Zinc Oxide Nanoparticles Alleviate Chilling Stress in Rice (Oryza Sativa L.) by Regulating Antioxidative System and Chilling Response Transcription Factors.

As one of the common abiotic stresses, chilling stress has negative effects on rice growth and development. Minimization of these adverse effects through various ways is vital for the productivity of rice. Nanoparticles (NPs) serve as one of the effective alleviation methods against abiotic stresses. In our research, zinc oxide (ZnO) NPs were utilized as foliar sprays on rice leaves to explore the mechanism underlying the effect of NPs against the negative impact of chilling stress on rice seedlings. We revealed that foliar application of ZnO NPs significantly alleviated chilling stress in hydroponically grown rice seedlings, including improved plant height, root length, and dry biomass. Besides, ZnO NPs also restored chlorophyll accumulation and significantly ameliorated chilling-induced oxidative stress with reduced levels of H2O2, MDA, proline, and increased activities of major antioxidative enzymes, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). We further found that foliar application of ZnO NPs induced the chilling-induced gene expression of the antioxidative system (OsCu/ZnSOD1, OsCu/ZnSOD2, OsCu/ZnSOD3, OsPRX11, OsPRX65, OsPRX89, OsCATA, and OsCATB) and chilling response transcription factors (OsbZIP52, OsMYB4, OsMYB30, OsNAC5, OsWRKY76, and OsWRKY94) in leaves of chilling-treated seedlings. Taken together, our results suggest that foliar application of ZnO NPs could alleviate chilling stress in rice via the mediation of the antioxidative system and chilling response transcription factors.

Chemical Screening of Nuclear Receptor Modulators.

Nuclear receptors are ligand-inducible transcriptional factors that control multiple biological phenomena, including proliferation, differentiation, reproduction, metabolism, and the maintenance of homeostasis. Members of the nuclear receptor superfamily have marked structural and functional similarities, and their domain functionalities and regulatory mechanisms have been well studied. Various modulators of nuclear receptors, including agonists and antagonists, have been developed as tools for elucidating nuclear receptor functions and also as drug candidates or lead compounds. Many assay systems are currently available to evaluate the modulation of nuclear receptor functions, and are useful as screening tools in the discovery and development of new modulators. In this review, we cover the chemical screening methods for nuclear receptor modulators, focusing on assay methods and chemical libraries for screening. We include some recent examples of the discovery of nuclear receptor modulators.

Molecular Cloning, Characterization, and Expression Regulation of Acyl-CoA Synthetase 6 Gene and Promoter in Common Carp Cyprinus carpio.

Omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), particularly docosahexaenoic acids (22:6n-3, DHA), have positive effects on multiple biologic and pathologic processes. Fish are the major dietary source of n-3 LC-PUFA for humans. Growing evidence supports acyl-coenzyme A (acyl-CoA) synthetase 6 (acsl6) being involved in cellular DHA uptake and lipogenesis in mammals, while its molecular function and regulatory mechanism remain unknown in fish. The present study focused on investigating the molecular characterization and transcription regulation of the acsl6 gene in the freshwater teleost common carp (Cyprinus carpio). First, the full length of acsl6 cDNA contained a coding region of 2148 bp for 715 amino acids, which possessed all characteristic features of the acyl-CoA synthetase (ACSL) family. Its mRNA expression was the highest in the brain, followed by in the heart, liver, kidney, muscle, and eyes, but little expression was detected in the ovary and gills. Additionally, a candidate acsl6 promoter region of 2058 bp was cloned, and the sequence from -758 bp to -198 bp was determined as core a promoter by equal progressive deletion and electrophoretic mobility shift assay. The binding sites for important transcription factors (TFs), including stimulatory protein 1 (SP1), CCAAT enhancer-binding protein (C/EBPα), sterol-regulatory element binding protein 1c (SREBP1c), peroxisome proliferator activated receptor α (PPARα), and PPARγ were identified in the core promoter by site-directed mutation and functional assays. Furthermore, the intraperitoneal injection of PPARγ agonists (balaglitazone) increased the expression of acsl6 mRNA, coupling with an increased proportion of DHA in the muscle, while opposite results were obtained in the injection of the SREBP1c antagonist (betulin). However, the expression of acsl6 and DHA content in muscle were largely unchanged by PPARα agonist (fenofibrate) treatment. These results indicated that acsl6 may play an important role for the muscular DHA uptake and deposition in common carp, and PPARγ and SREBP-1c are the potential TFs involved in the transcriptional regulation of acsl6 gene. To our knowledge, this is the first report of the characterization of acsl6 gene and its promoter in teleosts.

Novel Liver X Receptor Ligand GAC0001E5 Disrupts Glutamine Metabolism and Induces Oxidative Stress in Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is the predominant form of pancreatic cancer with a high mortality rate due to the lack of early detection and effective treatment options for advanced diseases. Metabolic reprogramming, a common hallmark of malignant transformation in pancreatic cancer, is critical for the growth and survival of cancer cells and a potential target mechanism for the treatment of pancreatic cancer. PDAC cells have upregulated glutamine metabolism to meet their biosynthetic and oxidative demands. Liver X receptors (LXRs) are ligand-dependent transcription factors involved in maintaining metabolic homeostasis. LXRs regulate critical cancer-related processes and pathways, including cholesterol, glucose and lipid metabolism, and inflammatory and immune responses. Analysis of transcriptomic data from PDAC clinical samples reveals overexpression of LXRs and their target genes in tumors as compared to normal tissue controls. Targeting LXRs with the novel LXR inverse agonist and degrader GAC0001E5 inhibited PDAC cell proliferation. Using a metabolomics approach, we discovered that 1E5 inhibits glutamine anaplerosis and induces oxidative stress, which are detrimental to PDAC cells. These findings highlight a novel role for LXR in regulating cancer metabolism and the potential application of LXR modulators in targeting cancer metabolism in pancreatic cancer and other malignancies.

Spermidine promotes Bacillus subtilis biofilm formation by activating expression of the matrix regulator slrR.

Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C-methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S-adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S-adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR Deletion of sinR or ectopic expression of slrR in the spermidine-deficient ΔspeD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR.

Anti-Cancer Effects of Radix Angelica Sinensis (Danggui) and N-Butylidenephthalide on Gastric Cancer: Implications for REDD1 Activation and mTOR Inhibition.

BACKGROUND/AIMS: Radix Angelica Sinensis (danggui in Chinese) is widely used in traditional chinese medicine (TCM). N-butylidenephthalide (BP), a bioactive compound in danggui, is a potential antitumor agent for various cancer types. However, its clinical effect and mechanism in the treatment of gastric cancer remain undetermined. METHODS: The in vivo protective effect of danggui in patients with gastric cancer were validated using data from Taiwan's National Health Insurance Research Database (NHIRD). The genes induced by BP-treatment were analyzed by whole transcriptome RNA sequencing (RNA-seq) and validated by real-time PCR, western blot and siRNA transfection. The effect of BP on AGS cell migration and invasion was evaluated in transwell assays. The antitumor effects of BP were evaluated in vivo in an AGS xenograft animal model. RESULTS: Danggui users were found to have an increased survival rate when compared with danggui nonusers (log-rank test p = 0.002) . The use of danggui highly associated with decreased mortality (the adjusted hazard ratio (HR) of danggui user was 0.72 [95 % CI, 0.57-0.92] (p = 0.009). The in vitro results showed that BP inhibited gastric cancer cell proliferation, and triggered cellular apoptosis depending on the activation of mitochondrial apoptotic pathway. Using RNA-seq analysis we found that REDD1 was the highest transcript induced by BP in gastric cancer cells. BP induce an increase of REDD1 expression that inhibits mTOR signaling, thus inhibiting gastric cancer growth. We used RNA interference to demonstrate that the knock-down of REDD1 attenuated the BP-induced mTORC1 activation and growth inhibition. BP suppressed the growth of AGS xenografts tumor in vivo. CONCLUSION: Danggui can prolong the survival rate of gastric cancer patients in Taiwan. BP caused gastric cancer cell death through the activation of mitochondria-intrinsic pathway and induced the REDD1 expression leading to mTOR signal pathway inhibition in gastric cancer cells. BP inhibited the in vivo growth of AGS xenograft tumors. These results may provide the basis for a new therapeutic approach toward the treatment of gastric cancer progression.

Histone Deacetylase 1 (HDAC1) Negatively Regulates Thermogenic Program in Brown Adipocytes via Coordinated Regulation of Histone H3 Lysine 27 (H3K27) Deacetylation and Methylation.

Inhibiting class I histone deacetylases (HDACs) increases energy expenditure, reduces adiposity, and improves insulin sensitivity in obese mice. However, the precise mechanism is poorly understood. Here, we demonstrate that HDAC1 is a negative regulator of the brown adipocyte thermogenic program. The Hdac1 level is lower in mouse brown fat (BAT) than white fat, is suppressed in mouse BAT during cold exposure or ß3-adrenergic stimulation, and is down-regulated during brown adipocyte differentiation. Remarkably, overexpressing Hdac1 profoundly blocks, whereas deleting Hdac1 significantly enhances, ß-adrenergic activation-induced BAT-specific gene expression in brown adipocytes. ß-Adrenergic activation in brown adipocytes results in a dissociation of HDAC1 from promoters of BAT-specific genes, including uncoupling protein 1 (Ucp1) and peroxisome proliferator-activated receptor γ co-activator 1α (Pgc1α), leading to increased acetylation of histone H3 lysine 27 (H3K27), an epigenetic mark of gene activation. This is followed by dissociation of the polycomb repressive complexes, including the H3K27 methyltransferase enhancer of zeste homologue (EZH2), suppressor of zeste 12 (SUZ12), and ring finger protein 2 (RNF2) from (and concomitant recruitment of H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) to) Ucp1 and Pgc1α promoters, leading to decreased H3K27 trimethylation, a histone transcriptional repression mark. Thus, HDAC1 negatively regulates the brown adipocyte thermogenic program, and inhibiting Hdac1 promotes BAT-specific gene expression through a coordinated control of increased acetylation and decreased methylation of H3K27, thereby switching the transcriptional repressive state to the active state at the promoters of Ucp1 and Pgc1α. Targeting HDAC1 may be beneficial in prevention and treatment of obesity by enhancing BAT thermogenesis.

PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease for which there is no cure. We have previously developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, we designed a drug screening strategy based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1200 FDA-approved compounds, we identified the PPARγ agonist pioglitazone as neuroprotective in Drosophila. Here, we show that pioglitazone can rescue TDP-43-dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS, we find that pioglitazone is also neuroprotective when FUS, but not SOD1, is expressed in motor neurons. Interestingly, survival analyses of TDP or FUS models show no increase in lifespan, which is consistent with recent clinical trials. Using a pharmacogenetic approach, we show that the predicted Drosophila PPARγ homologs, E75 and E78, are in vivo targets of pioglitazone. Finally, using a global metabolomic approach, we identify a set of metabolites that pioglitazone can restore in the context of TDP-43 expression in motor neurons. Taken together, our data provide evidence that modulating PPARγ activity, although not effective at improving lifespan, provides a molecular target for mitigating locomotor dysfunction in TDP-43 and FUS but not SOD1 models of ALS in Drosophila. Furthermore, our data also identify several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials.

(-)-Epigallocatechin-3-gallate stimulates myogenic differentiation through TAZ activation.

Muscle loss is a typical process of aging. Green tea consumption is known to slow down the progress of aging. Their underlying mechanisms, however, remain largely unknown. In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG), a polyphenolic compound of green tea, on myogenic differentiation and found that EGCG significantly increases myogenic differentiation. After EGCG treatment, the expression of myogenic marker genes, such as myosin heavy chain, are increased through activation of TAZ, a transcriptional coactivator with a PDZ-binding motif. TAZ-knockdown does not stimulate EGCG-induced myogenic differentiation. EGCG facilitates the interaction between TAZ and MyoD, which stimulates MyoD-mediated gene transcription. EGCG induces nuclear localization of TAZ through the dephosphorylation of TAZ at its Ser89 residue, which relieves 14-3-3 binding in the cytosol. Interestingly, inactivation of Lats kinase is observed after EGCG treatment, which is responsible for the production of dephosphorylated TAZ. Together, these results suggest that EGCG induces myogenic differentiation through TAZ, suggesting that TAZ plays an important role in EGCG induced muscle regeneration.

Carnitine-mediated antioxidant enzyme activity and Bcl2 expression involves peroxisome proliferator-activated receptor-γ coactivator-1α in mouse testis.

The protective effects of carnitine have been attributed to inhibition of apoptosis, alleviating oxidative stress and DNA repair mechanism by decreasing oxidative radicles. Carnitine also increases mitochondrial biogenesis via peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α). The role of carnitine in testicular PGC1α expression has not been documented. We hypothesised that the effects of carnitine as an antioxidant, inhibitor of apoptosis and controller of steroidogenesis in mouse testis may involve PGC1α as a regulator. The present study was designed to evaluate the localisation of PGC1α and the effects of carnitine treatment on the expression of PGC1α, Bcl2 and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) in mouse testis and serum testosterone concentrations. PGC1α was primarily immunolocalised to the Leydig cells and primary spermatocytes. Western blot analysis showed that carnitine (50mgkg-1 and 100mgkg-1 for 7 days) significantly increased PGC1α and Bcl2 expression in the testis in a dose-dependent manner. In addition, carnitine treatment significantly increased antioxidant enzyme (CAT, SOD and GPx) levels. The carnitine-induced changes in PGC1α in the testis were significantly correlated with changes in serum testosterone concentrations, as well as with changes in Bcl2 expression and antioxidant enzyme activity in the testis, as evaluated by electrophoresis. Therefore, the results of the present study suggest that carnitine treatment of mice increases PGC1α levels in the testis, which may, in turn, regulate steroidogenesis by increasing expression of Bcl2 and antioxidant enzymes.

5,6-Dehydrokawain from Alpinia zerumbet promotes osteoblastic MC3T3-E1 cell differentiation.

Bone homeostasis is maintained by balancing bone formation and bone resorption, but an imbalance between them is associated with various bone-related diseases such as osteoporosis and rheumatoid arthritis. We found that 5,6-dehydrokawain (DK) and dihydro-5,6-dehydrokawain (DDK), which were isolated as promising compounds from Alpinia zerumbet rhizomes, promote differentiation of osteoblastic MC3T3-E1 cells. DK and DDK increased the alkaline phosphatase activity and matrix mineralization of MC3T3-E1 cells. DK exerts larger effects than DDK. The gene expression of runt-related transcription factor 2 and osterix, which are essential transcription factors in the early period of osteoblast differentiation, was significantly increased by DK treatment. The mRNA level of distal-less homeobox 5 was also enhanced by DK treatment, and DK activated the p38 mitogen-activated protein kinase pathway. Therefore, DK may have clinical potential for preventing osteoporosis, and could be considered as a potential anabolic therapeutic agent.

Structural basis for native agonist and synthetic inhibitor recognition by the Pseudomonas aeruginosa quorum sensing regulator PqsR (MvfR).

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4-hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR.

Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: involvement of NFAT5.

PURPOSE: High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration. Neovascular retinal diseases are associated with edema. Various factors and channels, including vascular endothelial growth factor (VEGF) and aquaporins (AQPs), influence neovascularization and the development of edema. Therefore, we determined whether extracellular hyperosmolarity alters the expression of VEGF and AQPs in cultured human retinal pigment epithelial (RPE) cells. METHODS: Human RPE cells obtained within 48 h of donor death were prepared and cultured. Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. The levels of signaling proteins and nuclear factor of activated T cell 5 (NFAT5) were determined by western blotting. DNA binding of NFAT5 was determined with EMSA. NFAT5 was knocked down with siRNA. RESULTS: Extracellular hyperosmolarity stimulated VEGF gene transcription and the secretion of VEGF protein. Hyperosmolarity also increased the gene expression of AQP5 and AQP8, induced the phosphorylation of p38 MAPK and ERK1/2, increased the expression of HIF-1α and NFAT5, and induced the DNA binding of NFAT5. The hyperosmotic expression of VEGF was dependent on the activation of p38 MAPK, ERK1/2, JNK, PI3K, HIF-1, and NFAT5. The hyperosmotic induction of AQP5 was in part dependent on the activation of p38 MAPK, ERK1/2, NF-κB, and NFAT5. Triamcinolone acetonide inhibited the hyperosmotic expression of VEGF but not AQP5. The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia. CONCLUSIONS: Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells. The data suggest that high salt intake resulting in osmotic stress may aggravate neovascular retinal diseases and edema via the stimulation of VEGF production in RPE. The downregulation of AQP5 under hypoxic conditions may prevent the resolution of edema.

Beneficial Effect of Flavone Derivatives on Aß-Induced Memory Deficit Is Mediated by Peroxisome Proliferator-Activated Receptor γ Coactivator 1α: A Comparative Study.

In the present study, the neuroprotective effect of 5-hydroxy-6,7,4'-trimethoxyflavone (flavone 1), a natural flavone, was investigated in comparison with another flavone, 5,7,4'-trihydroxyflavone (flavone 2) on the hippocampus of amyloid beta (Aß)-injected rats. Rats were treated with the 2 flavones (1 mg/kg/d) for 1 week before Aß injection. Seven days after Aß administration, memory function of rats was assessed in a passive avoidance test (PAT). Changes in the levels of mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α), phospho-adenosine monophosphate (AMP)-activated protein kinase (pAMPK), AMPK, phospho-cAMP-responsive element-binding protein (CREB), CREB, and nuclear respiratory factor 1 (NRF-1) proteins were determined by Western blot analysis. Our results showed an improvement in memory in rats pretreated with flavonoids. At the molecular level, phosphorylation of CREB, known as the master modulator of memory processes, increased. On the other hand, the level of mitochondrial biogenesis factors, PGC-1α and its downstream molecules NRF-1 and TFAM significantly increased by dietary administration of 2 flavones. In addition, flavone 1 and flavone 2 prevented mitochondrial swelling and mitochondrial membrane potential reduction. Our results provided evidence that flavone 1 is more effective than flavone 2 presumably due to its O-methylated groups. In conclusion, it seems that in addition to classical antioxidant effect, flavones exert part of their protective effects through mitochondrial biogenesis.

TLR dependent XBP-1 activation induces an autocrine loop in rheumatoid arthritis synoviocytes.

X-box binding protein 1 (XBP1) is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes. XBP1 can also be activated in direct response to Toll-like receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Here we show that TLR-dependent XBP1 activation is operative in the synovial fibroblasts (SF) of patients with active rheumatoid arthritis (RA). We investigated the expression of ER stress response genes in patients with active RA and also in patients in remission. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission. Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SF by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent interleukin-6 and tumour necrosis factor (TNF) production. We also show that TNF itself induces sXBP1 in SF, thus generating a potential feedback loop for sustained SF activation. These data confirm the first link between TLR-dependent XBP1 activation and human inflammatory disease. sXBP1 appears to play a central role in this process by providing a convergence point for two different stimuli to maintain activation of SF.

Chronic exposure to low doses of pharmaceuticals disturbs the hepatic expression of circadian genes in lean and obese mice.

Drinking water can be contaminated with pharmaceuticals. However, it is uncertain whether this contamination can be harmful for the liver, especially during obesity. Hence, the goal of our study was to determine whether chronic exposure to low doses of pharmaceuticals could have deleterious effects on livers of lean and obese mice. To this end, lean and ob/ob male mice were treated for 4 months with a mixture of 11 drugs provided in drinking water at concentrations ranging from 10 to 106 ng/l. At the end of the treatment, some liver and plasma abnormalities were observed in ob/ob mice treated with the cocktail containing 106 ng/l of each drug. For this dosage, a gene expression analysis by microarray showed altered expression of circadian genes (e.g. Bmal1, Dbp, Cry1) in lean and obese mice. RT-qPCR analyses carried out in all groups of animals confirmed that expression of 8 different circadian genes was modified in a dose-dependent manner. For some genes, a significant modification was observed for dosages as low as 10²-10³ ng/l. Drug mixture and obesity presented an additive effect on circadian gene expression. These data were validated in an independent study performed in female mice. Thus, our study showed that chronic exposure to trace pharmaceuticals disturbed hepatic expression of circadian genes, particularly in obese mice. Because some of the 11 drugs can be found in drinking water at such concentrations (e.g. acetaminophen, carbamazepine, ibuprofen) our data could be relevant in environmental toxicology, especially for obese individuals exposed to these contaminants.

Glucocorticoid induced leucine zipper inhibits apoptosis of cardiomyocytes by doxorubicin.

Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found glucocorticoid-induced leucine zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes.

Characterization of the metabolic effects of irisin on skeletal muscle in vitro.

AIMS: This work explored the effects of irisin on metabolism, gene expression and mitochondrial content in cultured myocytes. METHODS: C2C12 myocytes were treated with various concentrations of irisin for various durations. Glycolysis and oxidative metabolism were quantified by measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and mitochondrial content was assessed by flow cytometry and confocal microscopy. RESULTS: Cells treated with irisin exhibited significantly increased oxidative metabolism. Irisin treatment also significantly increased mitochondrial uncoupling at various doses and durations. Lastly, treatment with irisin also significantly elevated metabolic gene expression including peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), irisin, glucose transporter 4 (GLUT4) and mitochondrial uncoupling protein 3 (UCP3) leading to increased mitochondrial biogenesis. CONCLUSIONS: Our observations are the first to document increased metabolism in myocytes through irisin-mediated induction of mitochondrial biogenesis and uncoupling with corresponding gene expression. These observations support the need for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin-based therapy.

High dietary taurine inhibits myocardial apoptosis during an atherogenic diet: association with increased myocardial HSP70 and HSF-1 but not caspase 3.

BACKGROUND AND AIM: Apoptosis is a major cause of myocyte death, and taurine is anti-apoptotic. Heat shock protein 70 (HSP70) (which is regulated by heat shock factor-HSF-1) is also anti-apoptotic, and caspase 3 stimulates the apoptotic pathway. This study investigated whether taurine affects atherogenic diet-induced myocardial apoptosis, and whether HSP70, HSF-1 and caspase 3 are involved. METHODS: New Zealand white rabbits were divided into 3 groups for 4 weeks according to their diet. Group 1 (control) was fed a normal rabbit diet; Group 2 (MC) received a normal rabbit diet with 1% methionine plus 0.5% cholesterol. Group 3 received MC diet + 2.5% taurine (MCT). RESULTS: The atherogenic diet did not affect myocardial HSP70 or HSF-1 protein, but increased myocardial apoptotic nuclei to 40% (p < 0.01) versus 7% in con and 12% in MCT (p < 0.01). However, in MCT, myocardial HSP70 expression increased by 42.7% versus con and MC (p = 0.016), HSF-1 by 12% versus con and MC (p < 0.05), and total nuclei count increased by 37% versus MC (p < 0.05). Caspase 3 subunits remained unchanged in all groups, and HSP70 was increased approximately twofold in endothelial layer of arterioles (p = 0.01). CONCLUSION: This study shows that taurine could reduce myocardial apoptotic nuclei and thus confer myocardial cytoprotection via stimulating myocardial HSP70 via HSF-1 and caspase 3-independent mechanisms.

Diesel exhaust particle-induced airway responses are augmented in obese rats.

Air pollutants and obesity are important factors that contribute to asthma. The aim of this study was to assess the airway responsiveness and inflammation in Otsuka-Long Evans Tokushima Fatty (OLETF) obese rats and Long Evans Tokushima-Otsuka (LETO) nonobese rats exposed to diesel exhaust particles (DEPs). Otsuka Long Evans Tokushima fatty rats and LETO rats were exposed intranasally to DEP and then challenged with aerosolized DEP on days 6 to 8. Body plethysmography, bronchoalveolar lavage (BAL), and histology were performed. Enhanced pause (Penh) was measured as an indicator of airway resistance on day 9 and samples were collected on day 10. After exposure to DEP, the OLETF group exhibited a greater increase in Penh compared to that in the LETO group. Moreover, the BAL fluid in mice showed an increase in the total and differential cell counts in the DEP-exposed OLETF group compared to that in the DEP-exposed LETO group. Histological assessment of lung tissue from each group revealed that the DEP-exposed OLETF group tended to have increased inflammatory cell infiltrations in the prebronchial area. Increased peroxisome proliferator-activated receptor γ, coactivator 1ß messenger RNA was observed in the lungs of obese rats compared to that in nonobese rats following DEP exposure. These data indicate that the DEP-exposed OLETF group had increased airway responses and inflammation compared to the DEP-exposed LETO group, indicating that diesel particulates and obesity may be co-contributors to asthma.