Acidic NAADP-releasable Ca compartments in the megakaryoblastic cell line MEG01

Background

A novel family of intracellular Ca²⁺-release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca²⁺ mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca²⁺ release from the acidic compartments.

Objectives

The present study is aimed to investigate NAADP-mediated Ca²⁺ release from intracellular stores in the megakaryoblastic cell line MEG01.

Methods

Changes in cytosolic and intraluminal free Ca²⁺ concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR.

Results

Treatment of MEG01 cells with the H⁺/K⁺ ionophore nigericin or the V-type H⁺-ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca²⁺ stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca²⁺ from acidic lysosomal-like Ca²⁺ stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca²⁺ efflux from the acidic lysosomal-like Ca²⁺ stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca²⁺-induced Ca²⁺ release (CICR) from the complementary Ca²⁺ compartment.

Conclusion

Our results show for the first time NAADP-evoked Ca²⁺ release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.     

MicroRNA mir-16 is anti-proliferative in enterocytes and exhibits diurnal rhythmicity in intestinal crypts

Background and aims

The intestine exhibits profound diurnal rhythms in function and morphology, in part due to changes in enterocyte proliferation. The regulatory mechanisms behind these rhythms remain largely unknown. We hypothesized that microRNAs are involved in mediating these rhythms, and studied the role of microRNAs specifically in modulating intestinal proliferation.

Methods

Diurnal rhythmicity of microRNAs in rat jejunum was analyzed by microarrays and validated by qPCR. Temporal expression of diurnally rhythmic mir-16 was further quantified in intestinal crypts, villi, and smooth muscle using laser capture microdissection and qPCR. Morphological changes in rat jejunum were assessed by histology and proliferation by immunostaining for bromodeoxyuridine. In IEC-6 cells stably overexpressing mir-16, proliferation was assessed by cell counting and MTS assay, cell cycle progression and apoptosis by flow cytometry, and cell cycle gene expression by qPCR and immunoblotting.

Results

mir-16 peaked 6 hours after light onset (HALO 6) with diurnal changes restricted to crypts. Crypt depth and villus height peaked at HALO 13-14 in antiphase to mir-16. Overexpression of mir-16 in IEC-6 cells suppressed specific G1/S regulators (cyclins D1-3, cyclin E1 and cyclin-dependent kinase 6) and produced G1 arrest. Protein expression of these genes exhibited diurnal rhythmicity in rat jejunum, peaking between HALO 11 and 17 in antiphase to mir-16.

Conclusions

This is the first report of circadian rhythmicity of specific microRNAs in rat jejunum. Our data provide a link between anti-proliferative mir-16 and the intestinal proliferation rhythm and point to mir-16 as an important regulator of proliferation in jejunal crypts. This function may be essential to match proliferation and absorptive capacity with nutrient availability.     

Macrophages from alpha 7 nicotinic acetylcholine receptor knockout mice demonstrate increased cholesterol accumulation and decreased cellular paraoxonase expression: A possible link between the nervous system and atherosclerosis development

Objective

The parasympathetic nervous system regulates inflammation in peripheral tissues through a pathway termed the “cholinergic anti-inflammatory reflex” (CAIR). Mice deficient in the alpha 7 nicotinic acetylcholine receptor (α7^−/−) have an impaired CAIR due to decreased signaling through this pathway. The purpose of this study was to determine if the increased inflammation in α7^−/− mice is associated with enhanced serum and macrophage atherogenicity.

Methods

We measured serum markers of inflammation and oxidative stress, and macrophage atherogenicity in mouse peritoneal macrophages harvested from α7^−/− mice on the background of C57BL/6 mice, as well as on the background of the atherosclerotic Apolipoprotein E-deficient (ApoE^−/−) mice.

Results

α7-Deficiency had no significant effects on serum cholesterol, or on markers of serum oxidative stress (TBARS and paraoxonase1 activities). However, α7-deficiency significantly increased serum CRP and IL-6 (p < 0.05) levels in atherosclerotic mice, confirming an anti-inflammatory role for the α7 receptor. Macrophage cholesterol mass was increased by 25% in both normal and atherosclerotic mice in the absence of the α7 receptor (p < 0.05). This was accompanied by conditional increases in oxidized LDL uptake and in macrophage total peroxide levels. Furthermore, α7-deficiency reduced macrophage paraoxonase2 mRNA and activity by 50-100% in normal and atherosclerotic mice (p < 0.05 for each), indicating a reduction in macrophage anti-oxidant capacity in the α7^−/− mice.

Conclusion

The above results suggest an anti-atherogenic role for the macrophage α7nAchr, through a mechanism that involves attenuated macrophage oxidative stress and decreased uptake of oxidized LDL.     

S-allyl cysteine in combination with clotrimazole downregulates Fas induced apoptotic events in erythrocytes of mice exposed to lead

Background

Chronic lead (Pb^2 +) exposure leads to the reduced lifespan of erythrocytes. Oxidative stress and K⁺ loss accelerate Fas translocation into lipid raft microdomains inducing Fas mediated death signaling in these erythrocytes. Pathophysiological-based therapeutic strategies to combat against erythrocyte death were evaluated using garlic-derived organosulfur compounds like diallyl disulfide (DADS), S allyl cysteine (SAC) and imidazole based Gardos channel inhibitor clotrimazole (CLT).

Methods

Morphological alterations in erythrocytes were evaluated using scanning electron microscopy. Events associated with erythrocyte death were evaluated using radio labeled probes, flow cytometry and activity gel assay. Mass spectrometry was used for detection of GSH-4-hydroxy-trans-2-nonenal (HNE) adducts. Fas redistribution into the lipid rafts was studied using immunoblotting technique and confocal microscopy.

Results

Combination of SAC and CLT was better than DADS and CLT combination and monotherapy with these agents in prolonging the survival of erythrocytes during chronic Pb^2 + exposure. Combination therapy with SAC and CLT prevented redistribution of Fas into the lipid rafts of the plasma membrane and downregulated Fas-dependent death events in erythrocytes of mice exposed to Pb^2 +.

Conclusion and general significance

Ceramide generation was a critical component of Fas receptor-induced apoptosis, since inhibition of acid sphingomyelinase (aSMase) interfered with Fas-induced apoptosis during Pb^2 + exposure. Combination therapy with SAC and CLT downregulated apoptotic events in erythrocytes by antagonizing oxidative stress and Gardos channel that led to suppression of ceramide-initiated Fas aggregation in lipid rafts. Hence, combination therapy with SAC and CLT may be a potential therapeutic option for enhancing the lifespan of erythrocytes during Pb^2 + toxicity.     

Essential role of ERK activation in neurite outgrowth induced by α-lipoic acid

Background

Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth.

Methods

To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for β-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth.

Results

LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth.

Conclusion

Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism.     

ABCG1 mediated oxidized LDL-derived oxysterol efflux from macrophages

Objectives

The uptake of oxidized LDL (oxLDL) by macrophages is a key initial event in atherogenesis, and the removal of oxidized lipids from artery wall via reverse cholesterol transport is considered antiatherogenic. The aims of this study were to investigate the pathways mediating the removal of oxysterols from oxLDL-loaded macrophages, and the subsequent uptake of the oxysterols by hepatocytes.

Methods

LDL was labeled with [3H]cholesterol, and LDL-[3H]cholesterol was oxidized by copper using a standard method. [3H]oxysterol formation in oxLDL was analyzed by thin layer chromatography. oxLDL-[3H]sterol was incubated with macrophages, allowing the uptake of [3H]sterol by macrophages. [3H]sterol efflux from macrophages mediated by ATP binding cassette transporters (ABCA1, ABCG1), or scavenger receptor class B type I (SR-BI) was measured. The subsequent uptake of the [3H]sterol by hepatocytes was also determined.

Results

7-Ketocholesterol was the major oxysterol formed in oxLDL, and it was significantly higher in oxLDL compared with that in native LDL (naLDL). oxLDL-derived sterol efflux to HDL from macrophages was significantly increased compared with naLDL-derived sterol, and it was mainly mediated by ABCG1, but not by ABCA1 or SR-BI. Moreover, although HDL dose-dependently induced sterol efflux from macrophages, only the exported sterol by ABCG1 pathway was efficiently taken up by hepatocytes.

Conclusions

ABCG1 mediates oxysterol efflux from oxLDL-loaded macrophages, and the exported oxysterol by ABCG1 pathway can be selectively taken up by hepatocytes.     

PEP-1-PEA-15 protects against toxin-induced neuronal damage in a mouse model of Parkinson's disease

Background

PEA-15 is abundantly expressed in both neurons and astrocytes throughout the brain. It is a multifunctional protein with the ability to increase cell survival via anti-apoptotic and anti-proliferative properties. However, the function of PEA-15 in neuronal diseases such as Parkinson's disease (PD) remains unclear. In this study, we investigated the protective effects of PEA-15 on neuronal damage induced by MPP⁺ in neuroblastoma SH-SY5Y and BV2 microglia cells and in a MPTP-induced PD mouse model using cell-permeable PEP-1-PEA-15.

Methods

PEP-1-PEA-15 was purified using affinity chromatography. Cell viability and DNA fragmentation were examined by MTT assay and TUNEL staining. Dopaminergic neuronal cell death in the animal model was examined by immunohistochemistry.

Results

PEP-1-PEA-15 transduced into the SH-SY5Y and BV2 cells in a time- and dose-dependent manner. Transduced PEP-1-PEA-15 protected against MPP⁺-induced toxicity by inhibiting intracellular ROS levels and DNA fragmentation. Further, it enhanced the expression levels of Bcl-2 and caspase-3 while reducing the expression levels of Bax and cleaved caspase-3. We found that PEP-1-PEA-15 transduced into the substantia nigra and prevented dopaminergic neuronal cell death in a MPTP-induced PD mouse. Also, we showed the neuroprotective effects in the model by demonstrating that treatment with PEP-1-PEA-15 ameliorated MPTP-induced behavioral dysfunctions and increased dopamine levels in the striatum.

Conclusions

PEP-1-PEA-15 can efficiently transduce into cells and protects against neurotoxin-induced neuronal cell death in vitro and in vivo.

General significance

These results demonstrate the potential for PEP-1-PEA-15 to provide a new strategy for protein therapy treatment of a variety of neurodegenerative diseases including PD.     

Localization of the Sodium-Taurocholate cotransporting polypeptide in membrane rafts and modulation of its activity by cholesterol in vitro

Background

The relevance of discrete localization of hepatobiliary transporters in specific membrane microdomains is not well known.

Aim

To determine whether the Na⁺/taurocholate cotransporting polypeptide (Ntcp), the main hepatic sinusoidal bile salt transporter, is localized in specific membrane microdomains.

Methods

Presence of Ntcp in membrane rafts obtained from mouse liver was studied by immunoblotting and immunofluorescence. HEK-293 cells stably transfected with rat Ntcp were used for in vitro studies. Expression, localization and function of Ntcp in these cells were assessed by immunoblotting, immunofluorescence and biotinylation studies and Na⁺-dependent taurocholate uptake assays, respectively. The effect of cholesterol depletion/repletion assays on Ntcp function was also investigated.

Results

Ntcp localized primarily to membrane rafts in in vivo studies and localized partially in membrane rafts in transfected HEK-293 cells. In these cells, membrane cholesterol depletion resulted in a shift of Ntcp localization into non-membrane rafts, which correlated with a 2.5-fold increase in taurocholate transport. Cholesterol repletion shifted back part of Ntcp into membrane rafts, and normalized taurocholate transport to values similar to control cells.

Conclusion

Ntcp localizes in membrane rafts and its localization and function are regulated by membrane cholesterol content. This may serve as a novel regulatory mechanism of bile salt transport in liver.     

Chemical composition and therapeutic effects of Lippia alba (Mill.) N. E. Brown leaves hydro-alcoholic extract in patients with migraine

Background

There is no universally accepted and effective prophylaxis of migraine headache episodes. Thus we aimed to investigate the effects of Lippia alba (Mill.) N. E. Brown, an herb with many effects on central nervous system, on pain frequency and intensity of migraine patients.

Methods

Patients were enrolled in a prospective, phase 2, non-controlled cohort study to orally receive hydro-alcoholic extract of L. alba leaves. Headache intensity and frequency of episodes were recorded before and after 30-60 days of treatment. We also studied the chemical composition of its essential oil by gas chromatography-mass spectrometry.

Results

We described for the first time a particular L. alba chemotype with geranial and carvenone as major compounds. With treatment, both frequency and intensity of pain episodes significantly decreased from baseline to first reassessment date. More than 80% of patients experienced a minimum 50% reduction on pain intensity and frequency. No side effects were reported.

Conclusions

Treatment with a geranial plus carvenone chemotype of L. alba hydro-alcoholic extract is a cheap, widely available, highly effective therapy to reduce both the intensity and the frequency of headache episodes of migraine patients with no side effects.     

Activated Rho/Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins

Background

We have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca²⁺ [Ca²⁺]_i. We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca²⁺]_i.

Methods

HSV were investigated in vitro either unfrozen within 12 h after removal or after storage at −196 °C in a cryomedium containing 1.8 M dimethyl sulfoxide and 0.1 M sucrose as cryoprotectant additives.

Results

Cryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30% whereas responses to KCl were attenuated by about 50%. Concentration-response curves for CaCl₂ on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (−)-(R) enantiomer of SDZ 202-791 whereas the Ca²⁺ channel activating (+)-(S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl₂ when tested after cryostorage. These functional effects were reflected by changes in [Ca²⁺]_i as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-(S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 μM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage.

Conclusions

Data suggested that cryopreservation modified [Ca²⁺]_i of venous smooth muscle cells (1) through depolarization-induced changes in Ca²⁺ influx and (2) through activation of Rho kinase signaling pathways.     

Glucocorticoids exert context-dependent effects on cells of the joint in vitro

Introduction

Glucocorticoids are known to attenuate bone formation in vivo leading to decreased bone volume and increased risk of fractures, whereas effects on the joint tissue are less characterized. However, glucocorticoids appear to have a reducing effect on inflammation and pain in osteoarthritis. This study aimed at characterizing the effect of glucocorticoids on chondrocytes, osteoclasts, and osteoblasts.

Experimental

We used four model systems to investigate how glucocorticoids affect the cells of the joint; two intact tissues (femoral head- and cartilage-explants), and two separate cell cultures of osteoblasts (2T3-pre-osteoblasts) and osteoclasts (CD14⁺-monocytes). The model systems were cultured in the presence of two glucocorticoids; prednisolone or dexamethasone. To induce anabolic and catabolic conditions, cultures were activated by insulin-like growth factor I/bone morphogenetic protein 2 and oncostatin M/tumor necrosis factor-α, respectively. Histology and markers of bone- and cartilage-turnover were used to evaluate effects of glucocorticoid treatment.

Results

Prednisolone treatment decreased collagen type-II degradation in immature cartilage, whereas glucocorticoids did not affect collagen type-II in mature cartilage. Glucocorticoids had an anti-catabolic effect on catabolic-activated cartilage from a bovine stifle joint and murine femoral heads. Glucocorticoids decreased viability of all bone cells, leading to a reduction in osteoclastogenesis and bone resorption; however, bone morphogenetic protein 2-stimulated osteoblasts increased bone formation, as opposed to non-stimulated osteoblasts.

Conclusions

Using highly robust in vitro models of bone and cartilage turnover, we suggest that effects of glucocorticoids highly depend on the activation and differential stage of the cell targeted in the joint. Present data indicated that glucocorticoid treatment may be beneficial for articular cartilage, although detrimental effects on bone should be taken into account.     

Epithelial transport and barrier function in occludin-deficient mice

Background and Aims

This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model.

Methods

Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (R^e, epithelial resistance). Conductance scanning differentiated transcellular (G^c) and tight junctional conductance (G^tj). The pH-stat technique quantified gastric acid secretion.

Results

In occludin+/+ mice, R^e was 23±5 Ω cm² in jejunum, 66±5 Ω cm² in distal colon and 33±6 Ω cm² in gastric corpus and was not altered in heterozygotic occludin+/− or homozygotic occludin−/− mice. Additionally, [³H]mannitol fluxes were unaltered. In the control colon, G^c and G^tj were 7.6±1.0 and 0.3±0.1 mS/cm² and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin−/− mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control R^t was 5.8±0.8 kΩ cm² in urinary bladder and 33±6 Ω cm² in stomach and not altered in occludin−/− mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin−/− mice.

Conclusion

Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.     

Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: Insight from molecular dynamics simulations

Background

Prion diseases are associated with a conformational switch for PrP from PrP^C to PrP^Sc. Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD.

Scope of review

MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native β-sheet and the conversion of S2-H2 loop to a 3₁₀ helix.

Major conclusions

Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrP^C to PrP^Sc.General significanceOur present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrP^Sc assemblies.     

Antioxidative activity of the olive oil constituent hydroxy-1-aryl-isochromans in cells and cell-free systems

Background

Hydroxy-1-aryl-isochromans (HAIC) are newly emerging natural polyphenolic antioxidants, enriched in extravirgin olive oil, whose antioxidative potency was only scarcely characterized using cell-free systems and cells.

Methods

We characterized the activity of HAIC to inactivate reactive oxygen species (ROS) generated by the xanthine/xanthine oxidase system, mitochondria (rat brain) and neural cells. ROS levels were estimated using ROS-sensitive probes, such as Amplex Red, MitoSOX^RED.

Results

HAIC (with 2, 3 or 4 hydroxyl substituents) effectively scavenge ROS released from mitochondria. EC₅₀ values estimated with mitochondria and submitochondrial particles were around 20 μM. Moreover, in PC12 and cultured neural primary cells, HAIC buffered cytosolic ROS. Although HAIC permeate biological membranes, HAIC fail to buffer matrix ROS in isolated mitochondria. We show that hydrogen peroxide was effectively abolished by HAIC, whereas the production of superoxide was not affected.

Conclusion

HAIC exert high antioxidative activity to reduce hydrogen peroxide. The antioxidative activity of HAIC is comparable with that of the stilbene-like, polyphenolic resveratrol, but much higher than that of trolox, N-acetylcysteine or melatonin.

General significance

Unlike resveratrol, HAIC do not impair mitochondrial ATP synthesis or Ca²⁺ retention by mitochondria. Thus, HAIC have the decisive advantage to be potent antioxidants with no detrimental side effects on mitochondrial functions.     

Haplotype frequency distribution for 7 microsatellites in chromosome 8 and 11 in relation to the metabolic syndrome in four ethnic groups: Tehran Lipid and Glucose Study

Introduction

Different variants of haplotype frequencies may lead to various frequencies of the same variants in individuals with drug resistance and disease susceptibility at the population level.

Materials and methods

In this study, the haplotype frequencies of 4 STR loci including the D8S1132, D8S1779, D8S514 and D8S1743, and 3 STR loci including D11S1304, D11S1998 and D11S934 were investigated in 563 individuals of four Iranian ethnic groups in the capital city of Iran, Tehran. One hundred thirty subjects had the metabolic syndrome. Haplotype frequencies of all markers were calculated.

Results

There were significant differences in the haplotype frequencies in short and long alleles between the metabolic affected subjects and controls. In addition, haplotype frequencies were significant in the four ethnic groups in both chromosomes 8 and 11.

Conclusion

Our findings show a relation between the short allele of D8S1743 in all related haplotype frequencies of subjects with metabolic syndrome. These findings may require more studies of some candidate genes, including the lipoprotein lipase gene, in this chromosomal region.     

Localization of nerve growth factor (NGF) receptors in the mitochondrial compartment: Characterization and putative role

Background

The neurotrophin NGF receptors trkA and p75NTR are expressed in the central and peripheral nervous system as well as in non-neuronal tissues; originally described to localize to the plasma membrane, recent studies have suggested other intracellular localizations for both NGF receptors.

Scope of review

In order to determine whether NGF receptors localize to the mitochondrial compartment mitochondria isolated from human kidney, rat tissues and a human podocyte as cell line before and after differentiation were used.

Major conclusions

Our results demonstrate that NGF receptors are localized in the mitochondrial compartment of undifferentiated human podocytes and in all tissues analyzed including rat central nervous system. In mitochondria p75NTR, but not trkA, co-immunoprecipitates with the adenine nucleotide translocator (ANT) and the phosphodiesterase 4 isoform A5 (PDE4A5). Moreover, NGF, via trkA, protects isolated mitochondria of rat brain cortex from mitochondrial permeability transition induced by Ca²⁺.

General significance

Although NGF receptors have been described as mainly citoplasmatic so far, we proved evidence of their expression at the mitochondrial level and their interaction with specific proteins. Our results demonstrating the expression of NGF receptors in the mitochondria provide new insights into the role of NGF at subcellular level, in different areas of the organism, including CNS.     

Effects of resveratrol on HepG2 cells as revealed by H-NMR based metabolic profiling

Background

Resveratrol, a polyphenol found in plant products, has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Several in vitro and in vivo studies have demonstrated the influence of resveratrol on multiple intracellular targets that may regulate metabolic homeostasis.

Methods

We analysed the metabolic modifications induced by resveratrol treatment in a human hepatoblastoma line, HepG2 cells, using a ¹H-NMR spectroscopy-based metabolomics approach that allows the simultaneous screening of multiple metabolic pathways.

Results

Results demonstrated that cells cultured in the presence or absence of resveratrol displayed different metabolic profiles: the treatment induced a decreased utilisation of glucose and amino acids for purposes of energy production and synthesis associated to a decreased release of lactate in the culture medium and an increase in succinate utilisation. At the same time, resveratrol treatment slowed the cell cycle in the S phase without inducing apoptosis, and increased Sirt1 expression, also affecting its intracellular localisation.

Conclusions

Our results show that the metabolomic analysis of the exometabolome of resveratrol-treated HepG2 cells indicates a metabolic switch from glucose and amino acid utilisation to fat utilisation for the production of energy, and seem in agreement with an effect mediated via AMPK- and Sirt1-activation.

General significance

NMR-based metabolomics has been applied in a hepatocyte cell culture model in relation to resveratrol treatment; such an approach could be transferred to evaluate the effects of nutritional compounds with health impact.