Unravelling the mechanisms that determine the uptake and metabolism of magnetic single and multicore nanoparticles in a Xenopus laevis model.

Multicore superparamagnetic nanoparticles have been proposed as ideal tools for some biomedical applications because of their high magnetic moment per particle, high specific surface area and long term colloidal stability. Through controlled aggregation and packing of magnetic cores it is possible to obtain not only single-core but also multicore and hollow spheres with internal voids. In this work, we compare toxicological properties of single and multicore nanoparticles. Both types of particles showed moderate in vitro toxicity (MTT assay) tested in Hep G2 (human hepatocellular carcinoma) and Caco-2 (human colorectal adenocarcinoma) cells. The influence of surface chemistry in their biological behavior was also studied after functionalization with O,O′-bis(2-aminoethyl) PEG (2000 Da). For the first time, these nanoparticles were evaluated in a Xenopus laevis model studying their whole organism toxicity and their impact upon iron metabolism. The degree of activation of the metabolic pathway depends on the size and surface charge of the nanoparticles which determine their uptake. The results also highlight the potential of Xenopus laevis model bridging the gap between in vitro cell-based assays and rodent models for toxicity assessment to develop effective nanoparticles for biomedical applications

[3-13C]Pyruvate: Useful Alternative to labeled Glucose for in vitro Metabolic Studies in Primary Mouse Hepatocytes

The application of stable isotope labeling in cell culture experiments is a widely used and potent model for the study and characterization of metabolic pathways and fluxes. In particular, incorporation of 13C-labels at certain carbon positions of a given metabolite may not only provide important information about pathways and fluxes but may even reveal unexpected metabolic phenomena without prior knowledge. Depending on the cellular model and the area of interest, one has to take into consideration which labeled substrate will give the most valuable information. Primary hepatocytes in cell culture – a widely used model to study liver physiology and diseases – are expected to use a variety of substrates for their cellular and energy metabolism. So far, 13C-NMR studies have been rarely performed in isolated hepatocytes. Labeled glucose is the most widely used substrate in cells isolated from various organs. However, we have shown in preliminary experiments that hepatocytes in primary culture do not readily metabolize glucose taken up from the medium. Since hepatocytes have a very high mitochondrial activity, we have therefore decided to use one- and two-dimensional multinuclear NMR-spectroscopy to characterize the metabolism of [3-13C]pyruvate and the metabolic isotopomers derived trough various pathways in these cells

Metabolic Characterization of Patients with Alcohol and Non-Alcoholic Steatohepatitis

Liver steatosis is an increasingly common cause of liver diseases. Today it is still difficult to differentiate patients with non-alcoholic steatohepatitis (NASH) from patients with alcoholic steatohepatitis (ASH) on the basis of clinical and biochemical evaluations. Since it is becoming urgent to develop new diagnostic methods for NASH, we applied high-resolution 1H- and (natural abundance) 13C-NMR spectroscopy to detect metabolic profiles in preparations of blood and urine in patients with NASH and ASH. 1H- and 13C-NMR spectra of urine and blood samples showed considerable and reproducible differences in specific metabolites involved in intermediary as well as lipid metabolism in patients with NASH and ASH compared to healthy controls. The results demonstrate that NMR applications on human body fluids are of great potential to characterize NASH, a disease displaying multiple interrelated metabolic factors. This approach could provide noninvasively data to characterize steatosis and give the possibility to distinguish between NASH and ASH

Cellular Metabolism and Apoptosis: Dexamethasone, a Promising new Candidate to Intervene on the Metabolic Level

Apoptosis – a regulated form of cell death – is the main mode of cell death in most liver injuries. Mitochondria, besides their recognized role as energy-providing metabolic centers, are also key regulatory centers during the genesis of apoptosis. Consequently, increasing interest has developed around the relations between cell metabolism and cell death/survival. We have recently shown that the initial phase of the apoptotic process is associated with alterations in specific glucose metabolic pathways. The synthetic glucocorticoid Dexamethasone (DEX) is a commonly used therapeutic agent in several inflammatory disorders. Besides its clinical use, DEX is known to inhibit apoptosis in vitro. So far, the exact underlying mechanisms of the anti-apoptotic effect of DEX are poorly understood and are currently being investigated.[1,2] Interestingly, with regards to our earlier observations DEX has been shown to modify metabolic pathways (in particular, anaplerosis and gluconeogenesis) both in vivo and in vitro

Metabolic Characterization of Patients with NASH and ASH by High-Resolution Multinuclear NMR Spectroscopy on Bodyfluids

INTRODUCTION: Liver steatosis is a common cause of liver disease. Non-alcoholic fatty liver disease (NASH or NAFLD) ranges from steatosis without inflammation to steatohepatitis, ballooning degeneration with or without liver fibrosis. Similar pathological findings are observed in patients with ASH (alcoholic steatohepatitis), and it is difficult to differentiate both pathologies by clinical and biochemical evaluations. High-resolution 1H-NMR has emerged as a powerful technique to simultaneously identify and quantify multiple metabolites of medical significance without a requirement for pre-selection or separation of metabolites. AIM: Since it is becoming urgent to develop new diagnostic methods for NAFLD, we applied novel multinuclear NMR methods to detect the metabolic profile in body fluids in patients with NASH and ASH. METHODS: Thirty non-alcoholic patients, 7 alcoholic steatotic patients and 30 age-matched control subjects were recruited. Urine samples were lyophilized. We used dual-extraction methods to investigate both water-soluble metabolites and lipophilic compounds involved in fatty acid- and lipid metabolism, in blood plasma and serum. Furthermore, to study metabolites not accessible by conventional 1H NMR analysis, natural abundance 13C NMR measurements were performed. To identify unknown metabolites in body fluids, two-dimensional 1H-1H} and {1H-13C experiments were used. RESULTS: 1H- and 13C-NMR spectra of urine and blood extracts clearly showed considerable differences in specific metabolites involved in liver intermediary metabolism in patients with NASH and ASH compared to healthy controls. Selective changes in patients with ASH compared to controls were detected urine (decreases of hippurate (to 46±13), and urea (to 37±21), increases of TMAO (to 157±28) and tyrosine (to 322±124) as well as accumulation of bile acids) and in blood plasma (increases of TMAO (to 314±58) and methionine (to 620±81) and decreases in branched-chain amino acids (to 32±5)). In patients with NASH, 13C-NMR spectra showed several and significant changes in metabolites involved in mitochondrial and lipid metabolism. CONCLUSIONS: New applications of NMR methods on human body fluids are of great potential to characterize NAFLD, a disease displaying multiple interrelated metabolic factors. This approach could provide new data to characterize steatosis, help to distinguish between NASH and ASH, and also give insights in the pathophysiology of both diseases

Dissecting the effect of cyclosporine on the sequence of events leading to hepatocellular apoptosis

INTRODUCTION: The immunosuppressant cyclosporine (Cyclosporin A, CsA) is known to have the capacity to prevent cellular apoptosis by inhibition of the mitochondrial permeability transition (MPT). We have recently shown that Fas receptor-mediated apoptosis in mouse liver involves early upregulations of specific glucose metabolic pathways and that pre-treatment with CsA prevented these early metabolic events. AIMS: A) In order to further characterize the protective effect of CsA on apoptotic liver injury, we analyzed the sequence of cellular events leading to hepatocyte cell death following anti-Fas injection. B) We then investigated the effects of pre-treatment with CsA on these characteristics that lead to hepatocellular cell death. METHODS: BALB/C mice were injected with anti-Fas antibody (0.5 µg/g, ip). CsA (50 mg/kg; Sandimmune®, ip) was injected 45 min prior to anti-Fas. Animals were sacrificed at five time points from 45min up to 7.5hrs after anti-Fas injection. Standard enzymatic assays were used for serum-ALT/AST and caspase-3 determinations. BID/tBID was analyzed by Western blot and GSH/GSSG by HPLC. RESULTS: A) Anti-Fas-induced liver injury followed a characteristic sequence of cellular events leading to death of the mice at around 8 hrs after anti-Fas injection. First the cleavage of BID was identified 2 hrs after injection, followed by elevation in caspase-3 activity at 3 hrs (26.8±5.3 U, P<0.001 vs. saline-treated controls). Liver cell damage was evident at 5 hrs. At this time serum ALT/AST were increased (7584±2240/5835±1158 U/L) and hepatocyte apoptosis and tissue hemorrhage were identified on histology. Intracellular GSH and GSSG started to decrease at 5 hrs but the ratio of GSH/GSSG remained constant at all time points. B) Pre-treatment with CsA significantly delayed the increase in caspase-3 activity (3 hrs: 7.6±2.1 U, P<0.01 vs. anti-Fas-only; 5 hrs: 43±6.4 U). Serum ALT were also significantly lower at 5 hrs (3100±1104 U/L, P<0.01 vs. anti-Fas-only). Histological evidence of apoptosis was markedly reduced at 7.5hrs. Interestingly, pre-treatment with the vehicle of the CsA formulation, Cremophor® EL (CrEL), offered a very similar protective effect on liver injury. CONCLUSIONS: Our results demonstrate that the protective effect of the CsA formulation occurs at all levels of the injury process. However, they also suggest that the inhibition of hepatocellular apoptosis observed with CsA has to be attributed mainly to the vehicle CrEL

Hepatocellular apoptosis in mice is associated with early upregulation of mitochondrial glucose metabolism

Hepatocyte death due to apoptosis is a hallmark of almost every liver disease. Manipulation of cell death regulatory steps during the apoptotic process is therefore an obvious goal of biomedical research. To clarify whether metabolic changes occur prior to the characteristic apoptotic events, we used ex vivo multinuclear NMR-spectroscopy to study metabolic pathways of [U-13C]glucose in mouse liver during Fas-induced apoptosis. We addressed whether these changes could be associated with protection against apoptosis afforded by Epidermal Growth Factor (EGF). Our results show that serum alanine and aspartate aminotransferase levels, caspase-3 activity, BID cleavage and changes in cellular energy stores were not observed before 3 h following anti-Fas injection. However, as early as 45 min after anti-Fas treatment, we observed upregulation of carbon entry (i.e. flux) from glucose into the Krebs-cycle via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) (up to 139 and 123 of controls, respectively, P < 0.001). This was associated with increased glutathione synthesis. EGF treatment significantly attenuated Fas-induced apoptosis, liver injury and the late decrease in energy stores, as well as the early fluxes through PDH and PC which were comparable to untreated controls. Using ex vivo multinuclear NMR-spectroscopic analysis, we have shown that Fas receptor activation in mouse liver time-dependently affects specific metabolic pathways of glucose. These early upregulations in glucose metabolic pathways occur prior to any visible signs of apoptosis and may have the potential to contribute to the initiation of apoptosis by maintaining mitochondrial energy production and cellular glutathione stores

Uptake of Fluorescent Iron Oxide Nanoparticles by Oligodendroglial OLN-93 Cells

To investigate the cellular accumulation and intracellular localization of dimercaptosuccinate-coated iron oxide nanoparticles (D-IONPs) in oligodendroglial cells, we have synthesized IONPs that contain the fluorescent dye BODIPY (BP) in their coat (BP-D-IONPs) and have investigated the potential effects of the absence or presence of this dye on the particle uptake by oligodendroglial OLN-93 cells. Fluorescent BP-D-IONPs and non-fluorescent D-IONPs had similar hydrodynamic diameters and -potentials of around 60 nm and -58 mV, respectively, and showed identical colloidal stability in physiological media with increasing particle size and positivation of the -potential in presence of serum. After exposure of oligodendroglial OLN-93 cells to BP-D-IONPs or D-IONPs in the absence of serum, the specific cellular iron content increased strongly to around 1,800 nmol/mg. This strong iron accumulation was lowered for both types of IONPs by around 50 % on exposure of the cells at 4 C and by around 90 % on incubation in presence of 10 % serum. The accumulation of both D-IONPs and BP-D-IONPs in the absence of serum was not affected by endocytosis inhibitors, whereas in the presence of serum inhibitors of clathrin-dependent endocytosis lowered the particle accumulation by around 50 %. These data demonstrate that oligodendroglial cells efficiently accumulate IONPs by an endocytotic process which is strongly affected by the temperature and the presence of serum and that BP-D-IONPs are a reliable tool to monitor by fluorescence microscopy the uptake and cellular fate of D-IONPs

PARK2 mutation causes metabolic disturbances and impaired survival of human iPSC-derived neurons

The protein parkin, encoded by the PARK2 gene, is vital for mitochondrial homeostasis, and although it has been implicated in Parkinson's disease (PD), the disease mechanisms remain unclear. We have applied mass spectrometry-based proteomics to investigate the effects of parkin dysfunction on the mitochondrial proteome in human isogenic induced pluripotent stem cell-derived neurons with and without PARK2 knockout (KO). The proteomic analysis quantified nearly 60% of all mitochondrial proteins, 119 of which were dysregulated in neurons with PARK2 KO. The protein changes indicated disturbances in oxidative stress defense, mitochondrial respiration and morphology, cell cycle control, and cell viability. Structural and functional analyses revealed an increase in mitochondrial area and the presence of elongated mitochondria as well as impaired glycolysis and lactate-supported respiration, leading to an impaired cell survival in PARK2 KO neurons. This adds valuable insight into the effect of parkin dysfunction in human neurons and provides knowledge of disease-related pathways that can potentially be targeted for therapeutic intervention