The novel NADPH oxidase 4 inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice.

In type 2 diabetes, it has been proposed that pancreatic beta-cell dysfunction is promoted by oxidative stress caused by NADPH oxidase (NOX) overactivity. Five different NOX enzymes (NOX1-5) have been characterized, among which NOX1 and NOX2 have been proposed to negatively affect beta-cells, but the putative role of NOX4 in type 2 diabetes-associated beta-cell dysfunction and glucose intolerance is largely unknown. Therefore, we presently investigated the importance of NOX4 for high-fat diet or HFD-induced glucose intolerance using male C57BL/6 mice using the new NOX4 inhibitor GLX351322, which has relative NOX4 selectivity over NOX2. In HFD-treated male C57BL/6 mice a two-week treatment with GLX351322 counteracted non-fasting hyperglycemia and impaired glucose tolerance. This effect occurred without any change in peripheral insulin sensitivity. To ascertain that NOX4 also plays a role for the function of human beta-cells, we observed that glucose- and sodium palmitate-induced insulin release from human islets in vitro was increased in response to NOX4 inhibitors. In long-term experiments (1-3 days), high-glucose-induced human islet cell reactive oxygen species (ROS) production and death were prevented by GLX351322. We propose that while short-term NOX4-generated ROS production is a physiological requirement for beta-cell function, persistent NOX4 activity, for example, during conditions of high-fat feeding, promotes ROS-mediated beta-cell dysfunction. Thus, selective NOX inhibition may be a therapeutic strategy in type 2 diabetes.

Acrylamide-induced effects on general and neurospecific cellular functions during exposure and recovery.

Basal cytotoxicity, morphological changes and alterations in cell physiological and neurochemical functions were studied in differentiated human neuroblastoma (SH-SY5Y) cells during exposure to acrylamide and during a subsequent recovery period after cessation of exposure. Acrylamide induced a 20% reduction in the number of neurites per cell at 0.21 mmol/L and 20% decrease in the protein synthesis rate at 0.17 mmol/L after 72 h of exposure. Furthermore, the basal level of intracellular calcium concentration ([Ca2+]i) and receptor-activated (carbachol, 0.1 mmol/L) Ca2+ fluxes increased by 49% and 21%, respectively, at 0.25 mmol/L. These observations were made at noncytotoxic acrylamide concentrations, signifying specific neurotoxic alterations. Forty-eight hours after cessation of acrylamide exposure, the SH-SY5Y cells had recovered, i.e., the number of neurites per cell as well as the basal level of [Ca2+]i and rate of protein synthesis were comparable to those of control cells. The general calpain inhibitor calpeptin decreased the acrylamide-induced (0.5 mmol/L) neurite degeneration, determined as reduction in number of neurites per cell, from 52% to 17% as compared to control cells, which further supports the hypothesis that an increased [Ca2+]i plays a significant role for acrylamide-induced axonopathy.

Insulin-like growth factor type 1 upregulates uncoupling protein 3.

In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor.

Expression and intracellular localization of leptin receptor long isoform-GFP chimera.

The leptin receptor (OBR) and its ligand leptin (OB) are key players in the regulation of body weight. The OBR is a member of the class I cytokine receptor family and is alternatively spliced into at least six different isoforms. The multiple forms are identical in their extracellular and transmembrane regions but differ in lengths. The two predominant isoforms include a long form (OBR(l)) with an intracellular domain of 303 amino acids and a shorter form (OBR(s)) with an intracellular domain of 34 amino acids. We have constructed a recombinant OBR(l) chimera with the green fluorescent protein (GFP) by fusing GFP to the C-terminus of the OBR(l). The OBR(l)-GFP chimera was transiently transfected and expressed in SHSY5Y and HEK293 cells. In a STAT-Luciferase assay we show that the GFP moiety in this chimera did not affect the signalling capacity of OBR(l)-GFP. In both SHSY5Y and HEK293 cells transfected with OBR(l)-GFP, a predominant intracellular green OBR(l)-GFP fluorescence was detected in vesicles also positive for internalized fluorophore conjugated leptin. We also found that treatment with the lysosomotropic reagent monensin did not relocalize OBR(l)-GFP together with the human transferrin receptor in recycling endosomes, indicating OBR(l)-GFP not to participate in this pathway. In biotinylation-streptavidin pulse chase experiments, using antibodies raised against GFP and OBR, we observed that the rate of early appearance of OBR(s) at the cell surface, upon leptin stimulation, was faster than that found for OBR(l)-GFP. Taken together, our results provide novel data concerning the intracellular trafficking of the two different isoforms of the leptin receptor.

Induction of VEGF and VEGF receptors in the spinal cord after mechanical spinal injury and prostaglandin administration.

Vascular endothelial growth factor (VEGF) is an angiogenetic factor that promotes endothelial cell proliferation during development and after injury to various types of tissue, including the central nervous system (CNS). Using immunohistochemical and in situ hybridization methods we have here demonstrated that VEGF and its receptors Flk-1, Flt-1 and Neuropilin-1 mRNAs and proteins are induced after incisions in the rat spinal cord. The inducible enzyme for prostaglandin synthesis cyclooxygenase-2 (COX-2) is known to be upregulated after spinal injury, cerebral ischemia and to stimulate angiogenesis. To test the hypothesis that prostaglandins may be involved in the VEGF response after lesion we investigated whether intraspinal microinjections of prostaglandin F2alpha (PGF2alpha) alters VEGF expression in the spinal cord. Such treatment was followed by a strong upregulation of VEGF mRNA and protein in the injection area. Finally, by use of an in vitro model with cell cultures of meningeal fibroblast and astrocyte origin, resembling the lesion area cellular content after spinal cord injury but devoid of inflammatory cells, we showed that VEGF is expressed in this in vitro model cell system after treatment with PGF2alpha and prostaglandin E2 (PGE2). These data suggest that cells within a lesion area in the spinal cord are capable of expressing VEGF and its receptors in response to mechanical injury and that prostaglandins may induce VEGF expression in such cells, even in the absence of inflammatory cells.

Characterization of leptin intracellular trafficking.

Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR) is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBR) with an intracellular domain of 303 amino acids and a shorter form (OBR) with an intracellular domain of 34 amino acids. Since OBR, is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3-transferrin containing compartments at 37 degrees C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF) and the lysosomal marker protein lamp-1. The transport of leptin was also shown to engage a monensin and bafilomycin sensitive degradation process in lysosomes. Together, our results provide novel data concerning the uptake, intracellular localization and transport of leptin.

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): validation and acute toxicity testing.

Scientific principles demand that before newly developed alternative methods for safety testing are fully embraced by the industrial or regulatory community, they reliably and reproducibly predict the designated toxic end point. The process used to determine reliability and reproducibility is termed validation, and it generally culminates with a highly controlled, blinded study using multiple chemicals and laboratories. It is imperative that the validation study is designed to confirm the previously established reproducibility and predictive power of the assay. Much has been learned recently about the practical aspects of validation through investigation of alternative methods for acute toxicity testing, i.e., those methods that assess acute systemic toxicity, skin irritation, and eye irritation. Although considerable progress has been made--many alternative tests are now commonly used in various industrial settings--there have been few tests that have successfully passed a complete validation. Some of the barriers to successful validation have been a) lack of high-quality, reproducible animal data; b) insufficient knowledge of the fundamental biologic processes involved in acute toxicity; and c) the development of truly robust in vitro assays that can accurately respond to materials with a wide range of chemical and physical characteristics. It is recommended that to progress in the areas of eye and skin irritation we need to expand our knowledge of toxic markers in humans and the biochemical basis of irritation; progress in the area of acute systemic toxicity will require the development of in vitro models to determine gastrointestinal uptake, blood-brain barrier passage, and biotransformation.

Acute oral toxicity.

The purposes of acute toxicity testing are to obtain information on the biologic activity of a chemical and gain insight into its mechanism of action. The information on acute systemic toxicity generated by the test is used in hazard identification and risk management in the context of production, handling, and use of chemicals. The LD50 value, defined as the statistically derived dose that, when administered in an acute toxicity test, is expected to cause death in 50% of the treated animals in a given period, is currently the basis for toxicologic classification of chemicals. For a classical LD50 study, laboratory mice and rats are the species typically selected. Often both sexes must be used for regulatory purposes. When oral administration is combined with parenteral, information on the bioavailability of the tested compound is obtained. The result of the extensive discussions on the significance of the LD50 value and the concomitant development of alternative procedures is that authorities today do not usually demand classical LD50 tests involving a large number of animals. The limit test, the fixed-dose procedure, the toxic class method, and the up-and-down methods all represent simplified alternatives using only a few animals. Efforts have also been made to develop in vitro systems; e.g., it has been suggested that acute systemic toxicity can be broken down into a number of biokinetic, cellular, and molecular elements, each of which can be identified and quantified in appropriate models. The various elements may then be used in different combinations to model large numbers of toxic events to predict hazard and classify compounds.

Neurite degeneration in differentiated human neuroblastoma cells.

We have studied neurite degeneration in differentiated human neuroblastoma (SH-SY5Y) cells. The axonopathy-inducing potency in vitro of caffeine, diazepam, methylmercury chloride (MeHg), triethyltin chloride (TET) and acrylamide (ACR) was elucidated. After 72 hours of exposure the neurite degeneration was determined (by morphological quantification) as well as the total protein content (general cytotoxicity). The concentrations that caused 20% reduction of number of neurites (ND(20)) for ACR (250+/-36 mum) and TET (0.097+/-0.03 mum) was significantly lower, 63% and 35%, respectively (P

Polygodial-induced noradrenaline release in human neuroblastoma SH-SY5Y cells.

Polygodial is a natural sesquiterpene which exhibits pronounced pungency and a powerful antifeedant activity. At low concentrations, which do not alter general cell membrane permeability, polygodial increases the intracellular concentration of free calcium ([Ca(2+)](i)). Sensory neurotransmission depends on noradrenaline (NA) release, and vesicular exocytosis, in turn, is dependent on an increase in [Ca(2+)](i). The nociceptive response induced by polygodial could therefore be directly linked to intracellular calcium levels. Consequently, the objective of this work was to investigate the effect of polygodial on NA release. The human neuroblastoma cell line SH-SY5Y was selected as an in vitro model for sensory neurones. Semiconfluent cells were preloaded with tritiated NA ([(3)H]NA). After 3 min exposure of polygodial to the cells, released and unreleased radioactivity were measured. Polygodial induced a significant [(3)H]NA release at concentrations between 0.1 and 0.5 mug/ml with a maximum effect at 0.2 mug/ml (40% increased release of [(3)H]NA as compared with unstimulated control cells). No polygodial-induced transmitter release was seen at 3.5 and 5 mug/ml. For comparison, carbachol (1 rim) increased [(3)H]NA release by 10% and the KCl-induced (100 mm) [(3)H]NA release increased by 8% as compared with unstimulated SH-SY5Y cells. In conclusion polygodial, at the concentrations 0.1-0.5 mug/ml (equal to 0.4-2 mum), induces NA release which is dependent on polygodial-induced increase in [Ca(2+)](i).

Polygodial induces inositol phosphate turnover in human neuroblastoma SH-SY5Y cells.

The pungent sesquiterpenoid unsaturated dialdehydes polygodial and isovelleral, have previously been shown to increase the intracellular free calcium concentration [Ca2+]i in human neuroblastoma SH-SY5Y cells, partly by a release from intracellular Ca2+ stores, whereas the non-pungent compound epipolygodial, had no effect on the [Ca2+]i. In this study, we investigated the effect of isovelleral, polygodial and epipolygodial on inositol phosphate (IP) formation on the assumption that there might be a correlation between the release of intracellular Ca2+ and pungency of the compounds. It was found that polygodial induced IP mobilization in a concentration dependent way, whereas isovelleral had no effect on the IP formation in the SH-SY5Y cells. Phosphoinositide (PPI) turnover was activated by epipolygodial, but only at concentrations 40-fold higher than for polygodial, which emphasizes the importance of the correct stereometry in the dialdehyde configuration for the biological activity of polygodial. The polygodial-induced formation of IP1 was reduced by 71% under extracellular calcium-free conditions, which suggests feedback interactions between the IP formation and the increase in [Ca2+]i to account for a periodic activation of phospholipase C(PLC).

Sodium-dependent glutamate uptake as an activator of oxidative metabolism in primary astrocyte cultures from newborn rat.

In the present report we describe the effect of glutamate on respiratory activity in primary cultures of astrocytes, derived from cerebral cortex of newborn rat. Glutamate (100 microM) caused an increased oxygen consumption. This effect could not be inhibited by antagonists to the NMDA or AMPA/kainate receptors. Neither trans-ACPD (an agonist to the metabotropic glutamate receptor) nor the Krebs cycle intermediate alpha-ketoglutarate had any effect on the respiratory rate. An uncontrolled influx of Na+, caused by gramicidin, could mimic the glutamate effect on respiratory activity. In addition, the glutamate effect was abolished by addition of ouabain or replacement of Na+ by Li+ in the perfusion buffer. We conclude that the co-transport of Na+, in the Na(+)-dependent high-affinity glutamate uptake system, mediated the glutamate-induced increase in oxygen consumption through an increased activity of Na+/K(+)-ATPases.