1 and FP1 in SN was measured by immunohistochemical staining and fluorescent quantitative polymerase chain reaction.

RESULTSRats with manganese-induced parkinsonism were successfully produced using the above method. Compared with that in the control group, the concentrations of Mn2+ in the SN of rats exposed to 5, 15, and 20 mg/kg Mn2+ were significantly higher (1.72?0.33 vs 0.56 ± 0.20 µg/g, P<0.01; 2.92±0.77 vs 0.56±0.20 µg/g, P<0.01; 5.65±1.60 vs 0.56±0.20 µg/g, P<0.01). The mean ODs of TH-positive cells in the SN of rats exposed to 5, 15, and 20 mg/kg Mn+ were significantly lower than that in the control group (0.054±0.008 vs 0.109±0.019, P<0.01; 0.016±0.004 vs 0.109±0.019, P<0.01; 0.003±0.001 vs 0.109±0.019, P<0.01). Compared with that in the control group, the mean optical densities (ODs) of DMT1-positive cells in the SN of rats exposed to 15, and 20 mg/kg Mn2+ were significantly higher (0.062±0.004 vs 0.015±0.007, P<0.01; 0.116±0.064 vs 0.015±0.007, P<0.01). The mean ODs of FP1-positive cells in the SN of rats exposed to 5, 15, and 20 mg/kg Mn2+ were significantly lower than that in the control group (0.092±0.011 vs 0.306±0.081, P<0.01; 0.048±0.008 vs 0.306±0.081, P<0.01; 0.008±0.002 vs 0.306±0.081, P< 0.01). Rats exposed to 15 and 20 mg/kg Mn2+ had significantly higher expression of DMT1 mRNA in the SN than those in the control group (0.052±0.0126 vs 0.001±0.0004, P<0.05; 0.124±0.0299 vs 0.001±0.0004, P<0.05). However, rats exposed to 5, 15, and 20 mg/kg Mn2 had significantly lower expression of FP1 mRNA in the SN than those in the control group (0.059±0.0076 vs 0.162±0.0463, P<0.05; 0.033±0.0094 vs 0.162±0.0463, P< 0.05; 0.002±0.0007 vs 0.162±0.0463, P<0.05).

CONCLUSIONThe increased expression of DMT1 and reduced expression of FP1 may be involved in the processes of Mn2+ accumulation in the SN and dopaminergic neuron loss in rats with manganese-induced parkinsonism.

Animals ; Cation Transport Proteins ; metabolism ; Disease Models, Animal ; Manganese ; adverse effects ; Parkinsonian Disorders ; chemically induced ; metabolism ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Substantia Nigra ; metabolism ; physiopathology