TRiC/CCT chaperonin is required for the folding and inhibitory effect of WDTC1 on adipogenesis.

Obesity has become a global pandemic. WDTC1 is a WD40-containing protein that functions as an anti-obesity factor. WDTC1 inhibits adipogenesis by working as an adaptor of the CUL4-DDB1 E3 ligase complex. It remains unclear about how WDTC1 is regulated. Here, we show that the TRiC/CCT functions as a chaperone to facilitate the protein folding of WDTC1 and proper function in adipogenesis. Through tandem purification, we identified the molecular chaperone TRiC/CCT as WDTC1-interacting proteins. WDTC1 bound the TRiC/CCT through its ADP domain, and the TRiC/CCT recognized WDTC1 through the CCT5 subunit. Disruption of the TRiC/CCT by knocking down CCT1 or CCT5 led to misfolding and lysosomal degradation of WDTC1. Furthermore, the knockdown of CCT1 or CCT5 eliminated the inhibitory effect of WDTC1 on adipogenesis. Our studies uncovered a critical role of the TRiC/CCT in the folding of WDTC1 and expanded our knowledge on the regulation of adipogenesis.

The Mediator subunit MED20 organizes the early adipogenic complex to promote development of adipose tissues and diet-induced obesity.

MED20 is a non-essential subunit of the transcriptional coactivator Mediator complex, but its physiological function remains largely unknown. Here, we identify MED20 as a substrate of the anti-obesity CRL4-WDTC1 E3 ubiquitin ligase complex through affinity purification and candidate screening. Overexpression of WDTC1 leads to degradation of MED20, whereas depletion of WDTC1 or CUL4A/B causes accumulation of MED20. Depleting MED20 inhibits adipogenesis, and a non-degradable MED20 mutant restores adipogenesis in WDTC1-overexpressing cells. Furthermore, knockout of Med20 in preadipocytes abolishes development of brown adipose tissues. Removing one allele of Med20 in preadipocytes protects mice from diet-induced obesity and reverses weight gain in Cul4a- or Cul4b-depleted mice. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis reveals that MED20 organizes the early adipogenic complex by bridging C/EBPß and RNA polymerase II to promote transcription of the central adipogenic factor, PPARγ. Our findings have thus uncovered a critical role of MED20 in promoting adipogenesis, development of adipose tissue and diet-induced obesity.

Opposite effects of alpha-lipoic acid on antioxidation and long-term potentiation in control and chronically lead-exposed rats.

Among the developmental changes identified in rats exposed to lead are impairments in long-term potentiation (LTP) in the hippocampus and changes in the levels of reactive oxygen species (ROS) in cells and some soft tissues. alpha-Lipoic acid (LA) has been reported to be highly effective in improving the thiol capacity of the cells and in reducing lead-induced oxidative stress. To explore the effects of LA on LTP in chronically lead-exposed rats and the relationship between ROS and LTP in both control and lead-exposed rats, we have compared LTP and oxidative stress parameters in groups of lead-exposed and control rats with or without LA treatment (10, 25, 50, and 100 mg/kg through intraperitoneal injection). The capacity of LA to decrease hippocampal lead levels in lead-exposed rats was examined. We found that LA had no effects in decreasing the level of lead in the hippocampus, but it did appear to have both antioxidant properties and a reparatory effect on LTP amplitude in rats developmentally exposed to lead for 2 weeks following birth. Interestingly, bell-shaped dose-response curves emerged. In the lower LA dosage groups (10, 25 mg/kg LA), there was an increasing LTP amplitude. The strongest protective effect in terms of the induction and amplitude of LTP in the lead-exposed group with at 25 mg/kg LA; when higher dosages were applied (50, 100 mg/kg LA), the LTP amplitude decreased as compared to the 25 mg/kg LA treatment group. The administration of LA to control animals resulted in a significant impairment of LTP amplitude, with the 100 mg/kg LA treatment having harmful effects on the oxidative parameters. These differential effects of LA on LTP in control and lead-exposed rats may be due to the different redox status of the control and lead-exposed rats.

Quercetin relieves chronic lead exposure-induced impairment of synaptic plasticity in rat dentate gyrus in vivo.

Increasing evidence suggests that lead (Pb) produces impairments partly through oxidative stress. Though many researchers have investigated protective effect of some antioxidant nutrients against Pb toxicity, little information is available about the effect of antioxidants on Pb-induced impairment of synaptic plasticity. Quercetin, a strong antioxidant and radical scavenger, is the representative natural flavonoid molecule abundant in fruits and vegetables. Previous studies have found that quercetin was neuroprotective in many cases. This study was designed to evaluate the effect of quercetin on chronic Pb exposure-induced impairment of synaptic plasticity in adult rat dentate gyrus (DG) area in vivo. The input/output (I/O) functions, paired-pulse reactions (PPR), excitatory postsynaptic potential (EPSP), and population spike (PS) amplitude were measured in the DG area of different groups of rats in response to stimulation applied to the lateral perforant path. The results showed that the depressed I/O, PPR, and long-term potentiation (LTP) of Pb-exposed group were significantly increased by quercetin treatment. In addition, hippocampal Pb concentration was partially reduced after quercetin treatment. These findings suggest that quercetin treatment could relieve chronic Pb exposure-induced impairment of synaptic plasticity and might be a potential therapeutic intervention to cure cognitive deficits induced by Pb.

Effects of EGCG on voltage-gated sodium channels in primary cultures of rat hippocampal CA1 neurons.

(-)-Epigallocatechin-3-gallate (EGCG), the main active component of green tea, is commonly known for its beneficial properties at low doses. On the other hand, little is known about the adverse effects of EGCG. Voltage-gated sodium channel (VGSC) is responsible for both initiation and propagation of action potentials of the neurons in the hippocampus and throughout the central nervous system (CNS). In this study, the effects of EGCG on voltage-gated sodium channel currents (I(Na)) were investigated in rat primary cultures of hippocampal CA1 neurons via the conventional whole-cell patch-clamp technique. We found that I(Na) was not affected by EGCG at the concentration of 0.1microM, but was completely blocked by EGCG at the concentration of 400microM and higher, and EGCG reduced the amplitudes of I(Na) in a concentration-dependent manner in the range of 0.1-400microM. Furthermore, our results also showed that at the concentration of 100microM, EGCG was known to have the following performances: (1) it decreased the activation threshold and the voltage at which the maximum I(Na) current was evoked, caused negative shifts of I(Na) steady-state activation curve. (2) It enlarged I(Na) tail-currents. (3) It induced a left shift of the steady-state inactivation. (4) It reduced fraction of available sodium channels. (5) It delayed the activation of I(Na) in a voltage-dependent manner. (6) It prolonged the time course of the fast inactivation of sodium channels. (7) It accelerated the activity-dependent attenuation of I(Na). On the basis of these findings, we propose that EGCG could impair certain physiological functions of VGSCs, which may contribute, directly or indirectly, to EGCG's effects in CNS.

Effects of Epigallocatechin-3-gallate on lead-induced oxidative damage.

Recent studies have shown that lead (Pb) could disrupt the prooxidant/antioxidant balance of tissue which leads to biochemical and physiological dysfunction. Epigallocatechin-3-gallate (EGCG), a catechin polyphenols component, is found to be an effective antioxidant. The present study investigated whether EGCG administration could reverse the changes on redox states in rat hippocampus caused by lead exposure. The association between redox status changes and long-term potentiation (LTP) in CA1 area of hippocampus were also examined. Wistar rats exposed to lead from postnatal day 1 were followed by 10 days of EGCG (10, 25 and 50 mg/kg) administration through intraperitoneally (ip), and the rats were sacrificed for experiments at the age of 21-23 days. The experimental results showed that glutathione (GSH) and superoxide dismutase (SOD) activity decreased accompanied with LTP amplitude decrease in CA1 area of hippocampus in the lead-exposed group. EGCG supplementation following lead intoxication resulted in increases in the GSH and SOD levels and increases in the LTP amplitude. Malondialdehyde (MDA) levels, a major lipid peroxidation byproduct, increased following lead exposure and decreased following EGCG treatment. In hippocampal neuron culture model, lead exposure (20 microM) significantly inhibited the viability of neurons which was followed by an accumulation of ROS and a decrease of mitochondrial membrane potential (delta Psi m). Treatment by EGCG (10-50 microM) effectively increased cell viability, decreased ROS formation and improved delta Psi m in hippocampal neurons exposed to lead. These observations suggest that EGCG is a potential complementary agent in the treatment of chronic lead intoxication through its antioxidative character.

The interaction of selenium and mercury in the accumulations and oxidative stress of rat tissues.

This study evaluates the interaction of selenium (Se) and mercury (Hg) in the accumulations and oxidative stress of rat tissues. Rats were divided into five groups including one control (n=9) and four treated groups including M-Hg (n=9), L-Hg+Se (n=11), M-Hg+Se (n=10), and H-Hg+Se (n=10) group. Treated groups of rats were instilled with different amounts of mercuric chloride (HgCl(2)) and dl-selenomethionine (SeMet) by gavage since pregnancy of their mothers. Atomic fluorescence spectroscopy (AFS) was applied for mercury and selenium quantification. Glutathione (GSH), malondialdehyde (MDA), and total superoxide dismutase (SOD) activity of tissues were detected using biochemical methods. Results showed that Hg was deposited mainly in kidney. Se could decrease Hg content in kidney but increase it in blood and liver. Hg decreased GSH and SOD and increased MDA levels in most detected tissues, while Se took on a counteraction effect in same tissues. This study suggests that interactions of Se and Hg affect their accumulation and Se may antagonize Hg-induced inhibition on organic activities.

Effects of nitrite exposure on haematological parameters, oxidative stress and apoptosis in juvenile turbot (Scophthalmus maximus).

Nitrite (NO2(-)) is commonly present as contaminant in aquatic environment and toxic to aquatic organisms. In the present study, we investigated the effects of nitrite exposure on haematological parameters, oxidative stress and apoptosis in juvenile turbot (Scophthalmus maximus). Fish were exposed to various concentrations of nitrite (0, 0.02, 0.08, 0.4 and 0.8mM) for 96 h. Fish blood and gills were collected to assay haematological parameters, oxidative stress and expression of genes after 0, 24, 48 and 96 h of exposure. In blood, the data showed that the levels of methemoglobin (MetHb), triglyceride (TG), potassium (K(+)), cortisol, heat shock protein 70 (HSP70) and glucose significantly increased in treatments with higher concentrations of nitrite (0.4 and/or 0.8mM) after 48 and 96 h, while the levels of haemoglobin (Hb) and sodium (Na(+)) significantly decreased in these treatments. In gills, nitrite (0.4 and/or 0.8mM) apparently reduced the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione (GSH), increased the formation of malondialdehyde (MDA), up-regulated the mRNA levels of c-jun amino-terminal kinase (JUK1), p53, caspase-3, caspase-7 and caspase-9 after 48 and 96 h of exposure. The results suggested caspase-dependent and JUK signaling pathways played important roles in nitrite-induced apoptosis in fish. Further, this study provides new insights into how nitrite affects the physiological responses and apoptosis in a marine fish.

3D clustering of GABAergic neurons enhances inhibitory actions on excitatory neurons in the mouse visual cortex.

Neocortical neurons with similar functional properties assemble into spatially coherent circuits, but it remains unclear how inhibitory interneurons are organized. We applied in vivo two-photon functional Ca(2+) imaging and whole-cell recording of synaptic currents to record visual responses of cortical neurons and analyzed their spatial arrangements. GABAergic interneurons were clustered in the 3D space of the mouse visual cortex, and excitatory neurons located within the clusters (insiders) had a lower amplitude and sharper orientation tuning of visual responses than outsiders. Inhibitory synaptic currents recorded from the insiders were larger than those of the outsiders. Single, isolated interneurons did not show such a location-tuning/amplitude relationship. The two principal subtypes of interneurons, parvalbumin- and somatostatin-expressing neurons, also formed clusters with only slightly overlapping each other and exhibited a different location-tuning relationship. These findings suggest that GABAergic interneurons and their subgroups form clusters to make their inhibitory function more effective than isolated interneurons.

Epigallocatechin-3-gallate induced primary cultures of rat hippocampal neurons death linked to calcium overload and oxidative stress.

Epigallocatechin-3-gallate (EGCG), a catechin polyphenols component, is the main ingredient of green tea extract. It has been reported that EGCG is a potent antioxidant and beneficial in oxidative stress-related diseases, but others and our previous study showed that EGCG has pro-oxidant effects at high concentration. Thus, in this study, we tried to examine the possible pathway of EGCG-induced cell death in cultures of rat hippocampal neurons. Our results showed that EGCG caused a rapid elevation of intracellular free calcium levels ([Ca(2+)](i)) in a dose-dependent way. Exposure to EGCG dose- and time-dependently increased the production of reactive oxygen species (ROS) and reduced mitochondrial membrane potential (Deltapsi(m)) as well as the Bcl-2/Bax expression ratio. Importantly, acetoxymethyl ester of 5,5'-dimethyl-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid, and vitamin E could attenuate EGCG-induced apoptotic responses, including ROS generation, mitochondrial dysfunction, and finally partially prevented EGCG-induced cell death. Furthermore, treatment of hippocampal neurons with EGCG resulted in an elevation of caspase-3 and caspase-9 activities with no significant accompaniment of lactate dehydrogenase release, which provided further evidence that apoptosis was the dominant mode of EGCG-induced cell death in cultures of hippocampal neurons. Taken together, these findings indicated that EGCG induced hippocampal neuron death through the mitochondrion-dependent pathway.