Sirt3 mediates the benefits of exercise on bone in aged mice.

Exercise in later life is important for bone health and delays the progression of osteoporotic bone loss. Osteocytes are the major bone cells responsible for transforming mechanical stimuli into cellular signals through their highly specialized lacunocanalicular networks (LCN). Osteocyte activity and LCN degenerate with aging, thus might impair the effectiveness of exercise on bone health; however, the underlying mechanism and clinical implications remain elusive. Herein, we showed that deletion of Sirt3 in osteocytes could impair the formation of osteocyte dendritic processes and inhibit bone gain in response to exercise in vivo. Mechanistic studies revealed that Sirt3 regulates E11/gp38 through the protein kinase A (PKA)/cAMP response element-binding protein (CREB) signaling pathway. Additionally, the Sirt3 activator honokiol enhanced the sensitivity of osteocytes to fluid shear stress in vitro, and intraperitoneal injection of honokiol reduced bone loss in aged mice in a dose-dependent manner. Collectively, Sirt3 in osteocytes regulates bone mass and mechanical responses through the regulation of E11/gp38. Therefore, targeting Sirt3 could be a novel therapeutic strategy to prevent age-related bone loss and augment the benefits of exercise on the senescent skeleton.

Legacy effect of high glucose on promoting survival of HCT116 colorectal cancer cells by reducing endoplasmic reticulum stress response.

Patients with diabetes have increased risk of cancer and poor response to anti-cancer treatment. Increased protein synthesis is associated with endoplasmic reticulum (ER) stress which can trigger the unfolded protein response (UPR) to restore homeostasis, failure of which can lead to dysregulated cellular growth. We hypothesize that hyperglycemia may have legacy effect in promoting survival of cancer cells through dysregulation of UPR. Using HCT116 colorectal cancer cells as a model, we demonstrated the effects of high glucose (25 mM) on promoting cell growth which persisted despite return to normal glucose medium (5.6 mM). Using the Affymetrix gene expression microarray in HCT116 cells programmed by high glucose, we observed activation of genes related to cell proliferation and cell cycle progression and suppression of genes implicated in UPR including BiP and CHOP. These gene expression changes were validated in HCT116 cancer cells using quantitative real-time PCR and Western blot analysis. We further examined the effects of thapsigargin, an anti-cancer prodrug, which utilized ER stress pathway to induce apoptosis. High glucose attenuated thapsigargin-induced UPR and growth inhibition in HCT116 cells, which persisted despite return to normal glucose medium. Western blot analysis showed activation of caspase-3 in thapsigargin-treated cells in both normal and high glucose medium, albeit with lower levels of cleaved caspase-3 in cells exposed to high glucose, suggesting reduced apoptosis. Flow cytometry analysis confirmed fewer apoptotic cells under thapsigargin treatment in cells exposed to high glucose. Our results suggested that hyperglycemia altered gene expression involved in UPR with increased cell proliferation and facilitated survival of HCT116 cells under thapsigargin-induced ER stress by reducing the apoptotic response.

A novel binary matrix consisting of graphene oxide and caffeic acid for the analysis of scutellarin and its metabolites in mouse kidney by MALDI imaging.

Although the in vivo metabolic pathways of scutellarin, a traditional Chinese medicine, have been investigated via different liquid chromatography techniques, studies on the distribution and location of scutellarin within organ tissue sections have not been reported. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can generate in situ spatial distribution profiles for scutellarin and its metabolites in a kidney section. However, the direct detection of a small molecule (m/z < 600) using conventional matrices often results in ion suppression and matrix interferences. In this study, we demonstrated a novel methodology using MALDI-MSI for the in situ spatial localization of scutellarin and its metabolites in kidney tissues by applying a binary matrix of graphene oxide (GO) and caffeic acid (CA). The results indicated that the binary matrix (GO/CA) significantly improved the detection efficiency of scutellarin and its metabolites with relatively high sensitivity, selectivity and reproducibility on tissue sections. This methodology was successfully applied to map scutellarin and its metabolites with MALDI-MSI in mouse kidney tissues. Specifically, scutellarin and scutellarein were found to be located in the cortex and medulla regions of the kidney with relatively high abundance, whereas the remaining metabolites appeared in the cortex with low abundance. We believe that the novel imaging methodology may also be used for the studies of cancerous tissues and inform the development of the future therapies of kidney tumors.

Derivatization strategy combined with parallel reaction monitoring for the characterization of short-chain fatty acids and their hydroxylated derivatives in mouse.

Short-chain fatty acids (SCFAs) and hydroxylated short-chain fatty acids (OH-SCFAs) are crucial intermediates related to a variety of diseases, such as bowel disease, cardiovascular disease, renal disease and cancer. A global profiling method to screen SCFAs and OH-SCFAs was developed by tagging these analytes with d0/d6-N, N-dimethyl-6,7-dihydro-5H-pyrrolo [3,4-d] pyrimidine-2-amine (d0/d6-DHPP) and using ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-MS/MS) in parallel reaction monitoring (PRM) mode. The derivatization procedure was simple and rapid. The targeted compounds could be derivatized within 3 min under mild condition and analyzed without the need of further purification. The derivatization significantly improved the chromatographic performance and mass spectrometry response. The d6-DHPP tagged standards were used as internal standards, which remarkably reduced the matrix effects. The use of high resolution PRM mode made it possible to locate unknown SCFA and OH-SCFA species, and greatly reduced the false positive identification results. The developed method was successfully applied to the analysis of mouse fecal, serum, and liver tissue samples harvested from the breast cancer nude mice that had been exposed with 2,2',4,4'-tetrabromodiphenyl ether (BDE-47). Results showed that 40 analytes (10 SCFAs and 30 OH-SCFAs) were characterized. Semi-quantitative analysis indicated that the exposure of BDE-47 to the mice altered the SCFA and OH-SCFA metabolism, especially in the high dose group. This study provides a high-throughput method to characterize SCFAs and OH-SCFAs in mouse samples.

Immunotoxic Potential of Bisphenol F Mediated through Lipid Signaling Pathways on Macrophages.

As a bisphenol A (BPA) alternative, bisphenol F (BPF) has been detected in various products, such as paper products, personal care products, and food. More importantly, the toxicity of BPF remains underexplored. We reported an integrated method to study the immunotoxic potentials and the underlying mechanisms of BPF on cell apoptosis, macrophage polarization, reactive oxygen species generation, expression and secretion of immune-related cytokines, and reprogramming of lipid signaling. More serious to BPA, BPF induced apoptosis in macrophages. The apoptosis was induced by activating both sphingomyelin-ceramide signaling pathway and oxidative stress, which included intrinsic (bax and caspase-9) and extrinsic apoptotic pathways (tumor necrosis factor receptor 1, caspase-8, and caspase-3). BPF exposure also induced the proinflammatory phenotype of the macrophage. This alternation was shown to be closely correlated with the modulation of biosynthesis and degradation of glycerophospholipids. This study demonstrated novel evidence that BPF as a substituent of BPA induced immunotoxic effects at environmentally relevant concentrations. We also showed that the reprogramming of lipidome plays a key role in the regulation of macrophage polarization and the induction of immunotoxicity of the BPA analogue.

In Situ Detection and Imaging of PFOS in Mouse Kidney by Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry.

Perfluorooctanesulfonic acid (PFOS) is an emerging environmental organic pollutant that has been widely used in daily life products in the last century. Numerous studies showed that the accumulation of PFOS in human through food chain would lead to various disease. However, there is currently no report about its in situ localization in the tissue. In present study, we aimed to develop a reproductive and less-cost method to quantitatively detect and determine the spatial distribution of PFOS in mouse kidney by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) with a commercially available matrix. α-Cyano-4-hydroxycinnamic acid (CHCA) matrix was optimized for PFOS detection in MALDI-IMS analysis. Compared to other organic matrices, CHCA used in negative ion mode showed less background interference and enhanced MS signal intensity and high spatial resolution (80 µm) for PFOS analysis. The use of a CHCA matrix with an autospray system led to successful identification of the PFOS ion signals on the perfusion kidney tissue. The detection limit was at the µg/mL level, with direct visualization from a MS image. The developed method with the optimized parameters was successfully employed to obtain the PFOS spatial distribution in the kidney collected from mice after the PFOS exposure for 14 days. PFOS was mainly distributed in the kidney cortex region, which was consistent with the histological analysis results. Taken together, a rapid, economic, and efficient method was developed for PFOS detection by MALDI-IMS using a CHCA matrix. Mapping the distribution of PFOS by MALDI-IMS with a CHCA matrix provides an innovative approach for the analysis of environmental pollutants in animal or human tissues.

Large-Scale Longitudinal Metabolomics Study Reveals Different Trimester-Specific Alterations of Metabolites in Relation to Gestational Diabetes Mellitus.

Despite the increasing research attention paid to gestational diabetes mellitus (GDM) due to its high prevalence, limited knowledge is available about its pathogenesis. In this study, 428 serum samples were collected from 107 pregnant women suffering from GDM and 107 matched healthy controls. The nontargeted metabolomics data of maternal serum samples from the first (T1, n = 214) and second trimesters (T2, n = 214) were acquired by using ultrahigh performance liquid chromatography coupled with Orbitrap mass spectrometry (MS). A total of 93 differential metabolites were identified on the basis of the accurate mass and MS/MS fragmentation. After false discovery rate correction, the levels of 31 metabolites in GDM group were significantly altered in the first trimester. The differential metabolites were mainly attributed to purine metabolism, fatty acid ß-oxidation, urea cycle, and tricarboxylic acid cycle pathways. The fold changes across pregnancy (T2/T1) of six amino acids (serine, proline, leucine/isoleucine, glutamic acid, tyrosine, and ornithine), a lysophosphatidylcholine (LysoPC(20:4)), and uric acid in GDM group were significantly different from those in the control groups, suggesting that these 8 metabolites might have contributed to the occurrence and progression of GDM. The findings revealed that the amino acid metabolism, lipid metabolism, and other pathways might be disturbed prior to GDM onset and during the period from the first to the second trimester of pregnancy.

Metabolic perturbation, proliferation and reactive oxygen species jointly contribute to cytotoxicity of human breast cancer cell induced by tetrabromo and tetrachloro bisphenol A.

Halogenated bisphenol A analogues (X-BPA) have been widely used in industrial production, such as flame retardant. Although BPA exposure was found to result in cytotoxicity, toxicity of X-BPA and molecular mechanism remain under-explored. In this study, we employed human breast cancer cell as a model to investigate the concentration-dependent toxicity and underlying mechanisms of tetrabromo bisphenol A (TBBPA) and tetrachloro bisphenol A (TCBPA). An integrated method involving molecular toxicology and mass spectrometry (MS)-based global metabolomics was applied to evaluate the toxicity of TCBPA and TBBPA on cell viability, reactive oxygen species (ROS), and metabolic alterations. The results demonstrated that low micromolar levels (0-10 µM) of TCBPA/TBBPA exposure induced cell proliferation and activated the energy metabolism of both glycolysis and amino acid. On the other hand, high micromolar levels (10-50 µM) of TCBPA/TBBPA exposure perturbed the balance between ROS and antioxidative defense process by promoting the ROS generation via the down-regulation of glutathione biosynthesis and up-regulation of nucleotide metabolism. This study, for the first time, provides evidence and mechanism for better understanding the cytotoxicity of TCBPA and TBBPA by regulating the specific metabolic pathways.

Integrative Chemical Proteomics-Metabolomics Approach Reveals Acaca/Acacb as Direct Molecular Targets of PFOA.

Identification of the direct molecular targets of environmental pollutants is of great importance for toxicity mechanism studies. Despite numerous studies have been conducted to investigate the toxicity mechanism of perfluorinated compounds (PFCs), their direct-binding protein targets which trigger downstream toxicity effects remain largely unknown. Herein, we present a systematic chemical proteomic study to profile the target proteins of PFCs by taking PFOA as a representative. Considering its electrophilicity, PFOA could preferentially bind to reactive cysteine-containing proteins. Therefore, two complementary cysteine-targeting probes, iodoacetamide alkyne (IAA) and ethynyl benziodoxolone azide (EBX), were selected to enrich the putative target proteins in the absence or presence of PFOA. Quantitative proteomic analysis of the enriched proteins identified Acaca and Acacb as novel target proteins of PFOA. We then applied parallel reaction monitoring (PRM)-based targeted proteomics study combined with thermal shift assay-based chemical proteomics to verify Acaca and Acacb as bona fide binding targets. These findings afford a plausible explanation for the PFOA-induced liver toxicity, especially regarding abnormal fatty acid metabolism that was validated by targeted metabolomics analysis. The present study documents an integrative chemical proteomics-metabolomics platform that facilitates the authentic identification of proteins that are targeted by small molecules and its potential to be applied for toxicity mechanism studies of environmental pollutants.

MALDI-MS Imaging Reveals Asymmetric Spatial Distribution of Lipid Metabolites from Bisphenol S-Induced Nephrotoxicity.

With the continuous exposure of environmental pollutants in organisms, determination of abundance variation and spatial distribution of lipids might expand our understanding of toxicological mechanisms occurring in the kidney. Herein, an integrated method involving mass spectrometry (MS)-based lipidomics and matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI) was developed for the study of nephrotoxicity in mice exposed to 10 and 100 µg bisphenol S (BPS)/kg body weight/day. The BPS exposure remarkable perturbed abundances of 91 potential markers that mainly involved in five metabolic pathways. We elucidated the lipids spatial heterogeneity by using morphological analysis, probabilistic latent semantic analysis, and coregistered multimodal three-dimensional (3D)-MSI. In morphological analysis, both 10 and 100 µg BPS induced significant nephrotoxicity to mice, including glomerular necrosis in renal cortex, cloudy swelling in renal medulla, and interstitial collapsing in renal pelvis. Significant differential signaling lipids such as sphingomyelin (SM) (d22:0/20:4), ceramide (Cer) (d18:2/24:1), and sphingosine (d18:0) related to inflammation were found to be up-regulated and colocalized in the renal cortex, medulla, and pelvis, respectively. Also, seven significant differential lipids, which are considered to be involved in membrane homeostasis and cellular function, were found to be colocalized in the renal cortex. The observed significant variations of morphology, lipid accumulation, and metabolism in the renal cortex implicated that lipids in the renal cortex were more sensitive to BPS exposure than those in the renal medulla and pelvis. Moreover, we reconstructed a 3D-MSI model of kidney and identified two heterogeneous-related substructures in the renal cortex and pelvis upon 100 µg BPS exposure. It might be used in novel specificity evaluation and early diagnosis for environmental pollutant-induced kidney diseases.

Role of inducible nitric oxide synthase in endothelium-independent relaxation to raloxifene in rat aorta.

BACKGROUND AND PURPOSE: Raloxifene can induce both endothelium-dependent and -independent relaxation in different arteries. However, the underlying mechanisms by which raloxifene triggers endothelium-independent relaxation are still incompletely understood. The purpose of present study was to examine the roles of NOSs and Ca2+ channels in the relaxant response to raloxifene in the rat isolated, endothelium-denuded aorta. EXPERIMENTAL APPROACH: Changes in isometric tension, cGMP, nitrite, inducible NOS protein expression and distribution in response to raloxifene in endothelium-denuded aortic rings were studied by organ baths, radioimmunoassay, Griess reaction, western blot and immunohistochemistry respectively. KEY RESULTS: Raloxifene reduced the contraction to CaCl2 in a Ca2+ -free, high K+ -containing solution in intact aortic rings. Raloxifene also acutely relaxed the aorta primarily through an endothelium-independent mechanism involving NO, mostly from inducible NOS (iNOS) in vascular smooth muscle layers. This effect of raloxifene involved the generation of cGMP and nitrite. Also, it was genomic in nature, as it was inhibited by a classical oestrogen receptor antagonist and inhibitors of RNA and protein synthesis. Raloxifene-induced stimulation of iNOS gene expression was partly mediated through activation of the NF-κB pathway. Raloxifene was more potent than 17ß-estradiol or tamoxifen at relaxing endothelium-denuded aortic rings by stimulation of iNOS. CONCLUSIONS AND IMPLICATIONS: Raloxifene-mediated vasorelaxation in rat aorta is independent of a functional endothelium and is mediated by oestrogen receptors and NF-κB. This effect is mainly mediated through an enhanced production of NO, cGMP and nitrite, via the induction of iNOS and inhibition of calcium influx through Ca2+ channels in rat aortic smooth muscle.

C-reactive protein promotes acute kidney injury by impairing G1/S-dependent tubular epithelium cell regeneration.

CRP (C-reactive protein) is regarded as an inflammatory biomarker in AKI (acute kidney injury), but its exact role in AKI remains unclear. Thus we sought to investigate the role of CRP in AKI. Clinically, elevated serum CRP levels were found to associate closely with increased serum creatinine and urea levels (P<0.01) in patients with AKI, which then fell after recovery from AKI. To determine the role of CRP in AKI, an ischaemia/reperfusion mouse model of AKI was developed using Tg (transgenic) mice that express human CRP. Compared with the WT (wild-type) mice, CRP Tg mice developed more severe renal injury at 24 h after ischaemia as determined by significantly increased serum creatinine and tubular necrosis. This was associated with an impaired TEC (tubular epithelium cell) regeneration as shown by an over 60% reduction in PCNA+ (proliferating-cell nuclear antigen) and BrdU+ (bromodeoxyuridine) TECs in CRP Tg mice with AKI. In vitro, the addition of CRP to a human TEC line (HK-2) also largely suppressed the proliferation of TECs. The functional role of CRP in AKI was demonstrated further by the blocking of CRP binding to the FcγRII (Fcγ receptor II) with a neutralizing anti-CD32 antibody, which restored TEC proliferation and prevented AKI in CRP Tg mice. Moreover, we found that impaired G1/S transition by suppression of the phosphorylation of CDK2 (cyclin-dependent kinase 2) and expression of cyclin E may be a key mechanism by which CRP inhibits TEC regeneration during the AKI repair process. In conclusion, CRP plays a pathogenic role in AKI by inhibiting G1/S-dependent TEC regeneration. The results of the present study suggest that targeting CRP signalling may offer a new therapeutic potential for AKI.