Ip3r-Grp75-Vdac and Relevant Ca2+ Signaling Regulate Dietary Palmitic Acid-Induced De Novo Lipogenesis by Mitochondria-Associated ER Membrane (MAM) Recruiting Seipin in Yellow Catfish.

BACKGROUND: The mitochondria-associated endoplasmic reticulum membrane (MAM) is the central hub for endoplasmic reticulum and mitochondria functional communication. It plays a crucial role in hepatic lipid homeostasis. However, even though MAM has been acknowledged to be rich in enzymes that contribute to lipid biosynthesis, no study has yet investigated the exact role of MAM on hepatic neutral lipid synthesis. OBJECTIVES: To address these gaps, this study investigated the systemic control mechanisms of MAM on neutral lipids synthesis by recruiting seipin, focusing on the role of the inositol trisphosphate receptor-1,4,5(Ip3r)-75 kDa glucose-regulated protein (Grp75)-voltage-dependent anion channel (Vdac) complex and their relevant Ca2+ signaling in this process. METHODS: To this end, a model animal for lipid metabolism, yellow catfish (Pelteobagrus fulvidraco), were fed 6 different diets containing a range of palmitic acid (PA) concentrations from 0-150 g/kg in vivo for 10 wk. In vitro, experiments were also conducted to intercept the MAM-mediated Ca2+ signaling in isolated hepatocytes by transfecting them with si-mitochondrial calcium uniporter (mcu). Because mcu was placed in the inner mitochondrial membrane (IMM), si-mcu cannot disrupt MAM's structural integrity. RESULTS: 1. Hepatocellular MAM subproteome analysis indicated excessive dietary PA intake enhanced hepatic MAM structure joined by activating Ip3r-Grp75-Vdac complexes. 2. Dietary PA intake induced hepatic neutral lipid accumulation through MAM recruiting Seipin, which activated lipid droplet biogenesis. Our findings also revealed a previously unidentified mechanism whereby MAM-recruited seipin and controlled hepatic lipid homeostasis, depending on Ip3r-Grp75-Vdac-controlled Ca2+ signaling and not only MAM's structural integrity. CONCLUSIONS: These results offer a novel insight into the MAM-recruited seipin in controlling hepatic lipid synthesis in a MAM structural integrity-dependent and Ca2+ signaling-dependent manner, highlighting the critical contribution of MAM in maintaining hepatic neutral lipid homeostasis.

Klf4-Sirt3/Pparα-Lcad pathway contributes to high phosphate-induced lipid degradation.

BACKGROUND: Phosphorus commonly reduces lipid deposition in the vertebrates. However, the underlying mechanisms involved in the process remain unclear. METHODS: Yellow catfish were given three experimental diets with dietary phosphate levels of 3.22, 6.47 and 7.99 g Pi kg- 1, respectively, for 8 weeks. The contents of triglyceride, non-esterified free fatty acids, adenosine triphosphate, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide, enzymatic activities, mRNA and protein expression were determined in the intestinal tissues. Hematoxylin and eosin, Oil Red O staining, and transmission electron microscope were performed for intestinal tissues. Primary intestinal epithelial cells were isolated from yellow catfish intestine. Western blot analysis, Immunoprecipitation assays, Immunofluorescence staining, and RNA extraction and quantitative real-time PCR were decided. Luciferase reporter assays and electrophoretic mobility shift assay were used to evaluate the function of Sirt3, PPARα and Lcad promoters. RESULTS: High dietary phosphate intake activated intestinal phosphate absorption and excretion, and reduced lipid deposition through increasing lipolysis in the intestine. Moreover, phosphate incubation increased the mRNA and protein expression of krüppel like factor 4 (klf4), silent mating-type information regulation 2 homolog 3 (sirt3), peroxisome proliferator activated receptor alpha (pparα) and long chain acyl-CoA dehydrogenase (lcad) in the intestinal epithelial cells (IECs), and klf4 knockdown attenuated the phosphate-induced increase of protein levels of Sirt3, Pparα and Lcad. Further investigation found that Klf4 overexpression increased the activity of sirt3 and pparα promoters, which in turn reduced the acetylation and protein level of Lcad. CONCLUSION: Dietary Pi excess induced lipid degradation by the activation of the Klf4-Sirt3/Pparα-Lcad pathway in the intestine and primary IECs. Video Abstract.

Sirt3-Sod2-mROS-Mediated Manganese Triggered Hepatic Mitochondrial Dysfunction and Lipotoxicity in a Freshwater Teleost.

Exposure to excessive manganese (Mn) is toxic to humans and animals. However, the toxic effects and mechanisms of excessive Mn influencing the vertebrates have been highly overlooked. In the present study, dietary Mn overload significantly increased hepatic lipid and Mn contents, decreased superoxide dismutase 2 (Sod2) activity, increased the Sod2 acetylation level, and induced mitochondrial dysfunction; Mn induced mitochondrial dysfunction through Mtf1/sirtuin 3 (Sirt3)-mediated acetylation of Sod2 at the sites K55 and K70. Meanwhile, mitochondrial oxidative stress was involved in Mn-induced lipotoxicity. Mechanistically, Mn-induced lipotoxicity was via oxidative stress-induced Hsf1 nucleus translocation and its DNA binding capacity to the regions of a peroxisome proliferator-activated receptor g (pparg) promoter, which in turn induced the transcription of lipogenic-related target genes. For the first time, our study demonstrated that Mn-induced hepatic lipotoxicity via a mitochondrial oxidative stress-dependent Hsf1/Pparg pathway and Mtf1/sirt3-mediated Sod2 acetylation participated in mitochondrial dysfunction. Considering that lipid metabolism and lipotoxicity are widely used as the biomarkers for environmental assessments of pollutants, our study provided innovative and important insights into Mn toxicological and environmental evaluation in aquatic environments.

Mitochondria-Dependent Oxidative Stress Mediates ZnO Nanoparticle (ZnO NP)-Induced Mitophagy and Lipotoxicity in Freshwater Teleost Fish.

Due to many special characteristics, zinc oxide nanoparticles (ZnO NPs) are widely used all over the world, leading to their wide distribution in the environment. However, the toxicities and mechanisms of environmental ZnO NP-induced changes of physiological processes and metabolism remain largely unknown. Here, we found that addition of dietary ZnO NPs disturbed hepatic Zn metabolism, increased hepatic Zn and lipid accumulation, downregulated lipolysis, induced oxidative stress, and activated mitophagy; N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, Zn2+ ions chelator) alleviated high ZnO NP-induced Zn and lipid accumulation, oxidative stress, and mitophagy. Mechanistically, the suppression of mitochondrial oxidative stress attenuated ZnO NP-activated mitophagy and ZnO NP-induced lipotoxicity. Taken together, our study elucidated that mitochondrial oxidative stress mediated ZnO NP-induced mitophagy and lipotoxicity; ZnO NPs could be dissociated to free Zn2+ ions, which partially contributed to ZnO NP-induced changes in oxidative stress, mitophagy, and lipid metabolism. Our study provides novel insights into the impacts and mechanism of ZnO NPs as harmful substances inducing lipotoxicity of aquatic organisms, and accordingly, metabolism-relevant parameters will be useful for the risk assessment of nanoparticle materials in the environment.

The ZIP6/ZIP10 heteromer is essential for the zinc-mediated trigger of mitosis.

Zinc has been known to be essential for cell division for over 40 years but the molecular pathways involved remain elusive. Cellular zinc import across biological membranes necessitates the help of zinc transporters such as the SLC39A family of ZIP transporters. We have discovered a molecular process that explains why zinc is required for cell division, involving two highly regulated zinc transporters, as a heteromer of ZIP6 and ZIP10, providing the means of cellular zinc entry at a specific time of the cell cycle that initiates a pathway resulting in the onset of mitosis. Crucially, when the zinc influx across this heteromer is blocked by ZIP6 or ZIP10 specific antibodies, there is no evidence of mitosis, confirming the requirement for zinc influx as a trigger of mitosis. The zinc that influxes into cells to trigger mitosis additionally changes the phosphorylation state of STAT3 converting it from a transcription factor to a protein that complexes with this heteromer and pS38Stathmin, the form allowing microtubule rearrangement as required in mitosis. This discovery now explains the specific cellular role of ZIP6 and ZIP10 and how they have special importance in the mitosis process compared to other ZIP transporter family members. This finding offers new therapeutic opportunities for inhibition of cell division in the many proliferative diseases that exist, such as cancer.

Dietary Choline Alleviates High-Fat Diet-Induced Hepatic Lipid Dysregulation via UPRmt Modulated by SIRT3-Mediated mtHSP70 Deacetylation.

The mitochondrial unfolded protein response (UPRmt) is known as a conservative mechanism in response to mitochondrial dysfunction. Thus, based on UPRmt, this study was conducted to determine the mechanism of a high-fat diet (HFD) inducing mitochondrial dysfunction and its role in stimulating hepatic lipid dysregulation. The choline-activated alleviating effect was also evaluated. In vivo, yellow catfish were fed three diets (control, HFD, and HFD + choline diet) for 10 weeks. In vitro, hepatocytes isolated from yellow catfish and the HepG2 cell line were cultured and incubated with fatty acid (FA) for 48 h. (1) HFD-induced mitochondrial dysfunction via SIRT3/mtHSP70-mediated UPRmt. HFD inhibited the subcellular localization of SIRT3 into the mitochondrion, resulting in the up-regulating of mtHSP70 acetylation via lysine residues 493 and 507. The mtHSP70 acetylation promoted the stability of mtHSP70, which then led to the UPRmt and further mitochondrial dysfunction. (2) SIRT3/mtHSP70-mediated UPRmt regulated HFD/FA-induced hepatic lipid dysregulation. SIRT3/mtHSP70-mediated UPRmt reduced FA ß-oxidation via mitochondrial dysfunction and then led to lipid dysregulation. Additionally, the mtHSP70-ACOX1 interaction was confirmed. (3) Choline alleviated HFD-induced UPRmt via up-regulating the localization of SIRT3 into the mitochondrion, which in turn led to the subsequent ameliorating effect on HFD-induced hepatic lipid dysregulation. Through SIRT3-mediated mtHSP70 deacetylation, dietary choline alleviates HFD-induced hepatic lipid dysregulation via UPRmt. This provides the first proof of acetylation regulating UPRmt and the crosstalk between UPRmt and FA ß-oxidation.

Effect of Water pH on the Uptake of Acidic (Ibuprofen) and Basic (Propranolol) Drugs in a Fish Gill Cell Culture Model.

Water pH is predicted to affect the uptake of ionizable pharmaceuticals in fish. The current study used an in vitro primary fish gill cell culture system to assess the effect of pH values in the range of 4.5-8.75 on the uptake rates of the base propranolol (pKa 9.42) and the acid ibuprofen (pKa 4.59). The rate-limiting step in the uptake was the diffusive supply flux of the unionized form from the water to the apical membrane, with subsequent rapid transfer across the epithelium. Computed uptake rate based on the unionized fraction best described the uptake of propranolol and ibuprofen over the range of pH values 5-8 and 6-8.75, respectively. For ibuprofen, the computed uptake rate overestimated the uptake below pH 6 where the unionized fraction increased from 4% at pH 6 to 55% at pH 4.5. As the unionized fraction increased, the uptake rate plateaued suggesting a saturation of the transport process. For both drugs, large variations in the uptake occur with only small fluctuations in pH values. This occurs between pH values 6 and 8, which is the pH range acceptable in regulatory test guidelines and seen in most of our freshwaters.

Zn Induces Lipophagy via the Deacetylation of Beclin1 and Alleviates Cu-Induced Lipotoxicity at Their Environmentally Relevant Concentrations.

In this study, the mechanisms of environmentally relevant doses of Cu and Zn mixtures influencing lipid deposition and metabolism were investigated in freshwater teleost yellow catfish Pelteobagrus fulvidraco (2 months old, 4.95 (t0.01 g, mean ± SEM). Our study indicated that waterborne Cu exposure increased lipid content, while Zn activated lipophagic flux and alleviated Cu-induced lipid accumulation. Yellow catfish hepatocytes treated with Zn or Zn + Cu activated autophagy-specific lipophagy, decreased lipid storage, and increased nonesterified fatty acid (NEFA) release, suggesting a causal relationship between lipophagy and lipid droplet (LD) breakdown under Zn and Zn + Cu conditions. Our further investigation found that Beclin1 deacetylation by sirtuin 1 (SIRT1) was required for Zn- and Zn + Cu-induced lipophagy and lipolysis, and lysine residues 427 and 434 were key sites for Beclin1 deacetylation. Taken together, these findings show that the Zn-induced deacetylation of Beclin1 promotes lipophagy as an important pathway to alleviate Cu-induced lipid accumulation in fish, which reveals a previously unidentified mechanism for understanding the antagonistic effects of Cu and Zn on metabolism at their environmentally relevant concentrations. Our results highlight the importance of combined exposure when the biological effects of heavy metals are evaluated during environmental risk assessments.

Lipophagy mediated carbohydrate-induced changes of lipid metabolism via oxidative stress, endoplasmic reticulum (ER) stress and ChREBP/PPARγ pathways.

High-carbohydrate diets (HCD) can induce the occurrence of nonalcoholic fatty liver disease (NAFLD), characterized by dramatic accumulation of hepatic lipid droplets (LDs). However, the potential molecular mechanisms are still largely unknown. In this study, we investigated the role of autophagy in the process of HCD-induced changes of hepatic lipid metabolism, and to examine the process of underlying mechanisms during these molecular contexts. We found that HCD significantly increased hepatic lipid accumulation and activated autophagy. Using primary hepatocytes, we found that HG increased lipid accumulation and stimulated the release of NEFA by autophagy-mediated lipophagy, and that lipophagy significantly alleviated high glucose (HG)-induced lipid accumulation. Oxidative and endoplasmic reticulum (ER) stress pathways played crucial regulatory roles in HG-induced lipophagy activation and HG-induced changes of lipid metabolism. Further investigation found that HG-activated lipophagy and HG-induced changes of lipid metabolism were via enhancing carbohydrate response element-binding protein (ChREBP) DNA binding capacity at PPARγ promoter region, which in turn induced transcriptional activation of the key genes related to lipogenesis and autophagy. The present study, for the first time, revealed the novel mechanism for lipophagy mediating HCD-induced changes of lipid metabolism by oxidative stress and ER stress, and ChREBP/PPARγ pathways. Our study provided innovative evidence for the direct relationship between carbohydrate and lipid metabolism via ChREBP/PPARγ pathway.

Hatching gland development and hatching in zebrafish embryos: A role for zinc and its transporters Zip10 and Znt1a.

Zinc transporters of the ZIP (Slc39, importers) and ZnT (Slc30, exporters) protein families have evolutionary conserved roles in biology. The aim of the present study was to explore the role of zinc, and zinc transporters Zip10 and Znt1a in zebrafish hatching gland development and larval hatching. In the study, knockdown of genes for Zip10 and Znt1a in zebrafish embryos was achieved using morpholino-modified oligonucleotides. A partial loss-of-function Znt1a mutant (Znt1asa17) allowed comparison with the Znt1a morphant. Free Zn2+ in embryos and apoptosis were investigated using fluorescent dyes whereas gene expression was investigated by whole-mount in situ hybridization (WISH). The results showed high levels of free Zn2+ in the hatching gland cells (HGC) along with abundant expression of zip10 and znt1a in normal embryo. Knockdown of zip10 reduced free Zn2+ in HGC, ceased their normal developmental apoptosis, and resulted in displacement and later disappearance of hatching glands and hatching enzymes he1a and catL1b, and inability to hatch. Conversely, knockdown of znt1a or the Znt1asa17 mutation accelerated hatching and coincided with high expression of hatching enzymes and free Zn2+ in the HGC. Thus, Zip10 and free Zn2+ in the HGC are required both for their development and function. This study also demonstrated the opposite functions of the two zinc transporters, ZIP10 and ZnT1 as well as shedding light on the role of Zn2+ in regulation of the human hatching enzyme homologue, ovastacin, which is activated by zinc and cleaves the zona pellucida protein, ZP2, to prevent polyspermy.

FXR-mediated inhibition of autophagy contributes to FA-induced TG accumulation and accordingly reduces FA-induced lipotoxicity.

BACKGROUND: Excessive dietary fat intake induces lipid deposition and contributes to the progress of nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanisms are still unclear. METHODS: Yellow catfish were given two experimental diets with dietary lipid levels of 11.3 and 15.4%, respectively, for 56 days, and the contents of triglyceride (TG), nonesterified free fatty acids (NEFA) and bile acid (BA), RNA-seq, enzymatic activities and mRNA expression were deteremined in the liver tissues. Hepatocytes from yellow catfish liver tissues were isolated and cultured. Fatty acids (FA) (palmitic acid: OA, oleic acid =1:1), pathway inhibitors (MA, autophagy inhibitor; guggulsterone, FXR inhibitor) and agonist (rapamyicn, autophagy agonist; GW4064, FXR agonist) were used to incubate the cells. TG and NEFA contents, ultrastructural observation, autophagic vesicles and intracellular LD,apoptosis,western blot and Co-IP, and Immunofluorescence analysis, enzymatic activities and Q-PCR were decided. RESULTS: Using RNA sequencing, we found that high fat diets induced changes in expression of many genes associated with the pathways of lipid metabolism and autophagy. The mRNA profiles of the differentially expressed genes (DEG) indicated that high dietary fat-induced lipid deposition was predominantly influenced by the inhibition of autophagy. Using primary hepatocytes, we found that fatty acids (FA) suppressed autophagy, which in turn reduced cellular free FA level by decreasing triglyceride (TG) breakdown. Moreover, our study indicated that farnesoid X receptor (FXR)-cyclic AMP-responsive element-binding protein (CREB) axis was the pivotal physiological switch regulating FA-induced changes of autophagy and lipid metabolism, which represented cellular defenses against FA-induced lipotoxicity. CONCLUSION: This discovery may provide new targets for treating pathological changes involved in the dysfunction of autophagy and metabolism, including NAFLD. Video Abstract.

Waterborne copper exposure up-regulated lipid deposition through the methylation of GRP78 and PGC1α of grass carp Ctenopharyngodon idella.

Early molecular events after the exposure of heavy metals, such as aberrant DNA methylation, suggest that DNA methylation was important in regulating physiological processes for animals and accordingly could be used as environmental biomarkers. In the present study, we found that copper (Cu) exposure increased lipid content and induced the DNA hypermethylation at the whole genome level. Especially, Cu induced hypermethylation of glucose-regulated protein 78 (grp78) and peroxisome proliferator-activated receptor gamma coactivator-1α (pgc1α). CCAAT/enhancer binding protein α (C/EBPα) could bind to the methylated sequence of grp78, whereas C/EBPß could not bind to the methylated sequence of grp78. These synergistically influenced grp78 expression and increased lipogenesis. In contrast, DNA methylation of PGC1α blocked the specific protein 1 (SP1) binding and interfered mitochondrial function. Moreover, Cu increased reactive oxygen species (ROS) production, activated endoplasmic reticulum (ER) stress and damaged mitochondrial function, and accordingly increased lipid deposition. Notably, we found a new toxicological mechanism for Cu-induced lipid deposition at DNA methylation level. The measurement of DNA methylation facilitated the use of these epigenetic biomarkers for the evaluation of environmental risk.

Zinc reduces hepatic lipid deposition and activates lipophagy via Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways.

Zinc (Zn) deficiency is the most consistently discovered nutritional manifestations of fatty liver disease. Although Zn is known to stimulate hepatic lipid oxidation, little is known about its underlying mechanism of action in lipolysis. Given the potential role of lipophagy in lipid metabolism, the purpose of this study was to test the hypothesis that Zn attenuates hepatic lipid accumulation by modulating lipophagy. The present study indicated that Zn is a potent promoter of lipophagy. Zn administration significantly alleviated hepatocellular lipid accumulation and increased the release of free fatty acids in association with enhanced fatty acid oxidation and inhibited lipogenesis, which was accompanied by activation of autophagy. Moreover, Zn reduced lipid accumulation and stimulated lipolysis by autophagy-mediated lipophagy. Zn-induced up-regulation of autophagy and lipid depletion is free Zn2+-dependent in the cytosols. Zn-induced autophagy and lipid turnover involved up-regulation of the calcium/calmodulin-dependent protein kinase kinase-ß (Ca2+/CaMKKß)/AMPK pathway. Meanwhile, Zn2+-activated autophagy and lipid depletion were via enhancing metal response element-binding transcription factor (MTF)-1 DNA binding at PPARα promoter region, which in turn induced transcriptional activation of the key genes related to autophagy and lipolysis. Zn activated the pathways of Zn2+/MTF-1/ Peroxisome proliferator-activated receptor (PPAR)α and Ca2+/CaMKKß/AMPK, resulting in the up-regulation of lipophagy and accordingly reduced hepatic lipid accumulation. Our study, for the first time, provided innovative evidence of the direct relationship between metal elements (Zn) and lipid metabolism. The present study also indicated the novel mechanism for Zn-induced lipolysis by the activation of Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways, which induced the occurrence of lipophagy. These results provide new insight into Zn nutrition and its potential beneficial effects on the prevention of fatty liver disease in vertebrates.-Wei, C.-C., Luo, Z., Hogstrand, C., Xu, Y.-H., Wu, L.-X., Chen, G.-H., Pan, Y.-X., Song, Y.-F. Zinc reduces hepatic lipid deposition and activates lipophagy via Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways.

The Use of Molecular Descriptors To Model Pharmaceutical Uptake by a Fish Primary Gill Cell Culture Epithelium.

Modeling approaches such as quantitative structure-activity relationships (QSARs) use molecular descriptors to predict the bioavailable properties of a compound in biota. However, these models have mainly been derived based on empirical data for lipophilic neutral compounds and may not predict the uptake of ionizable compounds. The majority of pharmaceuticals are ionizable, and freshwaters can have a range of pH values that affect speciation. In this study, we assessed the uptake of 10 pharmaceuticals (acetazolamide, beclomethasone, carbamazepine, diclofenac, gemfibrozil, ibuprofen, ketoprofen, norethindrone, propranolol, and warfarin) with differing modes of action and physicochemical properties (p Ka, log S, log D, log Kow, molecular weight (MW), and polar surface area (PSA)) by an in vitro primary fish gill cell culture system (FIGCS) for 24 h in artificial freshwater. Principal component analysis (PCA) and partial least-squares (PLS) regression was used to determine the molecular descriptors that influence the uptake rates. Ionizable drugs were taken up by FIGCS; a strong positive correlation was observed between log S and the uptake rate, and a negative correlation was observed between p Ka, log D, and MW and the uptake rate. This approach shows that models can be derived on the basis of the physicochemical properties of pharmaceuticals and the use of an in vitro gill system to predict the uptake of other compounds. There is a need for a robust and validated model for gill uptake that could be used in a tiered risk assessment to prioritize compounds for experimental testing.

ZnT8 Haploinsufficiency Impacts MIN6 Cell Zinc Content and ß-Cell Phenotype via ZIP-ZnT8 Coregulation.

The zinc transporter ZnT8 (SLC30A8) localises to insulin secretory granules of ß-cells where it facilitates zinc uptake for insulin crystallisation. ZnT8 abundance has been linked to ß-cell survival and functional phenotype. However, the consequences of ZnT8 haploinsufficiency for ß-cell zinc trafficking and function remain unclear. Since investigations in human populations have shown SLC30A8 truncating polymorphisms to decrease the risk of developing Type 2 Diabetes, we hypothesised that ZnT8 haploinsufficiency would improve ß-cell function and maintain the endocrine phenotype. We used CRISPR/Cas9 technology to generate ZnT8 haploinsufficient mouse MIN6 ß-cells and showed that ZnT8 haploinsufficiency is associated with downregulation of mRNAs for Slc39a8 and Slc39a14, which encode for the zinc importers, Znt- and Irt-related proteins 8 (ZIP8) and 14 (ZIP14), and with lowered total cellular zinc content. ZnT8 haploinsufficiency disrupts expression of a distinct array of important ß-cell markers, decreases cellular proliferation via mitogen-activated protein (MAP) kinase cascades and downregulates insulin gene expression. Thus, ZnT8 cooperates with zinc importers of the ZIP family to maintain ß-cell zinc homeostasis. In contrast to the hypothesis, lowered ZnT8 expression reduces MIN6 cell survival by affecting zinc-dependent transcription factors that control the ß-cell phenotype.

Role of Zinc and Magnesium Ions in the Modulation of Phosphoryl Transfer in Protein Tyrosine Phosphatase 1B.

While the majority of phosphatases are metalloenzymes, the prevailing model for the reactions catalyzed by protein tyrosine phosphatases does not involve any metal ion, yet both metal cations and oxoanions affect their enzymatic activity. Mg2+ and Zn2+ activate and inhibit, respectively, protein tyrosine phosphatase 1B (PTP1B). Molecular dynamics simulations, metadynamics, and quantum chemical calculations in combination with experimental investigations demonstrate that Mg2+ and Zn2+ compete for the same binding site in the active site only in the closed conformation of the enzyme in its phosphorylated state. The two cations have different effects on the arrangements and activities of water molecules that are necessary for the hydrolysis of the phosphocysteine intermediate in the second catalytic step of the reaction. Remarkable differences between the established structural enzymology of PTP1B investigated ex vivo and the function of PTP1B in vivo become evident. Different reaction pathways are viable when the presence of metal ions and their cellular concentrations are considered. The findings suggest that the substrate delivers the inhibitory Zn2+ ion to the active site. The inhibition and activation can be ascribed to the different coordination chemistries of Zn2+ and Mg2+ ions and the orientation of the metal-coordinated water molecules. Metallochemistry adds an additional dimension to the regulation of PTP1B and presumably other members of this enzyme family.

Biokinetic Modeling of Cd Bioaccumulation from Water, Diet and Sediment in a Marine Benthic Goby: A Triple Stable Isotope Tracing Technique.

Aquatic animals are often simultaneously exposed to metals through multiple routes in the natural environment. This study explored a triple stable isotope tracing method to quantify simultaneous cadmium (Cd) uptake biokinetics by yellow stripe goby from water (traced by 110Cd), sediment (traced by 111Cd), and diet (traced by 113Cd) when the fish were exposed to Cd for 24 h. The simultaneous uptake of Cd from multiple routes during 4 weeks was then predicted by the modified biokinetic model. The results demonstrated that the uptake rate constant of waterborne 110Cd, sediment-associated 111Cd, and dietary 113Cd was 3.1 L kg-1 d-1, 2.2 × 10-4 g g-1 d-1, and 3.3 × 10-3 g g-1 d-1 in the fish. Sedimentary Cd was less bioavailable than the waterborne and dietary Cd; however, sediment could become the predominant Cd source of the total Cd bioaccumulation when the partition coefficient of Cd between sediment and seawater ( Kd) is larger than 6 × 104 L kg-1. The simultaneous uptake of Cd from the three routes could be successfully predicted by the modified model. The model revealed that the Cd bioaccumulation generally increased with the increase of ambient Cd concentration in all the three routes. Overall, our findings demonstrated that the multiple stable isotopes tracing method and the modified biokinetic model have a wide generality and applicability for predicting Cd bioaccumulation under multiple routes of metal exposure scenario and may have application to other metals.

Zn Stimulates the Phospholipids Biosynthesis via the Pathways of Oxidative and Endoplasmic Reticulum Stress in the Intestine of Freshwater Teleost Yellow Catfish.

The hypothesis of our study was that waterborne Zn exposure evoked phospholipids (PL) biosynthesis to compensate for the loss of membrane integrity, and the pathways of oxidative stress and endoplasmic reticulum (ER) stress mediated the Zn-evoked changes of PL biosynthesis. Thus, we conducted RNA sequencing to analyze the differences in the intestinal transcriptomes between the control and Zn-treated P. fulvidraco. The 56-day Zn exposure increased the intestinal Zn accumulation, and mRNA levels of 816 genes were markedly up-regulated, while that of 263 genes were down-regulated. Many differentially expressed genes in the pathways of PL biosynthesis and protein processing in ER were identified. Their expression profiles indicated that waterborne Zn exposure injured protein metabolism, induced PL biosynthesis caused oxidative stress and ER stress, and activated the unfolded protein response. Then, using the primary enterocytes, we identified the mechanism of oxidative and ER stress mediating Zn-induced PL biosynthesis, and indicated that the activation of these pathways constituted adaptive mechanisms to reduce Zn toxicity. Our study demonstrated that Zn exposure via the water increased Zn accumulation and PL biosynthesis, and that oxidative stress and ER stress were interdependent and mediated the Zn-induced PL biosynthesis of the intestine in the freshwater teleost.

Expression of the ZIP/SLC39A transporters in ß-cells: a systematic review and integration of multiple datasets.

BACKGROUND: Pancreatic ß-cells require a constant supply of zinc to maintain normal insulin secretory function. Following co-exocytosis with insulin, zinc is replenished via the Zrt- and Irt-like (ZIP; SLC39A) family of transporters. However the ZIP paralogues of particular importance for zinc uptake, and associations with ß-cell function and Type 2 Diabetes remain largely unexplored. We retrieved and statistically analysed publically available microarray and RNA-seq datasets to perform a systematic review on the expression of ß-cell SLC39A paralogues. We complemented results with experimental data on expression profiling of human islets and mouse ß-cell derived MIN6 cells, and compared transcriptomic and proteomic sequence conservation between human, mouse and rat. RESULTS: The 14 ZIP paralogues have 73-98% amino sequence conservation between human and rodents. We identified 18 datasets for ß-cell SLC39A analysis, which compared relative expression to non-ß-cells, and expression in response to PDX-1 activity, cytokines, glucose and type 2 diabetic status. Published expression data demonstrate enrichment of transcripts for ZIP7 and ZIP9 transporters within rodent ß-cells and of ZIP6, ZIP7 and ZIP14 within human ß-cells, with ZIP1 most differentially expressed in response to cytokines and PDX-1 within rodent, and ZIP6 in response to diabetic status in human and glucose in rat. Our qPCR expression profiling data indicate that SLC39A6, -9, -13, and - 14 are the highest expressed paralogues in human ß-cells and Slc39a6 and -7 in MIN6 cells. CONCLUSIONS: Our systematic review, expression profiling and sequence alignment reveal similarities and potentially important differences in ZIP complements between human and rodent ß-cells. We identify ZIP6, ZIP7, ZIP9, ZIP13 and ZIP14 in human and rodent and ZIP1 in rodent as potentially biologically important for ß-cell zinc trafficking. We propose ZIP6 and ZIP7 are key functional orthologues in human and rodent ß-cells and highlight these zinc importers as important targets for exploring associations between zinc status and normal physiology of ß-cells and their decline in Type 2 Diabetes.

Dietary zinc addition influenced zinc and lipid deposition in the fore- and mid-intestine of juvenile yellow catfish Pelteobagrus fulvidraco.

The present study explored the mechanisms of dietary Zn influencing Zn and lipid deposition in the fore- and mid- intestine in yellow catfish Pelteobagrus fulvidraco, and investigated whether the mechanism was intestinal-region dependent. For this purpose, yellow catfish were fed three diets containing Zn levels of 8·83, 19·20 and 146·65 mg Zn/kg, respectively. Growth performance, intestinal TAG and Zn contents as well as activities and mRNA expression of enzymes and genes involved in Zn transport and lipid metabolism in the fore- and mid-intestine were analysed. Dietary Zn increased Zn accumulation as well as activities of Cu-, Zn-superoxide dismutase and ATPase in the fore- and mid-intestine. In the fore-intestine, dietary Zn up-regulated mRNA levels of ZnT1, ZnT5, ZnT7, metallothionein (MT) and metal response element-binding transcription factor-1 (MTF-1), but down-regulated mRNA levels of ZIP4 and ZIP5. In the mid-intestine, dietary Zn up-regulated mRNA levels of ZnT1, ZnT5, ZnT7, MT and MTF-1, but down-regulated mRNA levels of ZIP4 and ZIP5. Dietary Zn reduced TAG content, down-regulated activities of 6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME) and fatty acid synthase (FAS) activities, and reduced mRNA levels of 6PGD, G6PD, FAS, PPARγ and sterol-regulator element-binding protein (SREBP-1), but up-regulated mRNA levels of carnitine palmitoyltransferase IA, hormone-sensitive lipase (HSLa), adipose TAG lipase (ATGL) and PPARα in the fore-intestine. In the mid-intestine, dietary Zn reduced TAG content, activities of G6PD, ME, isocitrate dehydrogenase and FAS, down-regulated mRNA levels of 6PGD, G6PD, FAS, acetyl-CoA carboxylase a, PPARγ and SREBP-1, but up-regulated mRNA expression of HSLa, ATGL and PPARγ. The reduction in TAG content following Zn addition was attributable to reduced lipogenesis and increased lipolysis, and similar regulatory mechanisms were observed between the fore- and mid-intestine.