Transporter Protein Levels Research Articles

Does fresh or frozen embryo transfer affect imprinted gene expressions in human term placenta?

Our research aimed to compare the epigenetic alterations between placentae of in vitro fertilization (IVF) patients and spontaneous pregnancies. Additionally, the expression levels of proliferation markers (PCNA, Ki67) and glucose transporter proteins (GLUT1, GLUT3) were assessed in control and IVF placentae to examine the possible consequences of epigenetic alterations on placental development. Control group placentae were obtained from spontaneous pregnancies of healthy women (n = 16). IVF placentae were obtained from fresh (n = 16) and frozen (n = 16) embryo transfer pregnancies. A group of maternal and paternal imprint genes H19, IGF2, IGF2, IGF2R, PHLDA2, PLAGL1, MASH2, GRB10, PEG1, PEG3, and PEG10 were detected by Real-Time PCR. Additionally, PCNA, Ki67, GLUT1, and GLUT3 protein levels were assessed by immunohistochemistry and western blot. In the fresh embryo transfer placenta group (fETP), gene expression of paternal PEG1 and PEG10 was upregulated compared with the control group. Increased gene expression in paternal PEG1 and maternal IGFR2 genes was detected in the frozen embryo transfer placenta group (FET) compared with the control group. Conversely, expression levels of H19 and IGF2 genes were downregulated in the FET group. On the other hand, GLUT3 and PCNA expression was increased in FET group placentae. IVF techniques affect placental imprinted gene expressions which are important for proper placental development. Imprinted genes are differently expressed in fresh ET placentae and frozen ET placentae. In conclusion, these data indicate that altered imprinted gene expression may affect glucose transport and cell proliferation, therefore play an important role in placental development.

Alterations in erythrocyte membrane transporter expression levels in type 2 diabetic patients

Type 2 diabetes mellitus (T2DM) is one of the most common multifactorial diseases and several membrane transporters are involved in its development, complications and treatment. We have recently developed a flow-cytometry assay panel for the quantitative determination of red cell membrane protein levels with potential relevance in diseases. Here we report a detailed phenotypic analysis of a medium scale, clinically based study on the expression of T2DM-related membrane proteins, the GLUT1, GLUT3, MCT1, URAT1, ABCA1, ABCG2 and the PMCA4 transporters in erythrocytes. By comparing age-matched control subjects and three groups of T2DM patients (recently diagnosed, successfully managed, and patients with disease-related complications), we found significant differences in the membrane expression levels of the transporters in these groups. This is a first detailed analysis of T2DM related alterations in erythrocyte membrane transporter protein levels, and the results suggest significant changes in some of the transporter expression levels in various patient groups. By performing a further, more detailed analysis of the clinical and molecular biology parameters, these data may serve as a basis of establishing new, personalized diagnostic markers helping the prevention and treatment of type 2 diabetes.

Cabbage defense response provoked by Trichoderma Th-LAAO.

To investigate the molecular mechanism of Trichoderma L-amino acid oxidase (Th-LAAO) in protecting and in promoting growth of cabbage infected with Botrytis cinerea, a three-way interaction system was established. Cabbage leaves treated with purified Th-LAAO significantly constrained damaged leaf area caused by B. cinerea infection. In response to Th-LAAO treatment, the expression levels of genes involved in photosynthesis, such as ribulose-1,5-bisphosphate carboxylase oxygenase, Rubisco activase, and ATP synthase increased 2.54, 2.18, and 1.41 folds, respectively. The transcription levels of sucrose transport protein 1 increased 7.6 fold. As to the expression of defense-related genes, the transcription level of ascorbate peroxidase increased 1.46 fold. On the contrary, pathogenesis-related protein 1, chitinase, β-1,3 glucanase, and glutathione S-transferase decreased significantly. Overall, the results indicated that Th-LAAO may stimulate CO2 fixation and sucrose transport and elicit host defense responses in cabbage against B. cinerea, and this elicitation of defense response is likely to contribute to induced systemic resistance of host plant.

Effect of oxytocin on lipid accumulation under inflammatory conditions in human macrophages

Introduction and aimsOxytocin (OT) is a neuropeptide hormone secreted by the posterior pituitary gland. Deficits in OT action have been observed in patients with behavioral and mood disorders, some of which correlate with an increased risk of cardiovascular disease (CVD). Recent research has revealed a wider systemic role that OT plays in inflammatory modulation and development of atherosclerotic plaques. This study investigated the role that OT plays in cholesterol transport and foam cell formation in LPS-stimulated THP-1 human macrophages. MethodsTHP-1 differentiated macrophages were treated with media, LPS (100 ng/ml), LPS + OT (10 pM), or LPS + OT (100 pM). Changes in gene expression and protein levels of cholesterol transporters were analyzed by real time quantitative PCR (RT-qPCR) and Western blot, while oxLDL uptake and cholesterol efflux capacity were evaluated with fluorometric assays. ResultsRT-qPCR analysis revealed a significant increase in ABCG1 gene expression upon OT + LPS treatment, compared to LPS alone (p = 0.0081), with Western blotting supporting the increase in expression of the ABCG1 protein. Analysis of oxLDL uptake showed a significantly lower fluorescent value in LPS + OT (100pM) -treated cells when compared to LPS alone (p < 0.0001). While not statistically significant (p = 0.06), cholesterol efflux capacity increased with LPS + OT treatment. ConclusionWe demonstrate here that OT can attenuate LPS-mediated lipid accumulation in THP-1 macrophages. These findings support the hypothesis that OT could be used to reduce pro-inflammatory and potentially atherogenic changes observed in patients with heightened CVD risk. This study suggests further exploration of OT effects on monocyte and macrophage cholesterol handling in vivo.

Effects of psoralen on hepatic bile acid transporters in rats.

Fructus Psoraleae (FP), widely used in traditional medicine, is increasingly reported to cause serious hepatotoxicity in recent years. However, the main toxic constituents responsible for hepatotoxicity and the underlying mechanisms are poorly understood. In the present study, psoralen, a main and quality-control constituent of FP, was intragastrically administered to Sprague-Dawley rats at a dose of 60 mg/kg for 1, 3 and 7 days. Blood and selected tissue samples were collected and analyzed for biochemistry and histopathology to evaluate hepatotoxicity. The results showed that psoralen could induce hepatotoxicity by enhanced liver-to-body weight ratio and alterations of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total cholesterol after administration for 3 days. In addition, histopathological examinations also indicated the hepatotoxicity induced by psoralen. Furthermore, the mRNA and protein levels of hepatic bile acid transporters were significantly changed, in which MRP4, ABCG5 and ABCG8 were repressed, while the protein level of NTCP tended to increase in the rat liver. Taken together, psoralen caused liver injury possibly through affecting bile acid transporters, leading to the disorder of bile acid transport and accumulation in hepatocytes.

E-cigarette aerosols containing nicotine modulate nicotinic acetylcholine receptors and astroglial glutamate transporters in mesocorticolimbic brain regions of chronically exposed mice

Nicotine exposure increases the release of glutamate in part through stimulatory effects on pre-synaptic nicotinic acetylcholine receptors (nAChRs). To assess the impact of chronic electronic (e)-cigarette use on these drug dependence pathways, we exposed C57BL/6 mice to three types of inhalant exposures for 3 months; 1) e-cigarette aerosol generated from liquids containing nicotine (ECN), 2) e-cigarette aerosol generated from liquids containing vehicle chemicals without nicotine (Veh), and 3) air only (AC). We investigated the effects of daily e-cigarette exposure on protein levels of α7 nAChR and α4/β2 nAChR, gene expression and protein levels of astroglial glutamate transporters, including glutamate transporter-1 (GLT-1) and cystine/glutamate antiporter (xCT), in the frontal cortex (FC), striatum (STR) and hippocampus (HIP). We found that chronic inhalation of ECN increased α4/β2 nAChR in all brain regions, and increased α7 nAChR expression in the FC and STR. The total GLT-1 relative mRNA and protein expression were decreased in the STR. Moreover, GLT-1 isoforms (GLT-1a and GLT-1b) were downregulated in the STR in ECN group. However, inhalation of e-cigarette aerosol downregulated xCT expression in STR and HIP compared to AC and Veh groups. ECN group had increased brain-derived neurotrophic factor in the STR compared to control groups. Finally, mass spectrometry detected high concentrations of the nicotine metabolite, cotinine, in the FC and STR in ECN group. This work demonstrates that chronic inhalation of nicotine within e-cigarette aerosols significantly alters the expression of nAChRs and astroglial glutamate transporters in specific mesocorticolimbic brain regions.

Decreased mitochondrial electron transport proteins and increased complement mediators in plasma neural-derived exosomes of early psychosis

Potentially neurotoxic systems involved in traumatic and degenerative diseases of the brain were assessed in acute psychosis. Astrocyte-derived exosomes (ADEs) and neuron-derived exosomes (NDEs) were immunoprecipitated from plasma of ten untreated first-episode psychotics (FPs) and ten matched normal controls (Cs). Neural mitochondrial electron transport and complement proteins were extracted, quantified by ELISAs and normalized with levels of CD81 exosome marker. Levels of subunits 1 and 6 of NADH-ubiquinone oxidoreductase (complex I) and subunit 10 of cytochrome b-c1 oxidase (complex III), but not of subunit 1 of cytochrome C oxidase (complex IV) or superoxide dismutase 1 (SOD1) were significantly lower in ADEs and NDEs of FPs than Cs. This dysregulated pattern of electron transport proteins is associated with increased generation of reactive oxygen species. ADE glial fibrillary acidic protein levels were significantly higher in FPs than Cs, indicating a higher percentage of inflammatory astrocytes in FPs. ADE levels of C3b opsonin were significantly higher and those of C5b-9 attack complex was marginally higher in FPs than Cs. A significantly lower ADE level of the C3 convertase inhibitor CD55 may explain the higher levels of C3 convertase-generated C3b. ADE levels of the neuroprotective protein leukemia inhibitory factor (LIF) were significantly lower in FPs than Cs, whereas levels of IL-6 were no different. Plasma neural exosome levels of electron transport and complement proteins may be useful in predicting FP and guiding therapy. SOD mimetics, C3 convertase inhibitors and LIF receptor agonists also may have therapeutic benefits in FP.

Slow-Release H2S Donor Anethole Dithiolethione Protects Liver From Lipotoxicity by Improving Fatty Acid Metabolism.

“Lipotoxicity” induced by free fatty acids (FAs) plays a central role in the pathogenesis of many metabolic diseases, with few treatment options available today. Hydrogen sulfide (H2S), a novel gaseous signaling molecule, has been reported to have a variety of pharmacological properties, but its effect on FAs metabolism remains unclear. The purpose of this study was to investigate the effect and mechanisms of anethole dithiolethione (ADT, a sustained-release H2S donor) on hepatic FAs metabolism. ADT was administered daily for 4 weeks in male Syrian golden hamsters fed a high fat diet (HFD), and FAs profiles of liver tissues were analyzed using GC-MS. The results showed that in HFD-fed hamsters, ADT treatment significantly reduced the accumulation of toxic saturated and monounsaturated fatty acids (C16:0, C18:0, C16:1, and C18:1n9), while increased the content of n-6 and n-3 series polyunsaturated fatty acids (C20:3n6, C20:4n6, and C22:6n3). Mechanistically, ADT obviously inhibited the overexpression of acetyl-CoA carboxylase1 (ACC1), fatty acid synthase (FAS), and stearoyl-CoA desaturase1 (SCD1), and up-regulated the levels of fatty acid transport proteins (FATPs), liver fatty acid binding protein (L-FABP), carnitine palmitoyltransferase 1α (CPT1α), fatty acid desaturase (FADS)1 and FADS2. Notably, ADT administration significantly promoted Mitofusin1-mediated mitochondrial fusion and fatty acid β-oxidation. These findings suggest that ADT plays a beneficial role by regulating the synthesis, desaturation, β-oxidation, uptake, binding/isolation, and transport of FAs. In conclusion, ADT is effective in improving FAs metabolic disorders and liver injuries caused by HFD, which renders ADT a candidate drug for lipotoxicity-induced diseases.

Sodium butyrate ameliorates deoxycorticosterone acetate/salt-induced hypertension and renal damage by inhibiting the MR/SGK1 pathway.

Our recent work demonstrates that infusion of sodium butyrate (NaBu) into the renal medulla blunts angiotensin II-induced hypertension and improves renal injury. The present study aimed to test whether oral administration of NaBu attenuates salt-sensitive hypertension in deoxycorticosterone acetate (DOCA)/salt-treated rats. Uninephrectomized male Sprague-Dawley (SD) rats were treated with DOCA pellets (150 mg/rat) plus 1% NaCl drinking water for 2 weeks. Animals received oral administration of NaBu (1 g/kg) or vehicle once per day. Our results showed that NaBu administration significantly attenuated DOCA/salt-increased mean arterial pressure from 156 ± 4 mmHg to 136 ± 1 mmHg. DOCA/salt treatment markedly enhanced renal damage as indicated by an increased ratio of kidney weight/body weight, elevated urinary albumin, extensive fibrosis, and inflammation, whereas kidneys from NaBu-treated rats exhibited a significant reduction in these renal damage responses. Compared to the DOCA/salt group, the DOCA/salt-NaBu group had ~30% less salt water intake and decreased Na+ and Cl- excretion in urine but no alteration in 24-h urine excretion. Mechanistically, NaBu inhibited the protein levels of several sodium transporters stimulated by DOCA/salt in vivo, such as βENaC, γENaC, NCC, and NKCC-2. Further examination showed that NaBu downregulated the expression of mineralocorticoid receptor (MR) and serum and glucocorticoid-dependent protein kinase 1 (SGK1) in DOCA/salt-treated rats or aldosterone-treated human renal tubular duct epithelial cells. These results provide evidence that NaBu may attenuate DOCA/salt-induced hypertension and renal damage by inhibiting the MR/SGK1 pathway.

Proteomic Evaluation of Plasma Membrane Fraction Prepared from a Mouse Liver and Kidney Using a Bead Homogenizer: Enrichment of Drug-Related Transporter Proteins.

Quantifying the protein levels of drug transporters in plasma membrane fraction helps elucidate the function of these transporters. In this study, we conducted a proteomic evaluation of enriched drug-related transporter proteins in plasma membrane fraction prepared from mouse liver and kidney tissues using the membrane protein extraction kit and a bead homogenizer. Crude and plasma membrane fractions were prepared using either the Dounce or bead homogenizer, and protein levels were determined using quantitative proteomics. In liver tissues, the plasma membrane fractions were more enriched in transporter proteins than the crude membrane fractions; the average enrichment ratios of plasma-to-crude membrane fractions were 3.31 and 6.93 using the Dounce and bead homogenizers, respectively. The concentrations of transporter proteins in plasma membrane fractions determined using the bead homogenizer were higher than those determined using the Dounce homogenizer. Meanwhile, in kidney tissues, the plasma membrane fractions were enriched in transporters localized in the brush-border membrane to the same degree for both the homogenizers; however, the membrane fractions obtained using either homogenizer were not enriched in Na+/K+-ATPase and transporters localized in the basolateral membrane. These results indicate that fractionation, using the bead homogenizer, yielded transporter-enriched plasma membrane fractions from mouse liver and kidney tissues; however, no enrichment of basolateral transporters was observed in plasma membrane fractions prepared from kidney tissues.

Imipramine Influences Body Distribution of Supplemental Zinc Which May Enhance Antidepressant Action.

Zinc (Zn) was found to enhance the antidepressant efficacy of imipramine (IMI) in human depression and animal tests/models of depression. However, the underlying mechanism for this effect remains unknown. We measured the effect of intragastric (p.o.) combined administration of IMI (60 mg/kg) and Zn (40 mg Zn/kg) in the forced swim test (FST) in mice. The effect of Zn + IMI on serum, brain, and intestinal Zn concentrations; Zn transporter (ZnT, ZIP) protein levels in the intestine and ZnT in the brain; including BDNF (brain-derived neurotrophic factor) and CREB (cAMP response element-binding protein) protein levels in the brain were evaluated. Finally, the effect of IMI on Zn permeability was measured in vitro in colon epithelial Caco-2 cells. The co-administration of IMI and Zn induced antidepressant-like activity in the FST in mice compared to controls and Zn or IMI given alone. This effect correlated with increased BDNF and the ratio of pCREB/CREB protein levels in the prefrontal cortex (PFC) compared to the control group. Zn + IMI co-treatment increased Zn concentrations in the serum and brain compared to the control group. However, in serum, co-administration of IMI and Zn decreased Zn concentration compared to Zn alone treatment. Also, there was a reduction in the Zn-induced enhancement of ZnT1 protein level in the small intestine. Zn + IMI also induced an increase in the ZnT4 protein level in the PFC compared to the control group and normalized the Zn-induced decrease in the ZnT1 protein level in the hippocampus (Hp). The in vitro studies revealed enhanced Zn permeability (observed as the increased transfer of Zn through the intestinal cell membrane) after IMI treatment. Our data indicate that IMI enhances Zn transfer through the intestinal tract and influences the redistribution of Zn between the blood and brain. These mechanisms might explain the enhanced antidepressant efficacy of combined IMI/Zn treatment observed in the FST in mice.

Expression of ion transport genes in ionocytes isolated from larval zebrafish (Danio rerio) exposed to acidic or Na+-deficient water.

In zebrafish (Danio rerio), a specific ionocyte subtype, the H+-ATPase-rich (HR) cell, is presumed to be a significant site of transepithelial Na+ uptake/acid secretion. During acclimation to environments differing in ionic composition or pH, ionic and acid-base regulations are achieved by adjustments to the activity level of HR cell ion transport proteins. In previous studies, the quantitative assessment of mRNA levels for genes involved in ionic and acid-base regulations relied on measurements using homogenates derived from the whole body (larvae) or the gill (adult). Such studies cannot distinguish whether any differences in gene expression arise from adjustments of ionocyte subtype numbers or transcriptional regulation specifically within individual ionocytes. The goal of the present study was to use fluorescence-activated cell sorting to separate the HR cells from other cellular subpopulations to facilitate the measurement of gene expression of HR cell-specific transporters and enzymes from larvae exposed to low pH (pH 4.0) or low Na+ (5 μM) conditions. The data demonstrate that treatment of larvae with acidic water for 4 days postfertilization caused cell-specific increases in H+-ATPase (atp6v1aa), ca17a, ca15a, nhe3b, and rhcgb mRNA in addition to increases in mRNA linked to cell proliferation. In fish exposed to low Na+, expression of nhe3b and rhcgb was increased owing to HR cell-specific regulation and elevated numbers of HR cells. Thus, the results of this study demonstrate that acclimation to low pH or low Na+ environmental conditions is facilitated by HR cell-specific transcriptional control and by HR cell proliferation.

FengLiao affects gut microbiota and the expression levels of Na+/H+ exchangers, aquaporins and acute phase proteinsin mice with castor oil-induced diarrhea.

The severe side effects of chemosynthetic anti-diarrhea drugs have created an interest in low-toxic alternative plant-derived compounds. FengLiao consists of Polygonum hydropiper Linn. and Daphniphyllum calycinum Bench., and is widely used in China to treat diarrhea due to low levels of toxicity. In this study, the effects of FengLiao were analyzed in a castor oil-induced diarrhea model, using the anti-diarrhea drug, loperamide, as the positive control. The effects were evaluated using stool characteristics and the expression levels of various diarrhea-related factors in the jejunum and liver, as well as changes in the microbiota of the jejunum. The symptoms of diarrhea and stool consistency were improved through FengLiao and loperamide treatment. Furthermore, FengLiao down-regulated alpha 1-acid glycoprotein (AGP) and C-reactive protein (CRP) levels, and up-regulated transferrin (TRF) mRNA levels in the liver, and down-regulated Aquaporin 3 (AQP3) and Na+/H+ exchanger isoform 8 (NHE8) expression in the epithelial cells of the jejunum. It also increased the relative abundance of Bifidobacterium, Aerococcus, Corynebacterium_1 and Pseudomonas, and lowered the Firmicutes/Bacteroidetes (F/B) ratio, which maintained the balance between immunity and intestinal health. Taken together, FengLiao alleviated castor oil-induced diarrhea by altering gut microbiota, and levels of jejunum epithelial transport proteins and acute phase proteins.

Harnessing the potential of artificial neural networks for predicting protein glycosylation

Kinetic models offer incomparable insight on cellular mechanisms controlling protein glycosylation. However, their ability to reproduce site-specific glycoform distributions depends on accurate estimation of a large number of protein-specific kinetic parameters and prior knowledge of enzyme and transport protein levels in the Golgi membrane. Herein we propose an artificial neural network (ANN) for protein glycosylation and apply this to four recombinant glycoproteins produced in Chinese hamster ovary (CHO) cells, two monoclonal antibodies and two fusion proteins. We demonstrate that the ANN model accurately predicts site-specific glycoform distributions of up to eighteen glycan species with an average absolute error of 1.1%, correctly reproducing the effect of metabolic perturbations as part of a hybrid, kinetic/ANN, glycosylation model (HyGlycoM), as well as the impact of manganese supplementation and glycosyltransferase knock out experiments as a stand-alone machine learning algorithm. These results showcase the potential of machine learning and hybrid approaches for rapidly developing performance-driven models of protein glycosylation.

Region-specific changes in the mRNA and protein expression of LCPUFA biosynthesis enzymes and transporters in the placentae of women with preeclampsia

The biosynthesis and transport of long chain polyunsaturated fatty acids (LCPUFA) require the activity of fatty acid desaturase (FADS) enzymes, fatty acid transport proteins (FATP) and fatty acid binding proteins (FABP). In a previous study we have demonstrated region-specific changes in the LCPUFA levels in preeclampsia (PE) as compared to the normotensive control (NC) placentae. AimTo understand the region-specific changes in the mRNA levels and protein expression of biosynthesis enzymes and transporters of LCPUFA in PE and NC placentae. MethodsIn this cross-sectional study, 20 NC women and 44 women with PE (23 term (TPE) and 21 preterm PE (PTPE)) were recruited. The samples were collected from four regions of the placentae considering cord insertion as the center (CM, central maternal/basal; CF, central fetal/chorionic; PM, peripheral maternal/basal and PF, peripheral fetal/chorionic). The mRNA levels were estimated using qRT-PCR. Statistical analysis was done using both post hoc least significant difference (LSD) test and Benjamini Hochberg correction in the analysis of covariance. Preliminarily, localization and expression of proteins were studied by immunohistochemistry (n = 3/group). ResultsThe mRNA levels of FADS1, FADS2 and FATP1 were lower in the central regions (CM and CF) of the PE placentae (both TPE and PTPE) as compared to NC. These differences in the mRNA levels were observed by the LSD test and were not significant after the Benjamini Hochberg correction. Preliminary findings of IHC indicate that the protein expression of FADS1 and FATP4 was higher in the basal regions (CM and PM) of the PE placentae as compared to NC. FADS1, FADS2 and FATP4 proteins were localized in the syncytiotrophoblasts, cytotrophoblasts, mesenchymal cells, endothelial cells of the fetal capillaries and extravillous trophoblasts of the placenta. ConclusionFADS enzymes are detected in the placentae of Indian women. In PE placentae, there are region-specific alterations in the mRNA and protein levels of LCPUFA biosynthesis enzymes (FADS1 and FADS2) and transporters (FATP1, FATP4 and FABP3) as compared to term NC. These changes were more pronounced toward the basal side and region around the cord insertion.

Caco-2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations

BackgroundThe human Caco-2 cell line is a common in vitro model of the intestinal epithelial barrier. As the intestine is a major interface in cholesterol turnover and represents a non-biliary pathway for cholesterol excretion, Caco-2 cells are also a valuable model for studying cholesterol homeostasis, including cholesterol uptake and efflux. Currently available protocols are, however, either sketchy or not consistent among different laboratories. Our aim was therefore to generate a collection of optimized protocols, considering the different approaches of the different laboratories and to highlight possibilities and limitations of measuring cholesterol transport with this cell line.ResultsWe developed comprehensive and quality-controlled protocols for the cultivation of Caco-2 cells on filter inserts in a single tight monolayer. A cholesterol uptake as well as a cholesterol efflux assay is described in detail, including suitable positive controls. We further show that Caco-2 cells can be efficiently transfected for luciferase reporter gene assays in order to determine nuclear receptor activation, main transcriptional regulators of cholesterol transporters (ABCA1, ABCB1, ABCG5/8, NPC1L1). Detection of protein and mRNA levels of cholesterol transporters in cells grown on filter inserts can pose challenges for which we highlight essential steps and alternative approaches for consideration. A protocol for viability assays with cells differentiated on filter inserts is provided for the first time.ConclusionsThe Caco-2 cell line is widely used in the scientific community as model for the intestinal epithelium, although with highly divergent protocols. The herein provided information and protocols can be a common basis for researchers intending to use Caco-2 cells in the context of cellular cholesterol homeostasis.

Metformin is distributed to tumor tissue in breast cancer patients in vivo: A 11C-metformin PET/CT study.

Epidemiological studies and randomized clinical trials suggest that the antidiabetic drug, metformin, may have anti-neoplastic effects. The mechanism that mediates these beneficial effects has been suggested to involve direct action on cancer cells, but this will require distribution of metformin in tumor tissue. The present study was designed to investigate metformin distribution in vivo in breast and liver tissue in breast cancer patients. Seven patients recently diagnosed with ductal carcinoma were recruited. Using PET/CT, tissue distribution of metformin was determined in vivo for 90min after injection of a carbon-11-labeled metformin tracer. After surgery, tumor tissue was investigated for gene expression levels of metformin transporter proteins. Tumor tissue displayed a distinct uptake of metformin compared to normal breast tissue AUC0-90min (75.4 ± 5.5 vs 42.3 ± 6.3) g/ml*min (p = 0.01). Maximal concentration in tumor was at 1min where it reached approximately 30% of the activity in the liver. The metformin transporter protein with the highest gene expression in tumor tissue was multidrug and toxin extrusion 1 (MATE 1) followed by plasma membrane monoamine transporter (PMAT). This study confirms that metformin is transported into tumor tissue in women with breast cancer. This finding support that metformin may have direct anti-neoplastic effects on tumor cells in breast cancer patients. However, distribution of metformin in tumor tissue is markedly lower than in liver, an established metformin target tissue.

Inhibition of Androgen Receptor Decreases Fat Metabolism by Decreasing Carnitine Palmitoyltransferase I Levels in Skeletal Muscles of Trained Mice

PurposeAndrogen hormone levels are strongly associated with obesity in adult mammals, especially with advanced age. We investigated androgen receptor inhibition on fat metabolism and long‐chain fatty acid (LCFA) transport proteins in skeletal muscle during exercise.MethodsMale ICR mice were randomly divided into three groups: CON (control), EX (exercise), and EXIN (exercise + androgen receptor inhibition). EX and EXIN groups were trained on a treadmill five times a week. After four weeks, the fat metabolism of each group was measured using open‐circuit calorimetry during one hour of exercise. After the metabolism measurement, the expression levels of LCFA transport proteins (FAT/CD36, CPTI) were analyzed in skeletal muscle.ResultsWeight gain and final body weight were significantly lower in the EX group than in either the CON or EXIN groups. Conversely, food intake was significantly higher in the EX group than it was in the CON and EXIN groups. The total weight (CON; 2.07 ± 0.6, EX; 1.64 ± 0.2, EXIN; 1.95 ± 0.2) of the abdominal adipose tissue were significantly lower in the EX group than in the CON and EXIN groups (P &lt; 0.05). However, there was no different between the CON and EXIN group. Oxygen uptake and fat oxidation during exercise tended to be lower (12%) in the EXIN group than in the EX group. Total fat oxidation in the EXIN group was significantly lower during the initial 20‐min (P &lt; 0.003) and 40‐min (P &lt; 0.041) phases compared to that in the EX group. In addition, the level of FAT/CD36 protein in the EX and EXIN groups was approximately double that in the CON group (P &lt; 0.001, P &lt; 0.001). CPTI expression in the EX group was higher than that in the EX group (P &lt; 0.0069) as well as in the CON group.ConclusionExercise training increases the expression of LCFA transport proteins (FAT/CD36, CPTI). However, blocking androgen receptors decreases the expression of CPTI in the skeletal muscle, which reduces fat metabolism. Thus, reducing sex hormones or suppressing the sensitivity of AR receptors can inhibit energy efficiency and fat metabolism by suppressing CPTI.Support or Funding InformationThis work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea (NRF‐2015S1A5B5A01016088).

Hepatic Transporter Alterations by Nuclear Receptor Agonist T0901317 in Sandwich-Cultured Human Hepatocytes: Proteomic Analysis and PBPK Modeling to Evaluate Drug-Drug Interaction Risk

In vitro approaches for predicting drug-drug interactions (DDIs) caused by alterations in transporter protein regulation are not well established. However, reports of transporter regulation via nuclear receptor (NR) modulation by drugs are increasing. This study examined alterations in transporter protein levels in sandwich-cultured human hepatocytes (SCHH; n = 3 donors) measured by liquid chromatography-tandem mass spectrometry-based proteomic analysis after treatment with N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide (T0901317), the first described synthetic liver X receptor agonist. T0901317 treatment (10 μM, 48 hours) decreased the levels of organic cation transporter (OCT) 1 (0.22-, 0.43-, and 0.71-fold of control) and organic anion transporter (OAT) 2 (0.38-, 0.38-, and 0.53-fold of control) and increased multidrug resistance protein (MDR) 1 (1.37-, 1.48-, and 1.59-fold of control). The induction of NR downstream gene expression supports the hypothesis that T0901317 off-target effects on farnesoid X receptor and pregnane X receptor activation are responsible for the unexpected changes in OCT1, OAT2, and MDR1. Uptake of the OCT1 substrate metformin in SCHH was decreased by T0901317 treatment. Effects of decreased OCT1 levels on metformin were simulated using a physiologically-based pharmacokinetic (PBPK) model. Simulations showed a clear decrease in metformin hepatic exposure resulting in a decreased pharmacodynamic effect. This DDI would not be predicted by the modest changes in simulated metformin plasma concentrations. Altogether, the current study demonstrated that an approach combining SCHH, proteomic analysis, and PBPK modeling is useful for revealing tissue concentration-based DDIs caused by unexpected regulation of hepatic transporters by NR modulators. SIGNIFICANCE STATEMENT: This study utilized an approach combining sandwich-cultured human hepatocytes, proteomic analysis, and physiologically based pharmacokinetic modeling to evaluate alterations in pharmacokinetics (PK) and pharmacodynamics (PD) caused by transporter regulation by nuclear receptor modulators. The importance of this approach from a mechanistic and clinically relevant perspective is that it can reveal drug-drug interactions (DDIs) caused by unexpected regulation of hepatic transporters and enable prediction of altered PK and PD changes, especially for tissue concentration-based DDIs.

Experimental Traumatic Brain Injury Induces Chronic Glutamatergic Dysfunction in Amygdala Circuitry Known to Regulate Anxiety-Like Behavior.

Up to 50% of traumatic brain injury (TBI) survivors demonstrate persisting and late-onset anxiety disorders indicative of limbic system dysregulation, yet the pathophysiology underlying the symptoms is unclear. We hypothesize that the development of TBI-induced anxiety-like behavior in an experimental model of TBI is mediated by changes in glutamate neurotransmission within the amygdala. Adult, male Sprague-Dawley rats underwent midline fluid percussion injury or sham surgery. Anxiety-like behavior was assessed at 7 and 28 days post-injury (DPI) followed by assessment of real-time glutamate neurotransmission in the basolateral amygdala (BLA) and central nucleus of the amygdala (CeA) using glutamate-selective microelectrode arrays. The expression of anxiety-like behavior at 28 DPI coincided with decreased evoked glutamate release and slower glutamate clearance in the CeA, not BLA. Numerous factors contribute to the changes in glutamate neurotransmission over time. In two additional animal cohorts, protein levels of glutamatergic transporters (Glt-1 and GLAST) and presynaptic modulators of glutamate release (mGluR2, TrkB, BDNF, and glucocorticoid receptors) were quantified using automated capillary western techniques at 28 DPI. Astrocytosis and microglial activation have been shown to drive maladaptive glutamate signaling and were histologically assessed over 28 DPI. Alterations in glutamate neurotransmission could not be explained by changes in protein levels for glutamate transporters, mGluR2 receptors, astrocytosis, and microglial activation. Presynaptic modulators, BDNF and TrkB, were significantly decreased at 28 DPI in the amygdala. Dysfunction in presynaptic regulation of glutamate neurotransmission may contribute to anxiety-related behavior and serve as a therapeutic target to improve circuit function.