Expression Of Hepatic Transporters Research Articles

Metabolic role of the amino acids in the adaptation of Holstein dairy cows to two contrasting challenging situations

This thesis investigated the role of the amino acids (AA) during the transition period and lactation under grazing conditions, and heat stress (HS) as two contrasting challenging situations for the dairy cow. In the experiment 1, the branched-chain AA (BCAA) metabolism was studied in North American (NAH) vs. New Zealand (NZH) Holstein cows between -45 and 180 days in milk (DIM). Higher plasma concentrations of BCAA were confirmed in NAH vs. NZH in early lactation. The temporal profile of hepatic gene expression of specific BCAA transporters, especially LAT1 and Eeg1, differed between the genetic strains in association with the level of somatotropic axis uncoupling and insulin resistance in the liver. In the experiments 2 and 3, we studied the role of the metabolism of the AA against HS and its amelioration through nutritional strategies under controlled conditions. The plasma and milk metabolomics indicated that the HS triggered skeletal muscle mobilization and enhanced AA catabolism. Dietary supply of vitamin E and Se above requirements seemed to have modulated the skeletal muscle mobilization, while D3 and Ca did not show significant effects (experiment 2). The results of experiment 3 suggested that the partial amelioration of HS effects on productive and physiological parameters through high dietary supply of Met (+70 %), Lys (+65 %) and His (+21 %) were mediated by a recovery of phosphatidylcholines and decreased oxidation of Val, Trp and Leu due to enhanced one-carbon metabolism and Lys oxidation, respectively.

The effect of pregnancy-related hormones on hepatic transporters: studies with premenopausal human hepatocytes.

Pregnancy results in significant changes in drug pharmacokinetics (PK). While previous studies have elucidated the impact of pregnancy-related hormones (PRH) on mRNA or protein expression and activity of major hepatic metabolizing enzymes, their effect on hepatic drug transporters remains largely unexplored. Therefore, we investigated the effect of a cocktail of PRH on the mRNA expression and activity of hepatic transporters. Plated human hepatocytes (PHH) from 3 premenopausal donors were incubated, in triplicate, for 72h, with vehicle (DMSO < 0.01%), rifampin (10μM; positive control) or a cocktail of PRH consisting of estrone, estradiol, estriol, estetrol, progesterone, cortisol, testosterone, oxytocin, and placental growth hormone. The PRH concentrations replicated 0.1×, 1×, or 10× of the plasma concentrations of these hormones observed during each of the three trimesters of pregnancy. After treatment, mRNA expression (quantified by qPCR) of hepatic influx and efflux transporters as well as the activity of influx transporters was quantified (uptake of a selective substrate ± corresponding transporter inhibitor). The data were expressed relative to that in the control (vehicle) group. Significance was evaluated by ANOVA (followed by Dunn's multiple comparisons) or unpaired t-test when the within-lot data were analyzed, or repeated measures ANOVA (followed by Dunn's multiple comparisons) or paired t-test when data from all 3 lots were analyzed (p < 0.05). In general, a) PRH cocktails significantly induced transporter mRNA expression in the following order OAT2 ≈ NTCP ≈ OCT1 > OATP2B1 and repressed mRNA expression in the following order OATP1B3 > OATP1B1; b) these changes translated into significant induction of OAT2 (T1-T3) and NTCP (T2-T3, in only two lots) activity at the 1× PRH concentration. Compared with the influx transporters, the induction of mRNA expression of efflux transporters was modest, with mRNA expression of MRP2 and BSEP being induced the most. Once these data are verified through in vivo probe drug PK studies in pregnancy, they can be populated into physiologically based pharmacokinetic (PBPK) models to predict, for all trimesters of pregnancy, transporter-mediated clearance of any drug that is a substrate of the affected transporters.

Vincamine alleviates intrahepatic cholestasis in rats through modulation of NF-kB/PDGF/klf6/PPARγ and PI3K/Akt pathways

The defect in the hepatobiliary transport system results in an impairment of bile flow, leading to accumulation of toxic compounds with subsequent liver disorders. Vincamine, a plant indole alkaloid that is utilized as a dietary supplement, has been known for its promising pharmacological activities. For the first time, the present study was planned to estimate, at the molecular level, the potentiality of vincamine against alfa-naphthyl isothiocyanate (ANIT)-induced hepatic cholestasis. Liver function tests were analyzed. Hepatic activity of SOD and levels of GSH and MDA were assessed. Hepatic contents of bax, bcl2, NF-kB, PPARγ, catalase, heme-oxygenase-1, NTCP, and BSEP were evaluated using ELISA. mRNA levels of NF-kB, IL-1β, IL-6, TNFα, PDGF, klf6, PPARγ, and P53 were examined using qRT-PCR. PI3K, Akt and cleaved caspase-3 proteins were assessed using western blotting. Histopathological analyses were performed using hematoxylin & eosin staining. ANIT-induced hepatic cholestasis elevated liver function tests, including AST, ALT, GGT, ALP, and total bilirubin. ANIT reduced the protein expression of NTCP and BSEP hepatic transporters. It induced the expression of the inflammatory genes, TNFα, IL-6, IL-1β, and PDGF, and the expression of NF-kB at the genetic and protein level and suppressed the anti-inflammatory genes, klf6 and PPARγ. Also, antioxidant markers were reduced during ANIT induction such as GSH, SOD, catalase, heme-oxygenase-1 and PI3K/Akt pathway, while MDA levels were elevated. Furthermore, the expression of P53 gene, bax and cleaved caspase 3 proteins were activated, while bcl2 was inhibited. Also, the histopathological analysis showed degeneration of hepatocytes and inflammatory cellular infiltrates. However, vincamine treatment modulated all these markers. It improved liver function tests. It inhibited the expression of NF-kB, TNFα, IL-6, IL-1β and PDGF and activated the expression of klf6 and PPARγ. Furthermore, vincamine reduced MDA levels and induced GSH, SOD, catalase, heme-oxygenase-1 and PI3K/Akt pathway. Additionally, it inhibited expression of P53 gene, bax and cleaved caspase 3 proteins. More interestingly, vincamine showed better outcomes on the hepatic histopathological analysis and improved the alterations induced by ANIT. Vincamine alleviated hepatic dysfunction during ANIT-induced intrahepatic cholestasis through its anti-inflammatory and antioxidant efficacies by the modulation of NF-kB/PDGF/klf6/PPARγ and PI3K/Akt pathways.

Non-alcoholic fatty liver disease changes the expression and activity of hepatic drug-metabolizing enzymes and transporters in rats

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, which can cause serious complications and gradually increase the mortality rate. However, the effects of NAFLD on drug-metabolizing enzymes and transporters remain unclear, which may cause some confusion regarding patient medication. In this study, a NAFLD rat model was constructed by feeding rats with methionine and choline deficiency diets for 6 weeks, and the mRNA and protein levels of drug-metabolizing enzymes and transporter were analyzed by real-time fluorescent quantitative PCR and Western blot, respectively. The activity of drug-metabolizing enzymes was detected by cocktail methods. In the NAFLD rat model, the mRNA expression of phase I enzymes, phase II enzymes, and transporters decreased. At the protein level, only CYP1A1, CYP1B1, CYP2C11, and CYP2J3 presented a decrease. In addition, the activities of CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP3A2, UGT1A1, UGT1A3, UGT1A6, and UGT1A9 decreased. These changes may be caused by the alteration of FXR, HNF4α, LXRα, LXRβ, PXR, and RXR. In conclusion, NAFLD changes the expression and activity of hepatic drug-metabolizing enzymes and transporters in rats, which may affect drug metabolism and pharmacokinetics. In clinical medication, drug monitoring should be strengthened to avoid potential risks.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

Influence of Slco2b1-knockout and SLCO2B1-humanization on coproporphyrin I and III levels in rats.

Coproporphyrin (CP) I and III are byproducts of haem synthesis currently investigated as biomarkers for drug-drug interactions involving hepatic organic anion transporting polypeptide (OATP) 1B transporters. Another hepatically expressed OATP-member is OATP2B1. The aim of this study was to test the impact of OATP2B1, which specifically transports CPIII, on CP serum levels, applying novel rat models. CPIII transport kinetics and the interplay between OATP2B1 and multidrug resistance-associated proteins (MRPs) were determined in vitro using the vTF7 expression system. Novel rSlco2b1-/- and SLCO2B1+/+ rat models were characterized for physiological parameters and for CP serum levels. Hepatic and renal expression of transporters involved in CP disposition were determined by real-time qPCR, Western blot analysis, and immunohistochemistry. In vitro experiments revealed differences in transport kinetics comparing human and rat OATP2B1 and showed a consistent, species-specific interplay with hMRP3/rMRP3. Deletion of rOATP2B1 was associated with a trend towards lower CPI serum levels compared with wildtype rats, while CPIII remained unchanged. Comparing SLCO2B1+/+ with knockout rats revealed an effect of sex: only in females the genetic modification influenced CP serum levels. Analysis of hepatic and renal transporters revealed marginal, but in part, statistically significant differences in rMRP2 abundance, which may contribute to the observed changes in CP serum levels. Our findings support that factors other than OATP1B transporters are of relevance for basal CP levels. Only in female rats, humanization of SLCO2B1 affects basal CPI and CPIII serum levels, despite isomer selectivity of OATP2B1.

Representative Rodent Models for Renal Transporter Alterations in Human Nonalcoholic Steatohepatitis.

Alterations in renal elimination processes of glomerular filtration and active tubular secretion by renal transporters can result in adverse drug reactions. Nonalcoholic steatohepatitis (NASH) alters hepatic transporter expression and xenobiotic elimination, but until recently, renal transporter alterations in NASH were unknown. This study investigates renal transporter changes in rodent models of NASH to identify a model that recapitulates human alterations. Quantitative protein expression by surrogate peptide liquid chromatography-coupled mass spectrometry (LC-MS/MS) on renal biopsies from NASH patients was used for concordance analysis with rodent models, including methionine/choline deficient (MCD), atherogenic (Athero), or control rats and Leprdb/db MCD (db/db), C57BL/6J fast-food thioacetamide (FFDTH), American lifestyle-induced obesity syndrome (ALIOS), or control mice. Demonstrating clinical similarity to NASH patients, db/db, FFDTH, and ALIOS showed decreases in glomerular filtration rate (GFR) by 76%, 28%, and 24%. Organic anion transporter 3 (OAT3) showed an upward trend in all models except the FFDTH (from 3.20 to 2.39 pmol/mg protein), making the latter the only model to represent human OAT3 changes. OAT5, a functional ortholog of human OAT4, significantly decreased in db/db, FFDTH, and ALIOS (from 4.59 to 0.45, 1.59, and 2.83 pmol/mg protein, respectively) but significantly increased for MCD (1.67 to 4.17 pmol/mg protein), suggesting that the mouse models are comparable to human for these specific transport processes. These data suggest that variations in rodent renal transporter expression are elicited by NASH, and the concordance analysis enables appropriate model selection for future pharmacokinetic studies based on transporter specificity. These models provide a valuable resource to extrapolate the consequences of human variability in renal drug elimination. SIGNIFICANCE STATEMENT: Rodent models of nonalcoholic steatohepatitis that recapitulate human renal transporter alterations are identified for future transporter-specific pharmacokinetic studies to facilitate the prevention of adverse drug reactions due to human variability.

Emerging Roles of Uremic Toxins and Inflammatory Cytokines in the Alteration of Hepatic Drug Disposition in Patients with Kidney Dysfunction.

Patients with kidney dysfunction exhibit distinct pharmacokinetic profiles compared to those with normal kidney function. Hence, it is desirable to monitor the drug efficacy and toxicity caused by fluctuations in plasma drug concentrations associated with kidney dysfunction. Recently, pharmacokinetic information of drugs excreted mainly through the urine of patients with kidney dysfunction has been reported via drug-labeling information. Pharmacokinetic changes in drugs mainly eliminated by the liver cannot be overlooked as drug metabolism and/or transport activity in the liver may also be altered in patients with kidney dysfunction; however, the underlying mechanisms remain unclear. To plan an appropriate dosage regimen, it is necessary to clarify the underlying processes of functional changes in pharmacokinetic proteins. In recent years, uremic toxins have been shown to reduce the activity and/or expression of renal and hepatic transporters. This inhibitory effect has been reported to be time-dependent. In addition, inflammatory cytokines, such as interleukin-6, released from immune cells activated by uremic toxins and/or kidney injury can reduce the expression levels of drug-metabolizing enzymes and transporters in human hepatocytes. In this mini-review, we have summarized the renal and hepatic pharmacokinetic changes as well as the potential underlying mechanisms in kidney dysfunction, such as the chronic kidney disease and acute kidney injury. SIGNIFICANCE STATEMENT: Patients with kidney dysfunction exhibit distinct pharmacokinetic profiles compared to those with normal kidney function. Increased plasma concentrations of uremic toxins and inflammatory cytokines during kidney disease may potentially affect the activities and/or expression levels of drug-metabolizing enzymes and transporters in the liver and kidneys.

Paeoniflorin alleviates 17α-ethinylestradiol-induced cholestasis via the farnesoid X receptor-mediated bile acid homeostasis signaling pathway in rats.

Cholestasis, characterized by disturbance of bile formation, is a common pathological condition that can induce several serious liver diseases. As a kind of trigger, estrogen-induced cholestasis belongs to drug-induced cholestasis. Paeoniflorin is the most abundant bioactive constituent in Paeonia lactiflora Pall., Paeonia suffruticosa Andr., or Paeonia veitchii Lynch, a widely used herbal medicine for treating hepatic disease over centuries in China. However, the pharmacologic effect and mechanism of paeoniflorin on estrogen-induced cholestasis remain unclear. In this experiment, the pharmacological effect of paeoniflorin on EE-induced cholestasis in rats was evaluated comprehensively for the first time. Ultra-high-performance liquid chromatography coupled with Q-Exactive orbitrap mass spectrometer was used to monitor the variation of bile acid levels and composition. It was demonstrated that paeoniflorin alleviated 17α-ethinylestradiol (EE)-induced cholestasis dose-dependently, characterized by a decrease of serum biochemical indexes, recovery of bile flow, amelioration of hepatic and ileal histopathology, and reduction of oxidative stress. In addition, paeoniflorin intervention restored EE-disrupted bile acid homeostasis in enterohepatic circulation. Further mechanism studies using western blot, quantitative Real-Time PCR, and immunohistochemical showed that paeoniflorin could upregulate hepatic efflux transporters expression but downregulate hepatic uptake transporter expression. Meanwhile, paeoniflorin reduced bile acids synthesis by repressing cholesterol 7α-hydroxylase in hepatocytes. Paeoniflorin affected the above transporters and enzyme via activation of a nuclear receptor, farnesoid X receptor (FXR), which was recognized as a vital regulator for maintaining bile acid homeostasis. In conclusion, paeoniflorin alleviated EE-induced cholestasis and maintained bile acid homeostasis via FXR-mediated regulation of bile acids transporters and synthesis enzyme. The findings indicated that paeoniflorin might exert a potential therapeutic medicine for estrogen-induced cholestasis.

Hypercholesterolemia reduces the expression and function of hepatic drug metabolizing enzymes and transporters in rats

Hypercholesterolemia, one of the most common lipid metabolic diseases, may cause severe complications and even death. However, the effect of hypercholesterolemia on drug-metabolizing enzymes and transporters remains unclear. In this report, we established a rat model of diet-induced hypercholesterolemia. Quantitative real-time PCR and Western blot analysis were used to study the mRNA and protein expression of drug-metabolizing enzymes and transporters. The functions of these enzymes and transporters were evaluated by the cocktail assay. In hypercholesterolemic rats, the expression of phase I enzymes (CYP1A2, CYP2C11, CYP2E1, CYP3A1/2, CYP4A1 and FMO1/3) and phase II enzymes (UGT1A1/3, PROG, AZTG, SULT1A1, NAT1 and GSTT1) decreased. In addition, the mRNA levels of drug transporter Slco1a1/2, Slco1b2, Slc22a5, Abcc2, Abcb1a and Abcg2 decreased in rats with hypercholesterolemia, while Abcb1b and Abcc3 increased. The decreased expression of hepatic phase I and II enzymes and transporters may be caused by the changes of CAR, FXR, PXR, and Hnf4α levels. In conclusion, diet-induced hypercholesterolemia changes the expression and function of hepatic drug-metabolizing enzymes and transporters in rats, thereby possibly affecting drug metabolism and pharmacokinetics. In clinical hyperlipidemia, patients should strengthen drug monitoring to avoid possible drug exposure mediated risks.

Endoplasmic reticulum stress induces degradation of glucose transporter proteins during hyperglycemic hepatotoxicity: Role of PERK-eIF2α-ATF4 axis

Hyperglycemia induced reactive oxygen species oxidize macromolecules including cellular proteins leading to their accumulation in Endoplasmic Reticulum (ER) lumen which in turn activates unfolded protein response (UPR) sensors including, PERK (Protein Kinase RNA-Like ER Kinase). Activated PERK induces ER associated degradation of misfolded proteins to lower the ER stress. In the present study, we hypothesized that ER stress leads to the degradation of glucose transporter proteins resulting in complex glucose metabolism. In vivo studies were carried out in the experimental model of hyperglycemia using streptozotocin/nicotinamide induced diabetic male Wistar rats. High glucose (30 mM) treated HepG2 cells were used to perform the mechanistic study at different time points. PERK gene knockdown (siRNA transfection) and inhibition by ISRIB (Integrated Stress Response Inhibitor, a potent inhibitor of PERK signaling) confirmed the involvement of PERK axis in regulating the expression and translocation of hepatic glucose transporters. Co-immunoprecipitation and dual immunostaining studies further demonstrated increased degradation of GLUT proteins under high glucose conditions. Moreover, Morin (3,5,7,2′,4′ pentahydroxyflavone) treatment prevented PERK-eIF2α-ATF4 mediated degradation of glucose transporters and enhanced glucose uptake in both, HepG2 cells and diabetic rats. Targeting aberrant regulation of the expression and translocation of facilitative glucose transporter proteins (GLUT proteins) may provide novel therapeutic strategies for the better management of diabetes.

Effect of activation of liver X receptor alpha on cardiac & hepatic ABCC10 and SLC17A5 drug transporters in hypercholesterolemic rat model

BackgroundLiver x receptor α (LXRα) is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. Oxysterols (endogenous oxidized cholesterol derivatives) are the most potent endogenous LXRα-agonist. LXRα has a direct impact on several members of drug transporter superfamilies; ATP-binding cassette (ABC) and solute linked carrier (SLC). ObjectiveThe current study aimed to investigate the effect of LXRα-activation by either endogenous oxysterols or a synthetic LXRα-agonist (LXRa) such as TO901317 on hepatic and cardiac gene expression of ABCC10 and SLC17A5 drug transporters in an experimentally hypercholesterolemic rat model. Methods48 male rats were divided randomly into four groups (n = 12); control group rats received vehicle; hypercholesterolemic group (HCH group) rats received diet contain 2.5% cholesterol &deoxycholic acid for 8 weeks; (LXRa group) rats were fed standard pellet chow for 8 weeks, then a single dose of LXRa was administered (IP) at a dose of 10 mg/kg; (HCH + LXRa group) rats received diet contain 2.5% cholesterol &deoxycholic acid for 8 weeks, then a single dose of LXRa was administered (IP) at a dose of 10 mg/kg. ResultsOur findings revealed that hypercholesterolemia and LXRa significantly activated LXRα to varying degrees in both hepatic and cardiac tissues with subsequent alteration of LXRα and ABCC10 gene expression. Whereas, SLC17A5 gene expression was primarily affected by elevated serum cholesterol level and unmediated via LXRα-activation. ConclusionsAccordingly, it was concluded that ABCC10 is a specific LXRα-target gene and that LXRα autoregulates its own expression in rats.

Gardenia jasminoides Ellis polysaccharide ameliorates cholestatic liver injury by alleviating gut microbiota dysbiosis and inhibiting the TLR4/NF-κB signaling pathway.

Gardenia jasminoides Ellis is a well-known herbal medicine. In this study, the effect of G. jasminoides Ellis polysaccharide (GPS) on liver injury in an alpha-naphthylisothiocyanate (ANIT)-induced cholestatic mouse model and the associated molecular mechanisms were investigated. GPS administration dose-dependently ameliorated impaired hepatic function, including a 2-7-fold decrease in aminotransferase levels, ameliorating tissue damage, upregulating the expression of farnesoid X receptor (FXR) and pregnane X receptor (PXR) and their downstream efflux transporters, and decreasing the levels of 12 bile acids (BAs), in cholestatic mice. Furthermore, GPS ameliorated gut microbiota dysbiosis, improved intestinal barrier function, and reduced serum and hepatic lipopolysaccharide levels 1.5-fold. GPS also inhibited the Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling, decreased the expression of inflammatory factor genes, and ameliorated hepatic inflammation. Notably, fecal microbiota transplantation from GPS-fed mice also increased the hepatic expression of FXR, PXR, and efflux transporters; decreased the levels of 12 BAs; restored intestinal barrier function; and decreased hepatic inflammation mediated by the TLR4/NF-κB pathway. In conclusion, GPS has a protective effect against cholestatic liver injury through modulation of gut microbiota and inhibition of the TLR4/NF-κB pathway. Regulating gut microbiota using herbal medicine polysaccharides may hold unique therapeutic promise for cholestatic liver diseases.

Gut microbiota promotes cholesterol gallstone formation by modulating bile acid composition and biliary cholesterol secretion

Cholesterol gallstone disease is a worldwide common disease. Cholesterol supersaturation in gallbladder bile is the prerequisite for its pathogenesis, while the mechanism is not completely understood. In this study, we find enrichment of gut microbiota (especially Desulfovibrionales) in patients with gallstone disease. Fecal transplantation of gut microbiota from gallstone patients to gallstone-resistant strain of mice can induce gallstone formation. Carrying Desulfovibrionales is associated with enhanced cecal secondary bile acids production and increase of bile acid hydrophobicity facilitating intestinal cholesterol absorption. Meanwhile, the metabolic product of Desulfovibrionales, H2S increase and is shown to induce hepatic FXR and inhibit CYP7A1 expression. Mice carrying Desulfovibrionales present induction of hepatic expression of cholesterol transporters Abcg5/g8 to promote biliary secretion of cholesterol as well. Our study demonstrates the role of gut microbiota, Desulfovibrionales, as an environmental regulator contributing to gallstone formation through its influence on bile acid and cholesterol metabolism.

Sodium salicylate rewires hepatic metabolic pathways in obesity and attenuates IL-1β secretion from adipose tissue: The implications for obesity-impaired reverse cholesterol transport

IntroductionHigh-fat diet (HFD)-induced obesity impairs clearance of cholesterol through the Reverse Cholesterol Transport (RCT) pathway, with downregulation in hepatic expression of cholesterol and bile acid transporters, namely ABCG5/8 and ABCB11, and reduced high-density lipoprotein (HDL) cholesterol efflux capacity (CEC). In the current study, we hypothesized that the development of hepatosteatosis, secondary to adipose-tissue dysfunction, contributes to obesity-impaired RCT and that such effects could be mitigated using the anti-inflammatory drug sodium salicylate (NaS). Materials and methodsC57BL/6J mice, fed HFD ± NaS or low-fat diet (LFD) for 24 weeks, underwent glucose and insulin tolerance testing. The 3H-cholesterol movement from macrophage-to-feces was assessed in vivo. HDL-CEC was determined ex vivo. Cytokine secretion from adipose-derived stromal vascular fraction (SVF) cells was measured ex vivo. Liver and HDL proteins were determined by mass spectrometry and analyzed using Ingenuity Pathway Analysis. ResultsNaS delayed HFD-induced weight gain, abrogated priming of pro-IL-1β in SVFs, attenuated insulin resistance, and prevented steatohepatitis (ectopic fat accumulation in the liver). Prevention of hepatosteatosis coincided with increased expression of PPAR-alpha/beta-oxidation proteins with NaS and reduced expression of LXR/RXR-induced proteins including apolipoproteins. The latter effects were mirrored within the HDL proteome in circulation. Despite remarkable protection shown against steatosis, HFD-induced hypercholesterolemia and repression of the liver-to-bile cholesterol transporter, ABCG5/8, could not be rescued with NaS. Discussions and conclusionsThe cardiometabolic health benefits of NaS may be attributed to the reprogramming of hepatic metabolic pathways to increase fatty acid utilization in the settings of nutritional overabundance. Reduced hepatic cholesterol levels, coupled with reduced LXR/RXR-induced proteins, may underlie the lack of rescue of ABCG5/8 expression with NaS. This remarkable protection against HFD-induced hepatosteatosis did not translate to improvements in cholesterol homeostasis.

Ontogeny of renal, hepatic, and placental expression of ATP-binding cassette and solute carrier transporters in the rat and the rabbit

Species differences in developmental toxicity can be due to varying expression of xenobiotic transporters. Hence, knowledge on the ontogeny of these transporters, especially in human, rat and rabbit, is pivotal. Two superfamilies of transporters, the ATP-binding cassette (ABC) and the solute carrier (SLC) transporters, are well known for their role in the absorption, distribution and/or elimination of xenobiotics and endogenous substances. The aim of this study was to compare the expression levels of these xenobiotic transporters in liver, kidney and placenta of man, Wistar rat and New Zealand White rabbit during pre- and postnatal development. For this purpose, qPCR experiments were performed for rat and rabbit tissues and the gene expression profiles were compared with literature data from man, rat and rabbit. Data analysis showed large differences in transporter expression in development and between species. These results can be used to better understand developmental toxicity findings in non-clinical species and their relevance for man.

Bioprinting of Human Liver-Derived Epithelial Organoids for Toxicity Studies.

There is a need for long-lived hepatic in vitro models to better predict drug induced liver injury (DILI). Human liver-derived epithelial organoids are a promising cell source for advanced in vitro models. Here, organoid technology is combined with biofabrication techniques, which holds great potential for the design of in vitro models with complex and customizable architectures. Here, porous constructs with human hepatocyte-like cells derived from organoids are generated using extrusion-based printing technology. Cell viability of bioprinted organoids remains stable for up to ten days (88-107% cell viability compared to the day of printing). The expression of hepatic markers, transporters, and phase I enzymes increased compared to undifferentiated controls, and is comparable to non-printed controls. Exposure to acetaminophen, a well-known hepatotoxic compound, decreases cell viability of bioprinted liver organoids to 21-51% (p < 0.05) compared to the start of exposure, and elevated levels of damage marker miR-122 are observed in the culture medium, indicating the potential use of the bioprinted constructs for toxicity testing. In conclusion, human liver-derived epithelial organoids can be combined with a biofabrication approach, thereby paving the way to create perfusable, complex constructs which can be used as toxicology- and disease-models.

Altered cisplatin pharmacokinetics during nonalcoholic steatohepatitis contributes to reduced nephrotoxicity

Disease-mediated alterations to drug disposition constitute a significant source of adverse drug reactions. Cisplatin (CDDP) elicits nephrotoxicity due to exposure in proximal tubule cells during renal secretion. Alterations to renal drug transporter expression have been discovered during nonalcoholic steatohepatitis (NASH), however, associated changes to substrate toxicity is unknown. To test this, a methionine- and choline-deficient diet-induced rat model was used to evaluate NASH-associated changes to CDDP pharmacokinetics, transporter expression, and toxicity. NASH rats administered CDDP (6 mg/kg, i.p.) displayed 20% less nephrotoxicity than healthy rats. Likewise, CDDP renal clearance decreased in NASH rats from 7.39 to 3.83 mL/min, renal secretion decreased from 6.23 to 2.80 mL/min, and renal CDDP accumulation decreased by 15%, relative to healthy rats. Renal copper transporter-1 expression decreased, and organic cation transporter-2 and ATPase copper transporting protein-7b increased slightly, reducing CDDP secretion. Hepatic CDDP accumulation increased 250% in NASH rats relative to healthy rats. Hepatic organic cation transporter-1 induction and multidrug and toxin extrusion protein-1 and multidrug resistance-associated protein-4 reduction may contribute to hepatic CDDP sequestration in NASH rats, although no drug-related toxicity was observed. These data provide a link between NASH-induced hepatic and renal transporter expression changes and CDDP renal clearance, which may alter nephrotoxicity.

Curcumin Reduces Hepatic and White Adipose Tissue Inflammation and Expression of Specific Zinc Transporters in Diet-Induced Obese Mice

Abstract Objectives Obesity is a complex metabolic disease, that is often associated with non-alcoholic fatty liver disease (NAFLD). Inflammation is a common feature of both diseases. Curcumin, a traditionally used spice in Asia, exerts anti-inflammatory effects in liver and white adipose tissue (WAT) of diet-induced obese (DIO) mice. However, mechanisms involved in these beneficial effects remain obscure. Zinc is an important micronutrient involved in inflammatory responses. Whole-body zinc homeostasis plays a critical role in decreasing tissue specific inflammation. Zinc homeostasis is maintained mainly by zinc transporters known as ZnT (zinc transporters) and Zip (Zrt and Irt-like proteins) family. We propose that zinc transporters may contribute to curcumin's protective metabolic effects. Thus, the objective of this research was to determine curcumin's effects on inflammatory markers and zinc transporters in liver and WAT from DIO mice. Methods Male B6 mice were fed a HFD (45% kcal fat) or HFD supplemented with 0.4% (w/w) curcumin (HFC) for fourteen weeks. Serum triglycerides (TG) and free fatty acid (FFA) levels were measured. mRNA levels for inflammatory markers and zinc transporters were determined in WAT and liver by qRT-PCR. Results No significant changes were observed in body weight, serum TG and FFA levels with curcumin supplementation. However, gene expression of inflammatory markers, including Stat1, and Nf-KB subunit p65 were significantly reduced in liver and WAT from HFC group compared to HF (P &amp;lt; 0.05). Furthermore, curcumin reduced hepatic zinc transporters Zip10, Zip14, ZnT10 but increased ZnT9 expression. In WAT, curcumin significantly reduced mRNA levels for Zip1, Zip14, ZnT1, and ZnT7 (P &amp;lt; 0.05). Conclusions Our results indicate that zinc transporters may in part mediate the anti-inflammatory properties of curcumin, particularly Zip14, in WAT and liver of DIO mice. Future mechanistic studies are necessary to establish whether zinc transporters are required for curcumin's anti-inflammatory effects in obesity and associated NAFLD. Funding Sources AHA grant# 19AIREA34450279.

Hepatocyte membrane potential regulates serum insulin and insulin sensitivity by altering hepatic GABA release.

SUMMARYHepatic lipid accumulation in obesity correlates with the severity of hyperinsulinemia and systemic insulin resistance. Obesity-induced hepatocellular lipid accumulation results in hepatocyte depolarization. We have established that hepatocyte depolarization depresses hepatic afferent vagal nerve firing, increases GABA release from liver slices, and causes hyperinsulinemia. Preventing hepatic GABA release or eliminating the ability of the liver to communicate to the hepatic vagal nerve ameliorates the hyperinsulinemia and insulin resistance associated with diet-induced obesity. In people with obesity, hepatic expression of GABA transporters is associated with glucose infusion and disposal rates during a hyperinsulinemic euglycemic clamp. Single-nucleotide polymorphisms in hepatic GABA re-uptake transporters are associated with an increased incidence of type 2 diabetes mellitus. Herein, we identify GABA as a neuro-hepatokine that is dysregulated in obesity and whose release can be manipulated to mute or exacerbate the glucoregulatory dysfunction common to obesity.