Low doses of acetyl trihexyl citrate plasticizer promote adipogenesis in hepatocytes and mice.

Accumulating epidemiological evidence underscores the association between pervasive environmental factors and an increased risk of metabolic diseases. Environmental chemicals, recognized disruptors of endocrine and metabolic processes, may contribute to the global prevalence of metabolic disorders, including obesity. Acetyl tributyl citrate (ATHC), categorized as a citric acid ester plasticizer, serves as a substitute for di-(2-ethylhexyl) phthalate (DEHP) in various everyday products. Despite its widespread use and the increasing risk of exposure in humans and animals due to its high leakage rates, information regarding the safety of exposure to environmentally relevant doses of ATHC remains limited. This study aimed to investigate the potential impact of ATHC exposure on metabolic homeostasis. Both in vivo and in vitro exposure models were used to characterize the effects induced by ATHC exposure. C57BL/6 J male mice were subjected to a diet containing ATHC for 12 weeks, and metabolism-related parameters were monitored and analyzed throughout and after the exposure period. Results indicated that sub-chronic dietary exposure to ATHC induced an increase in body fat percentage, elevated serum lipid levels, and increased lipid content in the liver tissue of mice. Furthermore, the effect of ATHC exposure on murine hepatocytes were examined and results indicated that ATHC significantly augmented lipid levels in AML12 hepatocytes, disrupting energy homeostasis and altering the expression of genes associated with fatty acid synthesis, uptake, oxidation, and secretion pathways. Conclusively, both in vivo and in vitro results suggest that exposure to low levels of ATHC may be linked to an elevated risk of obesity and fatty liver in mice. The potential implications of ATHC on human health warrant comprehensive evaluation in future studies.

RIP1 kinase inactivation protects against LPS-induced acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome (ARDS) is characterized by lung tissue oedema and inflammatory cell infiltration, with limited therapeutic interventions available. Receptor-interacting protein kinase 1 (RIPK1), a critical regulator of cell death and inflammation implicated in many diseases, is not fully understood in the context of ARDS. In this study, we employed RIP1 kinase-inactivated (Rip1K45A/K45A) mice and two distinct RIPK1 inhibitors to investigate the contributions of RIP1 kinase activity in lipopolysaccharide (LPS)-induced ARDS pathology. Our results indicated that RIPK1 kinase inactivation, achieved through both genetic and chemical approaches, significantly attenuated LPS-induced ARDS pathology, as demonstrated by reduced polymorphonuclear neutrophil percentage (PMN%) in alveolar lavage fluid, expression of inflammatory and fibrosis-related factors in lung tissues, as well as histological examination. Results by tunnel staining and qRT-PCR analysis indicated that RIPK1 kinase activity played a role in regulating cell apoptosis and inflammation induced by LPS administration in lung tissue. In summary, employing both pharmacological and genetic approaches, this study demonstrated that targeted RIPK1 kinase inactivation attenuates the pathological phenotype induced by LPS inhalation in an ARDS mouse model. This study enhances our understanding of the therapeutic potential of RIPK1 kinase modulation in ARDS, providing insights for the pathogenesis of ARDS.

[Reversal Effect of NVP-BEZ235 on Doxorubicin-Resistance in Burkitt Lymphoma RAJI Cell Line].

OBJECTIVE: To study the reversal effect of NVP-BEZ235 on doxorubicin resistance in Burkitt lymphoma RAJI cell line. METHODS: The doxorubicin-resistant cell line was induced by treating RAJI cells with a concentration gradient of doxorubicin. The levels of Pgp, p-AKT, and p-mTOR in cells were detected by Western blot. Cell viability was detected by MTT assay. IC50 was computed by SPSS. RESULTS: The doxorubicin-resistant Burkitt lymphoma cell line, RAJI/DOX, was established successfully. The expression of Pgp and the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line were both higher than those in RAJI cell line. NVP-BEZ235 downregulated the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line. NVP-BEZ235 inhibited the proliferation of RAJI/DOX cell line, and the effect was obvious when it was cooperated with doxorubicin. CONCLUSION: The constitutive activation of PI3K/AKT/mTOR pathway of RAJI/DOX cell line was more serious than RAJI cell line. NVP-BEZ235 reversed doxorubicin resistance of RAJI/DOX cell line by inhibiting the PI3K/AKT/mTOR signal pathway.

Plasticizer acetyl triethyl citrate (ATEC) induces lipogenesis and obesity.

Environmental chemicals, such as plasticizers, have been linked to increased rates of obesity, according to epidemiological studies. Acetyl triethyl citrate (ATEC) is a plasticizer that is commonly utilized in pharmaceutical products and food packaging as a non-phthalate alternative. Due to its direct contact with the human body and high leakage rate from the polymers, assessment of the potential risk of ATEC exposure at environmentally relevant low doses to human health is needed. Male C57BL/6 J mice were fed diets containing ATEC at doses of either 0.1 or 10 µg/kg per day in a period of 12 weeks to mimic the real exposure environment. The findings suggest that in C57BL/6 J mice, ATEC exposure resulted in increased body weight gain, body fat percentage, and benign hepatocytes, as well as adipocyte size. Consistent with in vivo models, ATEC treatment obviously stimulated the increase of intracellular lipid load in both mouse and human hepatocytes. Mechanically, ATEC induced the transcriptional expression of genes involved in de novo lipogenesis and lipid uptake. Using both enzyme inhibitor and small interfering RNA (siRNA) transfection, we found that stearoyl-coenzyme A desaturase 1 (SCD1) played a significant role in ATEC-induced intracellular lipid accumulation. This study for the first time provided initial evidence suggesting the obesogenic and fatty liver-inducing effect of ATEC at low doses near human exposure levels, and ATEC might be a potential environmental obesogen and its effect on human health need to be further evaluated.

Characterizing the obesogenic and fatty liver-inducing effects of Acetyl tributyl citrate (ATBC) plasticizer using both in vivo and in vitro models.

The global incidence of obesity and non-alcoholic fatty liver disease (NAFLD) is rising rapidly in recent years. Environmental factors including usage of plastics and exposure to chemicals have been proposed as important contributors to the obesity pandemic. Acetyl tributyl citrate (ATBC) is a non-phthalate plasticizer widely used in food packaging, personal care products, medical devices and children's toys etc. Due to its high leakage rate from plastics, exposure risk of ATBC keeps increasing. Although there are some studies investigating the safety of ATBC on human health, these studies mainly focused on high dosages and information regarding ATBC safety at environmental-relevant low levels is still limited. In this study, we aimed to evaluate the safety of subchronic exposure to environmentally-relevant concentrations of ATBC. C57BL/6J mice were orally exposed to ATBC for 6 or 14 weeks. Results indicated that ATBC exposure increased the body weight gain, the body fat content and the size of adipocytes, induced liver steatosis in mice. Consistent with in vivo effects, ATBC treatment increased the intracellular lipid accumulation in vitro hepatocytes. Transcriptome sequencing, qRT-PCR analysis and western blotting revealed that ATBC exposure affected the expression of genes involved in de novo lipogenesis and lipid uptake. Therefore, based on our subchronic and in vitro results, it suggested that ATBC might be a potential environmental obesogen with metabolism-disturbing and fatty liver-inducing risk, and its application in many consumer products should be carefully re-evaluated.

Assessment of lipid metabolism-disrupting effects of non-phthalate plasticizer diisobutyl adipate through in silico and in vitro approaches.

Diisobutyl adipate (DIBA), as a novel non-phthalate plasticizer, is widely used in various products. However, little effort has been made to investigate whether DIBA might have adverse effects on human health. In this study, we integrated an in silico and in vitro strategy to assess the impact of DIBA on cellular homeostasis. Since numerous plasticizers could activate peroxisome proliferator-activated receptor γ (PPARγ) pathway to interrupt metabolism systems, we first utilized molecular docking to analyze interaction between DIBA and PPARγ. Results indicated that DIBA had strong affinity with the ligand-binding domain of PPARγ (PPARγ-LBD) at Histidine 499. Afterwards, we used cellular models to investigate in vitro effects of DIBA. Results demonstrated that DIBA exposure increased intracellular lipid content in murine and human hepatocytes, and altered transcriptional expression of genes related to PPARγ signaling and lipid metabolism pathways. At last, target genes regulated by DIBA were predicted and enriched for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Protein-protein interaction (PPI) network and transcriptional factors (TFs)-genes network were established accordingly. Target genes were enriched in Phospholipase D signaling pathway, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and Epidermal growth factor receptor (EGFR) signaling pathway which were related to lipid metabolism. These findings suggested that DIBA exposure might disturb intracellular lipid metabolism homeostasis via targeting PPARγ. This study also demonstrated that this integrated in silico and in vitro methodology could be utilized as a high throughput, cost-saving and effective tool to assess the potential risk of various environmental chemicals on human health.

Clostridium butyricum Inhibits Fat Deposition via Increasing the Frequency of Adipose Tissue-Resident Regulatory T Cells.

SCOPE: Clostridium butyricum (CB) exerts beneficial actions in several disorders. However, the impact and molecular cues of CB in fat metabolism remain elusive. This study demonstrates the CB inhibition of fat deposition by increasing the relative number of adipose tissue-resident Treg cells (aTregs). METHODS AND RESULTS: CB is administered orally to wild type (WT) mice fed with chow diet, which decrease fat deposition and adipogenic gene expression, associating with elevated serum levels of butyrate. Sodium butyrate (SB) feeding mimics the CB suppression of fat accumulation. Of note, the frequency of aTregs in both the CB and SB treatments, analyzed by flow cytometry, is markedly increased, accompanied by activated Wnt10b expression in white adipose tissues. However, CB and SB fail to inhibit fat deposition in Wnt10b-KO mice. Intriguingly, CB and SB are able to alleviate the obesity, fatty liver, and glucose abnormalities in high fat diet (HFD)-fed WT mice. CONCLUSIONS: These findings suggest that CB, through its metabolite butyrate, inhibits fat deposition via potentiating aTreg cell generation, and support the option of CB and SB for therapeutic interventions in obesity and related disorders.

Discovery of novel 2,8-diazaspiro[4.5]decan-1-one derivatives as potent RIPK1 kinase inhibitors.

Necroptosis, a key form of programmed lytic cell death, has gained recognition as an important driver in various inflammatory diseases. Inhibition of kinase activity of receptor interaction protein kinase 1 (RIPK1), which block the activation of the necroptosis pathway has shown therapeutic potential in many human diseases. In order to find new chemotypes of RIPK1 inhibitors, a virtual screening workflow was performed and led to the discovery of 8-benzoyl-3-benzyl-1,3,8-triazaspiro[4.5]decane-2,4-dione (compound 8) as a hit compound. Further structural optimization identified a series of 2,8-diazaspiro[4.5]decan-1-one derivatives as potent RIPK1 inhibitors. Among them, compound 41 exhibited prominent inhibitory activity against RIPK1 with an IC50 value of 92 nM. Meanwhile, compound 41 showed a significant anti-necroptotic effect in a necroptosis model in U937 cells. Therefore, compound 41 could be employed as a lead compound of RIPK1 inhibitors for further structural optimization.

Angiogenin and MMP-2 as potential biomarkers in the differential diagnosis of gestational trophoblastic diseases.

BACKGROUND: Gestational trophoblastic diseases (GTDs) are characterized by vascular abnormalities of the trophoblast, but their pathogenesis is unknown. Angiogenin (ANG) and matrix metalloproteinase (MMP)-2, which are molecules implicated in the angiogenic process, may play some role in this process. MATERIAL AND METHODS: We determined ANG and MMP-2 in the placental tissues of 26 patients who had a benign mole (BM), 12 patients with gestational trophoblast neoplasia (GTN) (1 invasive hydatidiform mole, 10 choriocarcinomas, and 1 placental-site trophoblastic tumor), and 28 normal chorionic villi (NCV) subjects using immunohistochemistry staining. We obtained the serum samples from 20 patients with GTDs and 20 early pregnant women and evaluated them by the enzyme linked immunosorbent assay. RESULTS: ANG expression in GTN (66.7%) and BM (100%) samples were both significantly higher (strong/intermediate staining) than in NCV (60.7%) samples (P < .001). Similarly, the immunoreactivities of MMP-2 in the GTN (66.7%) and BM (80.8%) samples were significantly elevated compared to that of the NCV (57.1%) samples (P < .001). The levels of ANG and MMP-2 in the maternal serum of the GTN group were both significantly higher than those of the control group (P < .001). ANG and MMP-2 expressions were associated with gestation age, clinical stage, and FIGO stage. A positive correlation between ANG and MMP-2 expression was observed (rs = 0.725; P < .01). CONCLUSION: ANG and MMP-2 levels were significantly elevated in the placental tissues and maternal serum from patients with GTDs. Further studies with more patients may clarify the vascular abnormalities in GTDs and determine potential biomarkers in the differential diagnosis of GTDs.

B Cell-mediated Humoral Immunity in Chronic Hepatitis B Infection.

B cell-mediated humoral immunity plays a vital role in viral infections, including chronic hepatitis B virus (HBV) infection, which remains a critical global public health issue. Despite hepatitis B surface antigen-specific antibodies are essential to eliminate viral infections, the reduced immune functional capacity of B cells was identified, which was also correlated with chronic hepatitis B (CHB) progression. In addition to B cells, T follicular helper (Tfh) cells, which assist B cells to produce antibodies, might also be involved in the process of anti-HBV-specific antibody production. Here, we provide a comprehensive review of the role of various subsets of B cells and Tfh cells during CHB progression and discuss current novel treatment strategies aimed at restoring humoral immunity. Understanding the mechanism of dysregulated B cells and Tfh cells will facilitate the ultimate functional cure of CHB patients.

RIP1 kinase inactivation protects against acetaminophen-induced acute liver injury in mice.

Many studies have investigated the role of receptor-interacting protein 1 (RIP1) kinase in acetaminophen (APAP) overdose-induced acute liver injury. However, the results were not consistent and there still remain controversies. Importantly, in these previous studies, the usage of DMSO to dissolve the RIP1 kinase inhibitor Nec-1, resulted in misleading conclusion. Our study aimed to determine the role of RIP1 kinase in APAP-induced liver injury, via genetically or pharmaceutically inhibition of RIP1 kinase activity. Our results indicated that APAP-induced liver injury was significantly attenuated in RIP1 kinase-dead (Rip1K45A/K45A) mice compared to WT control. High dosage of APAP-induced mortality was also rescued by RIP1 kinase inactivation. In agreement, RIP1 kinase inhibitor, Nec-1 which was formulated with PEG400, could efficiently alleviate APAP-induced hepatotoxicity. For the underlying mechanism, our results suggested that RIP1 kinase inactivation did not influence the hepatic GSH depletion, but significantly reduced the hepatic cell death and inflammation induced by APAP treatment. Using bone marrow transplantation model, we also demonstrated that it was RIP1 kinase activity in tissue-resident hepatic cells other than hematopoietic-derived cells mainly responsible for APAP-induced liver injury. Our study confirmed the important role of RIP1 kinase activity in APAP-induced acute liver failure.

An insulin-independent mechanism for transcriptional regulation of Foxo1 in type 2 diabetic mice.

Hepatic gluconeogenesis is the major contributor to the hyperglycemia observed in both patients and animals with type 2 diabetes. The transcription factor FOXO1 plays a dominant role in stimulating hepatic gluconeogenesis. FOXO1 is mainly regulated by insulin under physiological conditions, but liver-specific disruption of Foxo1 transcription restores normal gluconeogenesis in mice in which insulin signaling has been blocked, suggesting that additional regulatory mechanisms exist. Understanding the transcriptional regulation of Foxo1 may be conducive to the development of insulin-independent strategies for the control of hepatic gluconeogenesis. Here, we found that elevated plasma levels of adenine nucleotide in type 2 diabetes are the major regulators of Foxo1 transcription. We treated lean mice with 5'-AMP and examined their transcriptional profiles using RNA-seq. KEGG analysis revealed that the 5'-AMP treatment led to shifted profiles that were similar to db/db mice. Many of the upregulated genes were in pathways associated with the pathology of type 2 diabetes including Foxo1 signaling. As observed in diabetic db/db mice, lean mice treated with 5'-AMP displayed enhanced Foxo1 transcription, involving an increase in cellular adenosine levels and a decrease in the S-adenosylmethionine to S-adenosylhomocysteine ratio. This reduced methylation potential resulted in declining histone H3K9 methylation in the promoters of Foxo1, G6Pc, and Pepck. In mouse livers and cultured cells, 5'-AMP induced expression of more FOXO1 protein, which was found to be localized in the nucleus, where it could promote gluconeogenesis. Our results revealed that adenine nucleotide-driven Foxo1 transcription is crucial for excessive glucose production in type 2 diabetic mice.

Prognostic role of the recepteur d'origine nantais (RON) expression in primary high-grade osteosarcoma.

BACKGROUND: Osteosarcoma is a common primary malignant bone tumor susceptible to distant metastasis. The clinical outcome for patients remains poor due to the resistance to chemotherapy and lacking effective therapeutic targets. Recepteur d'origine nantais (RON), a transmembrane protein of the c-MET proto-oncogene family, has been reported to contribute to the malignant progression and bone metastasis in several tumors. The present study aimed to explore the prognostic significance of RON in primary high-grade osteosarcoma. METHODS: Immunohistochemistry (IHC) and western blotting (WB) were used to investigate the protein expression of RON in 80 surgically resected specimens (50 high-grade osteosarcoma specimens and 30 non-neoplastic bone tissues) and 6 cell lines. The χ2 test or independent-sample Student's t-test was used to assess the significance of RON difference between osteosarcoma and non-neoplastic bone tissues. The χ2 test and Fisher's exact test were used to analyze the association of RON with the clinicopathological features of osteosarcoma patients. Kaplan-Meier method and Cox proportional hazards model were used to assess the significance of RON for the survival of osteosarcoma patients. RESULTS: The results of IHC and WB observed significant overexpression of RON in osteosarcoma specimens (P < 0.001) and osteosarcoma cell lines. Moreover, immunohistochemical high expression of RON was associated with a poor response to chemotherapy (P = 0.032) as well as worse progression-free (P = 0.003) and overall (P < 0.001) survival of osteosarcoma patients. Multivariate analysis revealed that high expression of RON was independently associated with reduced progression-free (P = 0.027, HR = 2.31) and overall survival (P = 0.004, HR = 5.06) time of osteosarcoma patients. CONCLUSIONS: The present study demonstrated that high expression of RON held independent value for unfavorable survival in primary high-grade osteosarcoma. Its potential role as a therapeutic target for osteosarcoma treatment deserves further research.

Tributyltin triggers lipogenesis in macrophages via modifying PPARγ pathway.

Tributyltin (TBT), a bioaccumulative and persistent environmental pollutant, has been proposed as a metabolism disruptor and obesogen through targeting peroxisome proliferator-activated receptor gamma (PPARγ) receptor pathway. However, it remains unknown whether this biological effect occurs in macrophage, a cell type which cooperates closely with hepatocytes and adipocytes to regulate lipid metabolism. This study for the first time investigated the effect of TBT on PPARγ pathway in macrophages. Our results indicated that nanomolar levels of TBT was able to strongly activate PPARγ in human macrophages. TBT treatment also markedly increased the intracellular lipid accumulation, and enhanced the expression of lipid metabolism-related genes in macrophages, while these effects were all significantly down-regulated in PPARγ-deficient macrophages, confirming the involvement of PPARγ in TBT-induced lipogenesis. Next, a mouse model that C57BL/6 mice were orally exposed to TBT with the doses (250 and 500 µg/kg body weight) lower than NOAEL (no observed adverse effect level) was used to further investigate the in vivo mechanisms. And the in vivo results were consistent with cellular assays, confirming the induction of PPARγ and the increased expression of lipogenesis-regulating and lipid metabolism-related genes by TBT in vivo. In conclusion, this study not only provided the first evidence that TBT stimulated lipogenesis, activated PPARγ and related genes in human macrophages, but also provided insight into the mechanism of TBT-induced metabolism disturbance and obesity through targeting PPARγ via both in vitro cellular assays and in vivo animal models.

RIP1 kinase activity promotes steatohepatitis through mediating cell death and inflammation in macrophages.

Hepatocyte cell death and liver inflammation have been well recognized as central characteristics of nonalcoholic steatohepatitis (NASH), however, the underlying molecular basis remains elusive. The kinase receptor-interacting protein 1 (RIP1) is a multitasking molecule with distinct functions in regulating apoptosis, necroptosis, and inflammation. Dissecting the role of RIP1 distinct functions in different pathophysiology has absorbed huge research enthusiasm. Wild-type and RIP1 kinase-dead (Rip1K45A/K45A) mice were fed with high-fat diet (HFD) to investigate the role of RIP1 kinase activity in the pathogenesis of NASH. Rip1K45A/K45A mice exhibited significantly alleviated NASH phenotype of hepatic steatosis, liver damage, fibrosis as well as reduced hepatic cell death and inflammation compared to WT mice. Our results also indicated that both in vivo lipotoxicity and in vitro saturated fatty acids (palmitic acid) treatment were able to induce the kinase activation of RIP1 in liver macrophages. RIP1 kinase was required for mediating inflammasome activation, apoptotic and necrotic cell death induced by palmitic acid in both bone marrow-derived macrophage and mouse primary Kupffer cells. Results from chimeric mice established through lethal irradiation and bone marrow transplantation further confirmed that the RIP1 kinase in hematopoietic-derived macrophages contributed mostly to the disease progression in NASH. Consistent with murine models, we also found that RIP1 kinase was markedly activated in human NASH, and the kinase activation mainly occurred in liver macrophages as indicated by immunofluorescence double staining. In summary, our study indicated that RIP1 kinase was phosphorylated and activated mainly in liver macrophages in both experimental and clinical NASH. We provided direct genetic evidence that the kinase activity of RIP1 especially in hematopoietic-derived macrophages contributes to the pathogenesis of NASH, through mediating inflammasome activation and cell death induction. Macrophage RIP1 kinase represents a specific and potential therapeutic target for NASH.

PER1 interaction with GPX1 regulates metabolic homeostasis under oxidative stress.

Metabolism serves mammalian feeding and active behavior, and is controlled by circadian clock. The molecular mechanism by which clock factors regulate metabolic homeostasis under oxidative stress is unclear. Here, we have characterized that the daily oxygen consumption rhythm was deregulated in Per1 deficient mice. Per1 deficiency impaired daily mitochondrial dynamics and deregulated cellular GPx-related ROS fluctuations in the peripheral organs. We identified that PER1 enhanced GPx activity through PER1/GPX1 interaction in cytoplasm, consequently improving the oxidative phosphorylation efficiency of mitochondria. Per1 expression was specifically elevated in the fasting peripheral organs for protecting mitochondrial from oxidation stress. These observations reveal that Per1-driven mitochondrial dynamics is a critical effector mechanism for the regulation of mitochondrial function in response to oxidation stress.

Estimating the effects of asymptomatic and imported patients on COVID-19 epidemic using mathematical modeling.

The epidemic of Coronavirus Disease 2019 has been a serious threat to public health worldwide. Data from 23 January to 31 March at Jiangsu and Anhui provinces in China were collected. We developed an adjusted model with two novel features: the asymptomatic population and threshold behavior in recovery. Unbiased parameter estimation identified faithful model fitting. Our model predicted that the epidemic for asymptomatic patients (ASP) was similar in both provinces. The latent periods and outbreak sizes are extremely sensitive to strongly controlled interventions such as isolation and quarantine for both asymptomatic and imported cases. We predicted that ASP serve as a more severe factor with faster outbreaks and larger outbreak sizes compared with imported patients. Therefore, we argued that the currently strict interventions should be continuously implemented, and unraveling the asymptomatic pool is critically important before preventive strategy such as vaccines.

Peroxisome proliferator-activated receptor gamma (PPARγ) activation and metabolism disturbance induced by bisphenol A and its replacement analog bisphenol S using in vitro macrophages and in vivo mouse models.

Bisphenol A (BPA) and its replacement analog, bisphenol S (BPS), have been proposed as environmental obesogen to disrupt the lipid metabolism through regulating peroxisome proliferator-activated receptor gamma (PPARγ) receptor. However, there is a dearth of information on whether this biological effect can occur in human macrophage, a cell type which closely interacts with adipocytes and hepatocytes to control lipid metabolism. Here, we for the first time investigate the activity of BPA and BPS on PPARγ pathway in human macrophages. The results demonstrated that BPA and BPS served as activators of PPARγ in human macrophage cell line, and significantly induced the expression of lipid metabolism-related genes, including fatty acid binding protein 4 (FABP4), cluster of differentiation 36 (CD36) and nuclear receptor subfamily 1 group H member 3 (NR1H3). In PPARγ knockout cells, expression of these genes was down-regulated, suggesting that these genes are dependent on PPARγ. The underlying mechanisms were further investigated using an in vivo mouse model, and the results confirmed the induction of PPARγ and its respective target genes in mice following exposure to BPA or BPS. Moreover, the observed alteration of PPARγ expression highly correlated with the disturbance of metabolism profiles in liver tissues as detected by 1H Nuclear Magnetic Resonance (NMR)-based metabonomics. Overall, this study provided the first evidence that BPA and BPS activated PPARγ and its target genes in human macrophages, and provided comprehensive information to confirm that BPA and BPS disturb the metabolism through targeting PPARγ via both in vitro assays and in vivo animal models.

Clinicopathological and prognostic values of ErbB receptor family amplification in primary osteosarcoma.

Osteosarcoma is a malignant bone tumor with extremely high invasion, metastasis and mortality. The prognosis of patients with osteosarcoma remains poor. The ErbB receptor family was found to be overexpressed in human cancers and associated with poor prognosis. However, the role of ErbB receptor family in osteosarcoma has not been fully understood. The present study aimed to investigate the clinicopathological and prognostic significances of ErbB receptors in primary osteosarcoma. Western blot (WB), reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescence in situ hybridization (FISH) were used to detect the protein and gene expression of ErbB receptors in 60 primary osteosarcoma specimens and 30 non-neoplastic bone tissues. WB and RT-qPCR analyses showed that the protein and mRNA expression levels of EGFR, ErbB3 and ErbB4 in osteosarcoma specimens were significantly higher than those in non-neoplastic bone tissues. Seventeen (28.33%), 15 (25.00%) and 15 (25.00%) osteosarcoma specimens presented with amplification of EGFR, ErbB3 and ErbB4 gene, respectively, which were significantly higher compared with non-neoplastic bone tissues. The amplification of ErbB3 and ErbB4 in osteosarcoma was associated with advanced surgical stage. The amplification of EGFR, ErbB3, ErbB4 and the co-amplification of EGFR-ErbB3, EGFR-ErbB4, ErbB3-ErbB4 was linked with poor response to chemotherapy and distant metastasis. The amplification of EGFR, ErbB3 and ErbB4, as well as their co-amplification demonstrated independent prognostic values for reduced survival time of osteosarcoma patients and may serve as potential therapeutic targets for osteosarcoma patients in the future.

Adenine nucleotide-mediated regulation of hepatic PTP1B activity in mouse models of type 2 diabetes.

AIMS/HYPOTHESIS: Plasma 5'-AMP (pAMP) is elevated in mouse models of type 2 diabetes. However, the metabolic regulatory role of adenine nucleotides in type 2 diabetes remains unclear. METHODS: Adenine nucleotides and their metabolites in plasma and liver were examined by HPLC. 1H NMR-based metabolomics analysis was performed to explore the changes of metabolites in mouse models of type 2 diabetes. Na+/K+ ATPase and Na+/H+ exchanger activity were measured in response to adenine nucleotide metabolites. Human recombinant protein tyrosine phosphatase 1B (PTP1B) was used for enzyme kinetic assays. Protein binding assays were performed with microscale thermophoresis. The intracellular pH of hepatocyte AML12 cell lines was measured using the BCECF-AM method. We also analysed pAMP levels in participants with type 2 diabetes. RESULTS: Elevation of pAMP was a universal phenomenon in all mouse models of type 2 diabetes including db/db vs lean mice (13.9 ± 2.3 µmol/l vs 3.7 ± 0.9 µmol/l; p < 0.01), ob/ob vs lean mice (9.1 ± 2.0 µmol/l vs 3.9 ± 1.2 µmol/l; p < 0.01) and high-fat diet/streptozotocin-induced vs wild-type mice (6.6 ± 1.5 µmol/l vs 4.1 ± 0.9 µmol/l; p < 0.05); this elevation was required for the occurrence of hyperglycaemia in obese mice. 1H NMR-based metabolomics study following HPLC analysis revealed that the metabolite profile in wild-type mice treated with 5'-AMP was similar to that in db/db diabetic mice, especially the accumulation of a large quantity of ATP and its metabolites. The glucose-lowering drug metformin reduced the severity of hyperglycaemia both in 5'-AMP-induced wild-type mice and db/db mice. Metformin decreased the accumulation of liver ATP but not its metabolites in these hyperglycaemic mice. ATP and metformin reciprocally change cellular pH homeostasis in liver, causing opposite shifts in liver activity of PTP1B, a key negative regulator of insulin signalling. Furthermore, pAMP levels were also elevated in individuals with type 2 diabetes (45.2 ± 22.7 nmol/l vs 3.1 ± 1.9 nmol/l; p < 0.01). CONCLUSIONS/INTERPRETATION: These results reveal an emerging role for adenine nucleotide in the regulation of hyperglycaemia and provide a potential therapeutic target in obesity and type 2 diabetes.