Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis.

BACKGROUND & AIMS: Aramchol is a fatty acid-bile acid conjugate that reduces liver fat content and is being evaluated in a phase III clinical trial for non-alcoholic steatohepatitis (NASH). Aramchol attenuates NASH in mouse models and decreases steatosis by downregulating the fatty acid synthetic enzyme stearoyl CoA desaturase 1 (SCD1) in hepatocytes. Although hepatic stellate cells (HSCs) also store lipids as retinyl esters, the impact of Aramchol in this cell type is unknown. METHODS: We investigated the effects of Aramchol on a human HSC line (LX-2), primary human HSCs (phHSCs), and primary human hepatocytes (phHeps). RESULTS: In LX-2 and phHSCs, 10 µM Aramchol significantly reduced SCD1 mRNA while inducing PPARG (PPARγ) mRNA, with parallel changes in the 2 proteins; ACTA2, COL1A1, ß-PDGFR (bPDGFR) mRNAs were also significantly reduced in LX-2. Secretion of collagen 1 (Col1α1) was inhibited by 10 µM Aramchol. SCD1 knockdown in LX-2 cells phenocopied the effect of Aramchol by reducing fibrogenesis, and addition of Aramchol to these cells did not rescue fibrogenic gene expression. Conversely, in LX-2 overexpressing SCD1, Aramchol no longer suppressed fibrogenic gene expression. The drug also induced genes in LX-2 that promote cholesterol efflux and inhibited ACAT2, which catalyses cholesterol synthesis. In phHeps, Aramchol also reduced SCD1 and increased PPARG mRNA expression. CONCLUSIONS: Aramchol downregulates SCD1 and elevates PPARG in HSCs, reducing COL1A1 and ACTA2 mRNAs and COL1A1 secretion. These data suggest a direct inhibitory effect of Aramchol in HSCs through SCD1 inhibition, as part of a broader impact on both fibrogenic genes as well as mediators of cholesterol homeostasis. These findings illustrate novel mechanisms of Aramchol activity, including potential antifibrotic activity in patients with NASH and fibrosis. LAY SUMMARY: In this study, we have explored the potential activity of Aramchol, a drug currently in clinical trials for fatty liver disease, in blocking fibrosis, or scarring, by hepatic stellate cells, the principal collagen-producing (i.e. fibrogenic) cell type in liver injury. In both isolated human hepatic stellate cells and in a human hepatic stellate cell line, the drug suppresses the key fat-producing enzyme, stearoyl CoA desaturase 1 (SCD1), which leads to reduced expression of genes and proteins associated with hepatic fibrosis, while inducing the protective gene, PPARγ. The drug loses activity when SCD1 is already reduced by gene knockdown, reinforcing the idea that inhibition of SCD1 is a main mode of activity for Aramchol. These findings strengthen the rationale for testing Aramchol in patients with NASH.

Arachidyl amido cholanoic acid improves liver glucose and lipid homeostasis in nonalcoholic steatohepatitis via AMPK and mTOR regulation.

BACKGROUND: Arachidyl amido cholanoic acid (Aramchol) is a potent downregulator of hepatic stearoyl-CoA desaturase 1 (SCD1) protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis. In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis (NASH), 52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c, an indicator of glycemic control. AIM: To assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model [induced with a 0.1% methionine and choline deficient diet (0.1MCD)] after treatment with Aramchol. METHODS: Isolated primary mouse hepatocytes were incubated with 20 µmol/L Aramchol or vehicle for 48 h. Subsequently, analyses were performed including Western blot, proteomics by mass spectrometry, and fluxomic analysis with 13C-uniformly labeled glucose. For the in vivo part of the study, male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk. Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites. RESULTS: Combination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes. This translated into changes in the content of their downstream targets including proteins involved in fatty acid (FA) synthesis and oxidation [P-ACCα/ß(S79), SCD1, CPT1A/B, HADHA, and HADHB], oxidative phosphorylation (NDUFA9, NDUFB11, NDUFS1, NDUFV1, ETFDH, and UQCRC2), tricarboxylic acid (TCA) cycle (MDH2, SUCLA2, and SUCLG2), and ribosome (P-p70S6K[T389] and P-S6[S235/S236]). Flux experiments with 13C-uniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes, as indicated by the increase in the number of rounds that malate remained in the TCA cycle. Finally, liver metabolomic analysis showed that glucose homeostasis was improved by Aramchol in 0.1MCD fed mice in a dose-dependent manner, showing normalization of glucose, G6P, F6P, UDP-glucose, and Rbl5P/Xyl5P. CONCLUSION: Aramchol exerts its effect on glucose and lipid metabolism in NASH through activation of AMPK and inhibition of mTORC1, which in turn activate FA ß-oxidation and oxidative phosphorylation.

Cathepsins Drive Anti-Inflammatory Activity by Regulating Autophagy and Mitochondrial Dynamics in Macrophage Foam Cells.

BACKGROUND/AIMS: Atherosclerosis underlies the majority of cardiovascular events, consequent to non-resolving inflammation. Considerable evidence implicates autophagy dysfunction at the core of this inflammatory condition, but the basis of this dysfunction is not fully understood. METHODS: Using an in vitro model of lipid-laden macrophages, activity-based probes and high-throughput techniques, we studied the role of the cysteine proteases cathepsins in autophagy. RESULTS: We showed that cathepsin activity is suppressed by oxidized lipids and that cathepsin has an indispensable role in the autophagy-lysosomal degradation pathway. Accordingly, loss of cathepsin function resulted in autophagy derangement. Shotgun proteomics confirmed autophagy dysfunction and unveiled a pivotal role of cathepsin L in a putative cathepsin degradation network. At the physiological level, cathepsin inhibition resulted in mitochondrial stress, which translated into impaired oxidative metabolism, excessive production of reactive oxygen species and activation of the cellular stress response, driven by ATF4-CHOP transcription factors. In addition, transcriptomic analysis of these cells uncovered some genetic similarities with the inflammatory macrophage phenotype (a.k.a M1 macrophages) and increased expression of inflammatory cytokines. CONCLUSION: Our data highlight the importance of cathepsins for mitochondrial quality control mechanisms and amelioration of vascular inflammation.

CT Imaging of Enzymatic Activity in Cancer Using Covalent Probes Reveal a Size-Dependent Pattern.

X-ray CT instruments are among the most available, efficient, and cost-effective imaging modalities in hospitals. The field of CT molecular imaging is emerging which relies mainly on the detection of gold nanoparticles and iodine-containing compounds directed to tagging a variety of abundant biomolecules. Here for the first time we attempted to detect enzymatic activity, while the low sensitivity of CT scanners to contrast reagents made this a challenging task. Therefore, we developed a new class of nanosized cathepsin-targeted activity-based probes (ABPs) for functional CT imaging of cancer. ABPs are small molecules designed to covalently modify enzyme targets in an activity-dependent manner. Using a CT instrument, these novel probes enable detection of the elevated cathepsin activity within cancerous tissue, thus creating a direct link between biological processes and imaging signals. We present the generation and biochemical evaluation of a library of ABPs tagged with different sized gold nanoparticles (GNPs), with various ratios of cathepsin-targeting moiety and a combination of different polyethylene glycol (PEG) protective layers. The most potent and stable GNP-ABPs were applied for noninvasive cancer imaging in mice. Surprisingly, detection of CT contrast from the tumor had reverse correlation to GNP size and the amount of targeting moiety. Interestingly, TEM images of tumor sections show intercellular lysosomal subcellular localization of the GNP-ABPs. In conclusion, we demonstrate that the covalent linkage is key for detection using low sensitive imaging modalities and the utility of GNP-ABPs as a promising tool for enzymatic-based CT imaging.

Molecular Imaging of Cancer Using X-ray Computed Tomography with Protease Targeted Iodinated Activity-Based Probes.

X-ray computed tomography (CT) is a robust, precise, fast, and reliable imaging method that enables excellent spatial resolution and quantification of contrast agents throughout the body. However, CT is largely inadequate for molecular imaging applications due mainly to its low contrast sensitivity that forces the use of large concentrations of contrast agents for detection. To overcome this limitation, we generated a new class of iodinated nanoscale activity-based probes (IN-ABPs) that sufficiently accumulates at the target site by covalently binding cysteine cathepsins that are exceptionally highly expressed in cancer. The IN-ABPs are comprised of a short targeting peptide selective to specific cathepsins, an electrophilic moiety that allows activity-dependent covalent binding, and tags containing dendrimers with up to 48 iodine atoms. IN-ABPs selectively bind and inhibit activity of recombinant and intracellular cathepsin B, L, and S. We compared the in vivo kinetics, biodistribution, and tumor accumulation of IN-ABPs bearing 18 and 48 iodine atoms each, and their control counterparts lacking the targeting moiety. Here we show that although both IN-ABPs bind specifically to cathepsins within the tumor and produce detectable CT contrast, the 48-iodine bearing IN-ABP was found to be optimal with signals over 2.1-fold higher than its nontargeted counterpart. In conclusion, this study shows the synthetic feasibility and potential utility of IN-ABPs as potent contrast agents that enable molecular imaging of tumors using CT.

Adaptation to a blood pressure telemetry system revealed by measures of activity, agility and operant learning in mice.

INTRODUCTION: Implantable telemetry enables continuous monitoring of physiological functions in freely moving animals and can greatly complement pharmacological research. Despite its miniaturization, a sensor/transmitter constitutes 5% or more of a mouse's bodyweight. The aim of the present study was to evaluate whether factors related to the presence of a probe/transmitter influence the ambulatory activity, strength, agility, or operant, motivated behaviors of this small rodent. METHODS: Adult male mice (C57BL/6N, 22-25g, 9-10weeks; implanted n=26, intact n=45) were evaluated during week-long tests, conducted three and eight weeks after surgical implantation of the PA-C10 blood pressure probe. An open field test, grip force measurement, Rotarod test were performed, followed by 7-day continuous monitoring of spontaneous wheel running activity and positively reinforced operant conditioning in an automated data collection system. RESULTS: An implanted blood pressure transmitter did not affect behavior of mice in the open field test, on the Rotarod or their grip force, compared to unoperated controls. Voluntary wheel running distance was reduced three, but not eight weeks after implantation. Three weeks after the surgery, performance in the positively reinforced operant conditioning in operated mice was slightly decreased compared to intact animals, while retention and acquisition of a 2nd, reversal-learning task eight weeks after the surgery were unaffected. DISCUSSION: We conclude that an implantable transmitter may have detectable effects in the first few weeks following implantation on some elements of mouse behavior. With sufficient recovery, mice perform comparably to unoperated controls in tests of strength, endurance, agility and learned operant behavior.

Mice in Bion-M 1 space mission: training and selection.

After a 16-year hiatus, Russia has resumed its program of biomedical research in space, with the successful 30-day flight of the Bion-M 1 biosatellite (April 19-May 19, 2013). The principal species for biomedical research in this project was the mouse. This paper presents an overview of the scientific goals, the experimental design and the mouse training/selection program. The aim of mice experiments in the Bion-M 1 project was to elucidate cellular and molecular mechanisms, underlying the adaptation of key physiological systems to long-term exposure in microgravity. The studies with mice combined in vivo measurements, both in flight and post-flight (including continuous blood pressure measurement), with extensive in vitro studies carried out shortly after return of the mice and in the end of recovery study. Male C57/BL6 mice group housed in space habitats were flown aboard the Bion-M 1 biosatellite, or remained on ground in the control experiment that replicated environmental and housing conditions in the spacecraft. Vivarium control groups were used to account for housing effects and possible seasonal differences. Mice training included the co-adaptation in housing groups and mice adaptation to paste food diet. The measures taken to co-adapt aggressive male mice in housing groups and the peculiarities of "space" paste food are described. The training program for mice designated for in vivo studies was broader and included behavioral/functional test battery and continuous behavioral measurements in the home-cage. The results of the preliminary tests were used for the selection of homogenous groups. After the flight, mice were in good condition for biomedical studies and displayed signs of pronounced disadaptation to Earth's gravity. The outcomes of the training program for the mice welfare are discussed. We conclude that our training program was effective and that male mice can be successfully employed in space biomedical research.

Rat imaging and in vivo stability studies using [11C]-dimethyl-diphenyl ammonium, a candidate agent for PET-myocardial perfusion imaging.

BACKGROUND: PET myocardial perfusion imaging (MPI) holds several advantages over SPECT for diagnosing coronary artery disease. The short half-lives of prevailing PET-MPI agents hamper wider clinical application of PET in nuclear cardiology; prompting the development of novel PET-MPI agents. We have previously reported on the potential of radiolabeled ammonium salts, and particularly on that of [(11)C]dimethyl-diphenyl-ammonium ([(11)C]DMDPA), for cardiac PET imaging. This study was designed to improve the radiosynthesis and increase the yield of [(11)C]DMDPA, characterize more meticulously the kinetics of radioactivity distribution after its injection via micro-PET/CT studies, and further explore its potential for PET-MPI. METHODS: The radiosynthetic procedure of [(11)C]DMDPA was improved with respect to the previously reported one. The kinetics of radioactivity distribution following injection of [(11)C]DMDPA were investigated in juvenile and young adult male SD rats using microPET/CT, and compared to those of [(13)N]NH3. Furthermore, the metabolic fate of [(11)C]DMDPA in vivo was examined after its injection into rats. RESULTS: Following a radiosynthesis time of 25-27 min, 11.9 ± 1.1 GBq of [(11)C]DMDPA was obtained, with a 43.7% ± 4.3% radiochemical yield (n = 7). Time activity curves calculated after administration of [(11)C]DMDPA indicated rapid, high and sustained radioactivity uptake in hearts of both juvenile and young adult rats, having a two-fold higher cardiac radioactivity uptake compared to [(13)N]NH3. Accordingly, at all time points after injection to both juvenile and young adult rats, image quality of the left ventricle was higher with [(11)C]DMDPA compared to [(13)N]NH3. In vivo stability studies of [(11)C]DMDPA indicate that no radioactive metabolites could be detected in plasma, liver and urine samples of rats up to 20 min after injection, suggesting that [(11)C]DMDPA is metabolically stable in vivo. CONCLUSIONS: This study further illustrates that [(11)C]DMDPA holds, at least in part, essential qualities required from a PET-MPI probe. Owing to the improved radiosynthetic procedure reported herein, [(11)C]DMDPA can be produced in sufficient amounts for clinical use.

Contribution of social isolation, restraint, and hindlimb unloading to changes in hemodynamic parameters and motion activity in rats.

The most accepted animal model for simulation of the physiological and morphological consequences of microgravity on the cardiovascular system is one of head-down hindlimb unloading. Experimental conditions surrounding this model include not only head-down tilting of rats, but also social and restraint stresses that have their own influences on cardiovascular system function. Here, we studied levels of spontaneous locomotor activity, blood pressure, and heart rate during 14 days under the following experimental conditions: cage control, social isolation in standard rat housing, social isolation in special cages for hindlimb unloading, horizontal attachment (restraint), and head-down hindlimb unloading. General activity and hemodynamic parameters were continuously monitored in conscious rats by telemetry. Heart rate and blood pressure were both evaluated during treadmill running to reveal cardiovascular deconditioning development as a result of unloading. The main findings of our work are that: social isolation and restraint induced persistent physical inactivity, while unloading in rats resulted in initial inactivity followed by normalization and increased locomotion after one week. Moreover, 14 days of hindlimb unloading showed significant elevation of blood pressure and slight elevation of heart rate. Hemodynamic changes in isolated and restrained rats largely reproduced the trends observed during unloading. Finally, we detected no augmentation of tachycardia during moderate exercise in rats after 14 days of unloading. Thus, we concluded that both social isolation and restraint, as an integral part of the model conditions, contribute essentially to cardiovascular reactions during head-down hindlimb unloading, compared to the little changes in the hydrostatic gradient.