Free Cholesterol Affects the Function and Localization of Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) and Organic Cation Transporter 1 (OCT1)

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are associated with obesity. They are accompanied by increased levels of free cholesterol in the liver. Most free cholesterol resides within the plasma membrane. We assessed the impact of adding or removing free cholesterol on the function and localization of two hepatocellular uptake transporters: the Na⁺/taurocholate cotransporting polypeptide (NTCP) and the organic cation transporter 1 (OCT1). We used a cholesterol–MCD complex (cholesterol) to add cholesterol and methyl-β-cyclodextrin (MCD) to remove cholesterol. Our results demonstrate that adding cholesterol decreases NTCP capacity from 132 ± 20 to 69 ± 37 µL/mg/min and OCT1 capacity from 209 ± 66 to 125 ± 26 µL/mg/min. Removing cholesterol increased NTCP and OCT1 capacity to 224 ± 65 and 279 ± 20 µL/mg/min, respectively. In addition, adding cholesterol increased the localization of NTCP within lipid rafts, while adding or removing cholesterol increased OCT1 localization in lipid rafts. These results demonstrate that increased cholesterol levels can impair NTCP and OCT1 function, suggesting that the free cholesterol content of the liver can alter bile acid and drug uptake into the liver. This could explain the increased plasma bile acid levels in NAFLD and NASH patients and potentially lead to altered drug disposition.     

NPC1L1 and SR-BI are Involved in Intestinal Cholesterol Absorption from Small-Size Lipid Donors

In the human intestinal content after a meal, cholesterol is dispersed in a complex mixture of emulsified droplets, vesicles, mixed micelles and precipitated material. The aim of this study was to determine the contribution of the main intestinal cholesterol transporters (NPC1L1, SR-BI) to the absorption processes, using different cholesterol-solubilizing donors. Cholesterol donors prepared with different taurocholate concentrations were added to an apical medium of differentiated TC7/Caco-2 cells. As the taurocholate concentrations increased, cholesterol donor size decreased (from 712 to 7 nm in diameter), which enhanced cholesterol absorption in a dose-dependent manner (38-fold). Two transport processes were observed: (1) absorption from large donors exhibited low-capacity transport with no noticeable transporter contribution; (2) efficient cholesterol absorption occurs from small lipid donors (相似文献   

Drosophila Solute Carrier 5A5 Regulates Systemic Glucose Homeostasis by Mediating Glucose Absorption in the Midgut

The small intestine is the initial site of glucose absorption and thus represents the first of a continuum of events that modulate normal systemic glucose homeostasis. A better understanding of the regulation of intestinal glucose transporters is therefore pertinent to our efforts in curbing metabolic disorders. Using molecular genetic approaches, we investigated the role of Drosophila Solute Carrier 5A5 (dSLC5A5) in regulating glucose homeostasis by mediating glucose uptake in the fly midgut. By genetically knocking down dSLC5A5 in flies, we found that systemic and circulating glucose and trehalose levels are significantly decreased, which correlates with an attenuation in glucose uptake in the enterocytes. Reciprocally, overexpression of dSLC5A5 significantly increases systemic and circulating glucose and trehalose levels and promotes glucose uptake in the enterocytes. We showed that dSLC5A5 undergoes apical endocytosis in a dynamin-dependent manner, which is essential for glucose uptake in the enterocytes. Furthermore, we showed that the dSLC5A5 level in the midgut is upregulated by glucose and that dSLC5A5 critically directs systemic glucose homeostasis on a high-sugar diet. Together, our studies have uncovered the first Drosophila glucose transporter in the midgut and revealed new mechanisms that regulate glucose transporter levels and activity in the enterocyte apical membrane.     

Early Postnatal Exposure to a Low Dose of Nanoparticulate Silver Induces Alterations in Glutamate Transporters in Brain of Immature Rats

Due to strong antimicrobial properties, silver nanoparticles (AgNPs) are used in a wide range of medical and consumer products, including those dedicated for infants and children. While AgNPs are known to exert neurotoxic effects, current knowledge concerning their impact on the developing brain is scarce. During investigations of mechanisms of neurotoxicity in immature rats, we studied the influence of AgNPs on glutamate transporter systems which are involved in regulation of extracellular concentration of glutamate, an excitotoxic amino acid, and compared it with positive control—Ag citrate. We identified significant deposition of AgNPs in brain tissue of exposed rats over the post-exposure time. Ultrastructural alterations in endoplasmic reticulum (ER) and Golgi complexes were observed in neurons of AgNP-exposed rats, which are characteristics of ER stress. These changes presumably underlie substantial long-lasting downregulation of neuronal glutamate transporter EAAC1, which was noted in AgNP-exposed rats. Conversely, the expression of astroglial glutamate transporters GLT-1 and GLAST was not affected by exposure to AgNPs, but the activity of the transporters was diminished. These results indicate that even low doses of AgNPs administered during an early stage of life create a substantial risk for health of immature organisms. Hence, the safety of AgNP-containing products for infants and children should be carefully considered.     

Molecular Regulation of Canalicular ABC Transporters

The ATP-binding cassette (ABC) transporters expressed at the canalicular membrane of hepatocytes mediate the secretion of several compounds into the bile canaliculi and therefore play a key role in bile secretion. Among these transporters, ABCB11 secretes bile acids, ABCB4 translocates phosphatidylcholine and ABCG5/G8 is responsible for cholesterol secretion, while ABCB1 and ABCC2 transport a variety of drugs and other compounds. The dysfunction of these transporters leads to severe, rare, evolutionary biliary diseases. The development of new therapies for patients with these diseases requires a deep understanding of the biology of these transporters. In this review, we report the current knowledge regarding the regulation of canalicular ABC transporters’ folding, trafficking, membrane stability and function, and we highlight the role of molecular partners in these regulating mechanisms.     

Urea-Peptide Hybrids as VEGF-A165/NRP-1 Complex Inhibitors with Improved Receptor Affinity and Biological Properties

Neuropilin-1 (NRP-1), the major co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2), may also independently act with VEGF-A₁₆₅ to stimulate tumour growth and metastasis. Therefore, there is great interest in compounds that can block VEGF-A165/NRP-1 interaction. Peptidomimetic type inhibitors represent a promising strategy in the treatment of NRP-1-related disorders. Here, we present the synthesis, affinity, enzymatic stability, molecular modeling and in vitro binding evaluation of the branched urea–peptide hybrids, based on our previously reported Lys(hArg)-Dab-Oic-Arg active sequence, where the Lys(hArg) branching has been modified by introducing urea units to replace the peptide bond at various positions. One of the resulting hybrids increased the affinity of the compound for NRP-1 more than 10-fold, while simultaneously improving resistance for proteolytic stability in serum. In addition, ligand binding to NRP-1 induced rapid protein stock exocytotic trafficking to the plasma membrane in breast cancer cells. Examined properties characterize this compound as a good candidate for further development of VEGF₁₆₅/NRP-1 inhibitors.     

Interaction Profiles of Central Nervous System Active Drugs at Human Organic Cation Transporters 1–3 and Human Plasma Membrane Monoamine Transporter

Many psychoactive compounds have been shown to primarily interact with high-affinity and low-capacity solute carrier 6 (SLC6) monoamine transporters for norepinephrine (NET; norepinephrine transporter), dopamine (DAT; dopamine transporter) and serotonin (SERT; serotonin transporter). Previous studies indicate an overlap between the inhibitory capacities of substances at SLC6 and SLC22 human organic cation transporters (SLC22A1–3; hOCT1–3) and the human plasma membrane monoamine transporter (SLC29A4; hPMAT), which can be classified as high-capacity, low-affinity monoamine transporters. However, interactions between central nervous system active substances, the OCTs, and the functionally-related PMAT have largely been understudied. Herein, we report data from 17 psychoactive substances interacting with the SLC6 monoamine transporters, concerning their potential to interact with the human OCT isoforms and hPMAT by utilizing radiotracer-based in vitro uptake inhibition assays at stably expressing human embryonic kidney 293 cells (HEK293) cells. Many compounds inhibit substrate uptake by hOCT1 and hOCT2 in the low micromolar range, whereas only a few substances interact with hOCT3 and hPMAT. Interestingly, methylphenidate and ketamine selectively interact with hOCT1 or hOCT2, respectively. Additionally, 3,4-methylenedioxymethamphetamine (MDMA) is a potent inhibitor of hOCT1 and 2 and hPMAT. Enantiospecific differences of R- and S-α-pyrrolidinovalerophenone (R- and S-α-PVP) and R- and S-citalopram and the effects of aromatic substituents are explored. Our results highlight the significance of investigating drug interactions with hOCTs and hPMAT, due to their role in regulating monoamine concentrations and xenobiotic clearance.     

Chikusetsu Saponin V Attenuates MPP+-Induced Neurotoxicity in SH-SY5Y Cells via Regulation of Sirt1/Mn-SOD and GRP78/Caspase-12 Pathways

Studies have shown that saponins from Panax japonicus (SPJ) possess neuroprotective effects. However, whether Chikusetsu saponin V (CsV), the most abundant member of SPJ, can exert neuroprotective effects against 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced cytotoxicity is not known. In this study, we aimed to investigate the neuroprotective effects of CsV on MPP⁺-induced cytotoxicity in human neuroblastoma SH-SY5Y cells and explore its possible mechanisms. Our results show that CsV attenuates MPP⁺-induced cytotoxicity, inhibits ROS accumulation, and increases mitochondrial membrane potential dose-dependently. We also found that levels of Sirt1 protein and Mn-SOD mRNA significantly decreased in MPP⁺-treated group but were restored with CsV treatment in a dose-dependent manner. Furthermore, GRP78 protein and Caspase-12 mRNA levels were elevated by MPP⁺ exposure but reversed by CsV treatment. CsV inhibited the MPP⁺-induced downregulation of Bcl-2 and up-regulation of Bax in a dose-dependent manner and, thus, increased the ratio of Bcl-2/Bax. Overall, these results suggest that Sirt1/Mn-SOD and GRP78/Caspase-12 pathways might be involved in the CsV-mediated neuroprotective effects.     

Modulation of Prostanoids Profile and Counter-Regulation of SDF-1α/CXCR4 and VIP/VPAC2 Expression by Sitagliptin in Non-Diabetic Rat Model of Hepatic Ischemia-Reperfusion Injury

Molecular mechanisms underlying the beneficial effect of sitagliptin repurposed for hepatic ischemia-reperfusion injury (IRI) are poorly understood. We aimed to evaluate the impact of IRI and sitagliptin on the hepatic profile of eicosanoids (LC-MS/MS) and expression/concentration (RTqPCR/ELISA) of GLP-1/GLP-1R, SDF-1α/CXCR4 and VIP/VPAC1, VPAC2, and PAC1 in 36 rats. Animals were divided into four groups and subjected to ischemia (60 min) and reperfusion (24 h) with or without pretreatment with sitagliptin (5 mg/kg) (IR and SIR) or sham-operated with or without sitagliptin pretreatment (controls and sitagliptin). PGI₂, PGE₂, and 13,14-dihydro-PGE₁ were significantly upregulated in IR but not SIR, while sitagliptin upregulated PGD₂ and 15-deoxy-12,14-PGJ₂. IR and sitagliptin non-significantly upregulated GLP-1 while Glp1r expression was borderline detectable. VIP concentration and Vpac2 expression were downregulated in IR but not SIR, while Vpac1 was significantly downregulated solely in SIR. IRI upregulated both CXCR4 expression and concentration, and sitagliptin pretreatment abrogated receptor overexpression and downregulated Sdf1. In conclusion, hepatic IRI is accompanied by an elevation in proinflammatory prostanoids and overexpression of CXCR4, combined with downregulation of VIP/VPAC2. Beneficial effects of sitagliptin during hepatic IRI might be mediated by drug-induced normalization of proinflammatory prostanoids and upregulation of PGD₂ and by concomitant downregulation of SDF-1α/CXCR4 and reinstating VIP/VCAP2 signaling.     

Inhibitors of Human Divalent Metal Transporters DMT1 (SLC11A2) and ZIP8 (SLC39A8) from a GDB-17 Fragment Library

Solute carrier proteins (SLCs) are membrane proteins controlling fluxes across biological membranes and represent an emerging class of drug targets. Here we searched for inhibitors of divalent metal transporters in a library of 1,676 commercially available 3D-shaped fragment-like molecules from the generated database GDB-17, which lists all possible organic molecules up to 17 atoms of C, N, O, S and halogen following simple criteria for chemical stability and synthetic feasibility. While screening against DMT1 (SLC11A2), an iron transporter associated with hemochromatosis and for which only very few inhibitors are known, only yielded two weak inhibitors, our approach led to the discovery of the first inhibitor of ZIP8 (SLC39A8), a zinc transporter associated with manganese homeostasis and osteoarthritis but with no previously reported pharmacology, demonstrating that this target is druggable.     

L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells

The l-type amino acid transporter 1 (LAT1) is a membranous transporter that transports neutral amino acids for cells and is dysregulated in various types of cancer. Here, we first observed increased LAT1 expression in pemetrexed-resistant non-small cell lung cancer (NSCLC) cells with high cancer stem cell (CSC) activity, and its mRNA expression level was associated with shorter overall survival in the lung adenocarcinoma dataset of the Cancer Genome Atlas database. The inhibition of LAT1 by a small molecule inhibitor, JPH203, or by RNA interference led to a significant reduction in tumorsphere formation and the downregulation of several cancer stemness genes in NSCLC cells through decreased AKT serine/threonine kinase (AKT)/mammalian target of rapamycin (mTOR) activation. The treatment of the cell-permeable leucine derivative promoted AKT/mTOR phosphorylation and reversed the inhibitory effect of JPH203 in the reduction of CSC activity in pemetrexed-resistant lung cancer cells. Furthermore, we observed that LAT1 silencing caused the downregulation of programmed cell death 1 ligand 1 (PD-L1) on lung cancer cells. The PD-L1⁺/LAT1⁺ subpopulation of NSCLC cells displayed great CSC activity with increased expression of several cancer stemness genes. These data suggest that LAT1 inhibitors can serve as anti-CSC agents and could be used in combination with immune checkpoint inhibitors in lung cancer therapy.     

Characteristics of TIMP1, CD63, and β1-Integrin and the Functional Impact of Their Interaction in Cancer

Tissue Inhibitor of Metalloproteases 1, also known as TIMP-1, is named for its well-established function of inhibiting the proteolytic activity of matrix metalloproteases. Given this function, many studies were carried out to verify if TIMP-1 was able to interrupt processes such as tumor cell invasion and metastasis. In contrast, many studies have shown that TIMP-1 expression is increased in several types of tumors, and this increase was correlated with a poor prognosis and lower survival in cancer patients. Later, it was shown that TIMP-1 is also able to modulate cell behavior through the induction of signaling pathways involved in cell growth, proliferation, and survival. The mechanisms involved in the regulation of the pleiotropic functions of TIMP-1 are still poorly understood. Thus, this review aimed to present literature data that show its ability to form a membrane complex with CD63 and β1-integrin, and point to N-glycosylation as a potential regulatory mechanism of the functions exerted by TIMP-1. This article reviewed the characteristics and functions performed individually by TIMP1, CD63, and β1-integrin, the roles of the TIMP-1/CD63/β1-integrin complex, both in a physiological context and in cancer, and the regulatory mechanisms involved in its assembly.     

The Role of Glucose Transporters in Brain Disease: Diabetes and Alzheimer��s Disease

The occurrence of altered brain glucose metabolism has long been suggested in both diabetes and Alzheimer’s diseases. However, the preceding mechanism to altered glucose metabolism has not been well understood. Glucose enters the brain via glucose transporters primarily present at the blood-brain barrier. Any changes in glucose transporter function and expression dramatically affects brain glucose homeostasis and function. In the brains of both diabetic and Alzheimer’s disease patients, changes in glucose transporter function and expression have been observed, but a possible link between the altered glucose transporter function and disease progress is missing. Future recognition of the role of new glucose transporter isoforms in the brain may provide a better understanding of brain glucose metabolism in normal and disease states. Elucidation of clinical pathological mechanisms related to glucose transport and metabolism may provide common links to the etiology of these two diseases. Considering these facts, in this review we provide a current understanding of the vital roles of a variety of glucose transporters in the normal, diabetic and Alzheimer’s disease brain.     

Tunicamycin depresses p-glycoprotein glycosylation without an effect on its membrane localization and drug efflux activity in l1210 cells

P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump.     

Genetic Variation in <Emphasis Type="Italic">ABC G5/G8</Emphasis> and <Emphasis Type="Italic">NPC1L1</Emphasis> Impact Cholesterol Response to Plant Sterols in Hypercholesterolemic Men

ATP-binding cassette hetero-dimeric transporters G5 and G8 (ABCG5/G8) have been postulated to mediate intestinal cholesterol efflux, whereas Niemann-Pick C1 Like 1 (NPC1L1) protein is believed to be essential for intestinal cholesterol influx. The individual or combined genetic markers, such as single nuclear polymorphisms (SNPs), of these two transporter genes may explain inter-individual variations in plasma cholesterol response following plant sterol (PS) intervention. The present study was aimed at investigating the association between ABCG5/G8 and NPC1L1 genotype SNPs with sterol absorption and corresponding plasma concentrations. The study used a 4-week crossover design with 82 hypercholesterolemic men characterized by high vs. low basal plasma PS concentrations consuming spreads with or without 2 g/day of PS. For the ABCG8 1289 C > A (T400 K) polymorphism, the A allele carriers with high basal plasma PS concentrations demonstrated a 3.9-fold greater reduction (p < 0.05) in serum low density lipoprotein cholesterol (LDL-C) than their low basal plasma PS counterparts. For the NPC1L1 haplotype of 872 C > G (L272L) and 3929 G > A (Y1291Y), individuals carrying mutant alleles showed a 2.4-fold greater (p < 0.05) reduction in LDL-C levels, compared to wild type counterparts. Results suggest that genetic and metabolic biomarkers together may predict inter-individual lipid level responsiveness to PS-intervention, and thus could be useful in devising individualized cholesterol lowering strategies.     

Involvement of the ERK/HIF-1α/EMT Pathway in XCL1-Induced Migration of MDA-MB-231 and SK-BR-3 Breast Cancer Cells

Chemokine–receptor interactions play multiple roles in cancer progression. It was reported that the overexpression of X-C motif chemokine receptor 1 (XCR1), a specific receptor for chemokine X-C motif chemokine ligand 1 (XCL1), stimulates the migration of MDA-MB-231 triple-negative breast cancer cells. However, the exact mechanisms of this process remain to be elucidated. Our study found that XCL1 treatment markedly enhanced MDA-MB-231 cell migration. Additionally, XCL1 treatment enhanced epithelial–mesenchymal transition (EMT) of MDA-MB-231 cells via E-cadherin downregulation and upregulation of N-cadherin and vimentin as well as increases in β-catenin nucleus translocation. Furthermore, XCL1 enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, the effects of XCL1 on cell migration and intracellular signaling were negated by knockdown of XCR1 using siRNA, confirming XCR1-mediated actions. Treating MDA-MB-231 cells with U0126, a specific mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, blocked XCL1-induced HIF-1α accumulation and cell migration. The effect of XCL1 on cell migration was also evaluated in ER^-/HER2⁺ SK-BR-3 cells. XCL1 also promoted cell migration, EMT induction, HIF-1α accumulation, and ERK phosphorylation in SK-BR-3 cells. While XCL1 did not exhibit any significant impact on the matrix metalloproteinase (MMP)-2 and -9 expressions in MDA-MB-231 cells, it increased the expression of these enzymes in SK-BR-3 cells. Collectively, our results demonstrate that activation of the ERK/HIF-1α/EMT pathway is involved in the XCL1-induced migration of both MDA-MB-231 and SK-BR-3 breast cancer cells. Based on our findings, the XCL1–XCR1 interaction and its associated signaling molecules may serve as specific targets for the prevention of breast cancer cell migration and metastasis.     

Tumor-Suppressor Role of the α1-Na/K-ATPase Signalosome in NASH Related Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide, with an estimate of 0.84 million cases every year. In Western countries, because of the obesity epidemic, non-alcoholic steatohepatitis (NASH) has become the major cause of HCC. Intriguingly, the molecular mechanisms underlying tumorigenesis of HCC from NASH are largely unknown. We hypothesized that the growing uncoupled metabolism during NASH progression to HCC, manifested by lower cell redox status and an apoptotic ‘switch’ activity, follows a dysregulation of α1-Na/K-ATPase (NKA)/Src signalosome. Our results suggested that in NASH-related malignancy, α1-NKA signaling causes upregulation of the anti-apoptotic protein survivin and downregulation of the pro-apoptotic protein Smac/DIABLO via the activation of the PI3K → Akt pro-survival pathway with concomitant inhibition of the FoxO3 circuit, favoring cell division and primary liver carcinogenesis. Signalosome normalization using an inhibitory peptide resets apoptotic activity in malignant cells, with a significant decrease in tumor burden in vivo. Therefore, α1-NKA signalosome exercises in HCC the characteristic of a tumor suppressor, suggesting α1-NKA as a putative target for clinical therapy.     

Protein Abundance of Drug Transporters in Human Hepatitis C Livers

Transmembrane drug transport in hepatocytes is one of the major determinants of drug pharmacokinetics. In the present study, ABC transporters (P-gp, MRP1, MRP2, MRP3, MRP4, BCRP, and BSEP) and SLC transporters (MCT1, NTCP, OAT2, OATP1B1, OATP1B3, OATP2B1, OCT1, and OCT3) were quantified for protein abundance (LC-MS/MS) and mRNA levels (qRT-PCR) in hepatitis C virus (HCV)-infected liver samples from the Child–Pugh class A (n = 30), B (n = 21), and C (n = 7) patients. Protein levels of BSEP, MRP3, MCT1, OAT2, OATP1B3, and OCT3 were not significantly affected by HCV infection. P-gp, MRP1, BCRP, and OATP1B3 protein abundances were upregulated, whereas those of MRP2, MRP4, NTCP, OATP2B1, and OCT1 were downregulated in all HCV samples. The observed changes started to be seen in the Child–Pugh class A livers, i.e., upregulation of P-gp and MRP1 and downregulation of MRP2, MRP4, BCRP, and OATP1B3. In the case of NTCP, OATP2B1, and OCT1, a decrease in the protein levels was observed in the class B livers. In the class C livers, no other changes were noted than those in the class A and B patients. The results of the study demonstrate that drug transporter protein abundances are affected by the functional state of the liver in hepatitis C patients.