Formononetin, an isoflavone, activates AMP-activated protein kinase/ß-catenin signalling to inhibit adipogenesis and rescues C57BL/6 mice from high-fat diet-induced obesity and bone loss.

Balance between adipocyte and osteoblast differentiation is the key link of disease progression in obesity and osteoporosis. We have previously reported that formononetin (FNT), an isoflavone extracted from Butea monosperma, stimulates osteoblast formation and protects against postmenopausal bone loss. The inverse relationship between osteoblasts and adipocytes prompted us to analyse the effect of FNT on adipogenesis and in vivo bone loss, triggered by high-fat diet (HFD)-induced obesity. The anti-obesity effect and mechanism of action of FNT was determined in 3T3-L1 cells and HFD-induced obese male mice. Our findings show that FNT suppresses the adipogenic differentiation of 3T3-L1 fibroblasts, through down-regulation of key adipogenic markers such as PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα) and sterol regulatory element-binding protein (SREBP) and inhibits intracellular TAG accumulation. Increased intracellular reactive oxygen species levels and AMP-activated protein kinase (AMPK) activation accompanied by stabilisation of ß-catenin were attributed to the anti-adipogenic action of FNT. In vivo, 12 weeks of FNT treatment inhibited the development of obesity in mice by attenuating HFD-induced body weight gain and visceral fat accumulation. The anti-obesity effect of FNT results from increased energy expenditure. FNT also protects against HFD-induced dyslipidaemia and rescues deterioration of trabecular bone volume by increasing bone formation and decreasing bone resorbtion caused by HFD. FNT's rescuing action against obesity-induced osteoporosis commenced at the level of progenitors, as bone marrow progenitor cells, obtained from the HFD mice group supplemented with FNT, showed increased osteogenic and decreased adipogenic potentials. Our findings suggest that FNT inhibits adipogenesis through AMPK/ß-catenin signal transduction pathways and protects against HFD-induced obesity and bone loss.

Effects of intratracheal exposure of 2-chloroethyl ethyl sulfide (CEES) on the activation of CCAAT-enhancer-binding protein (C/EBP) and its protection by antioxidant liposome.

Exposure of 2-chloroethyl ethyl sulfide (CEES) to guinea pigs causes lung injury by infiltration of neutrophils in interstitial lung spaces. A unique MAPK-regulated transcription factor, C/EBP (CCAAT-enhancer-binding protein), regulates the expression of intracellular adhesion molecule-1 (ICAM-1), involved in recruiting neutrophils in lung. The present study was to determine if CEES exposure causes activation of C/EBP, in particular the predominant ß-isoform and if so whether it can be prevented by intratracheal delivery of an antioxidant liposome containing N-acetyl cysteine and tocopherols. Lung injury was developed in guinea pigs by intratracheal exposure of CEES (0.5 mg/kg). The antioxidant liposome was given intratracheally 5 min after CEES exposure, and the animals were sacrificed after 30 days. CEES exposure caused a 2.3-fold increase in the activation of C/EBP accompanied with a 45% and 121% increase in the protein level of C/EBP ß and ICAM-1, respectively, and this effect was counteracted by the antioxidant liposome.

Prostacyclin receptors: Transcriptional regulation and novel signalling mechanisms.

The prostanoid Prostacyclin plays diverse physiologic roles within the vasculature and other systems, and is widely implicated in several cardiovascular, pulmonary and renal diseases. Despite this, knowledge of the factors regulating expression of the I prostanoid receptor (the IP) remained largely unknown. This review details recent advances in understanding the key transcriptional regulators determining expression of the PTGIR gene in the human vasculature and the identification of novel interacting partners of the IP that impact on its function therein. Included in this are the trans-acting factors that regulate expression of the PTGIR under basal- and regulated-conditions, particularly those determining its up-regulation in response to cellular differentiation, estrogen and low serum-cholesterol. Moreover, the functional implications of the interactions between the IP with PDZK1, a multi PDZ-domain containing protein essential for reverse-cholesterol transport and endothelialization, and the IP with IKEPP, the intestinal and kidney enriched PDZ protein, for the role of the prostacyclin-IP axis within the vasculature are reviewed.

Evidence that C/EBP-ß LAP Increases Fat Metabolism and Protects Against Diet-Induced Obesity in Response to mTOR Inhibition.

Aging and obesity are common risk factors for numerous chronic pathologies, and the compounding effects of old age and increased adiposity pose a serious threat to public health. Starting from the assumption that aging and obesity may have shared underpinnings, we investigated the antiobesogenic potential of a successful longevity intervention, the mTORC1 inhibitor rapamycin. We find that rapamycin prevents diet-induced obesity in mice and increases the activity of C/EBP-ß LAP, a transcription factor that regulates the metabolic shift to lipid catabolism observed in response to calorie restriction. Independent activation of C/EBP-ß LAP with the antiretroviral drug adefovir dipivoxil recapitulates the anti-obesogenic effects of rapamycin without reducing signaling through mTORC1 and increases markers of fat catabolism in the liver. Our findings support a model that C/EBP-ß LAP acts downstream of mTORC1 signaling to regulate fat metabolism and identifies a novel drug that may be exploited to treat obesity and decrease the incidence of age-related disease.

Gankyrin Promotes Tumor-Suppressor Protein Degradation to Drive Hepatocyte Proliferation.

Background & Aims: Uncontrolled liver proliferation is a key characteristic of liver cancer; however, the mechanisms by which this occurs are not well understood. Elucidation of these mechanisms is necessary for the development of better therapy. The oncogene Gankyrin (Gank) is overexpressed in both hepatocellular carcinoma and hepatoblastoma. The aim of this work was to determine the role of Gank in liver proliferation and elucidate the mechanism by which Gank promotes liver proliferation. Methods: We generated Gank liver-specific knock-out (GLKO) mice and examined liver biology and proliferation after surgical resection and liver injury. Results: Global profiling of gene expression in GLKO mice showed significant changes in pathways involved in liver cancer and proliferation. Investigations of liver proliferation after partial hepatectomy and CCl4 treatment showed that GLKO mice have dramatically inhibited proliferation of hepatocytes at early stages after surgery and injury. In control LoxP mice, liver proliferation was characterized by Gank-mediated reduction of tumor-suppressor proteins (TSPs). The failure of GLKO hepatocytes to proliferate is associated with a lack of down-regulation of these proteins. Surprisingly, we found that hepatic progenitor cells of GLKO mice start proliferation at later stages and restore the original size of the liver at 14 days after partial hepatectomy. To examine the proliferative activities of Gank in cancer cells, we used a small molecule, cjoc42, to inhibit interactions of Gank with the 26S proteasome. These studies showed that Gank triggers degradation of TSPs and that cjoc42-mediated inhibition of Gank increases levels of TSPs and inhibits proliferation of cancer cells. Conclusions: These studies show that Gank promotes hepatocyte proliferation by elimination of TSPs. This work provides background for the development of Gank-mediated therapy for the treatment of liver cancer. RNA sequencing data can be accessed in the NCBI Gene Expression Omnibus: GSE104395.

Experimental Nonalcoholic Steatohepatitis and Liver Fibrosis Are Ameliorated by Pharmacologic Activation of Nrf2 (NF-E2 p45-Related Factor 2).

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis. METHODS: Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals. RESULTS: TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice. CONCLUSIONS: Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress.

Insights into CYP2B6-mediated drug-drug interactions.

Mounting evidence demonstrates that CYP2B6 plays a much larger role in human drug metabolism than was previously believed. The discovery of multiple important substrates of CYP2B6 as well as polymorphic differences has sparked increasing interest in the genetic and xenobiotic factors contributing to the expression and function of the enzyme. The expression of CYP2B6 is regulated primarily by the xenobiotic receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) in the liver. In addition to CYP2B6, these receptors also mediate the inductive expression of CYP3A4, and a number of important phase II enzymes and drug transporters. CYP2B6 has been demonstrated to play a role in the metabolism of 2%-10% of clinically used drugs including widely used antineoplastic agents cyclophosphamide and ifosfamide, anesthetics propofol and ketamine, synthetic opioids pethidine and methadone, and the antiretrovirals nevirapine and efavirenz, among others. Significant inter-individual variability in the expression and function of the human CYP2B6 gene exists and can result in altered clinical outcomes in patients receiving treatment with CYP2B6-substrate drugs. These variances arise from a number of sources including genetic polymorphism, and xenobiotic intervention. In this review, we will provide an overview of the key players in CYP2B6 expression and function and highlight recent advances made in assessing clinical ramifications of important CYP2B6-mediated drug-drug interactions.

Activation of PPARγ2 by PPARγ1 through a functional PPRE in transdifferentiation of myoblasts to adipocytes induced by EPA.

PPARγ and Wnt signaling are central positive and negative regulators of adipogenesis, respectively. Here we identified that, eicosapentaenoic acid (EPA) could effectively induce the transdifferentiation of myoblasts into adipocytes through modulation of both PPARγ expression and Wnt signaling. During the early stage of transdifferentiation, EPA activates PPARδ and PPARγ1, which in turn targets ß-catenin to degradation and down-regulates Wnt/ß-catenin signaling, such that the myogenic fate of myoblasts could be switched to adipogenesis. In addition, EPA up-regulates the expression of PPARγ1 by activating RXRα, then PPARγ1 binds to the functional peroxisome proliferator responsive element (PPRE) in the promoter of adipocyte-specific PPARγ2 to continuously activate the expression of PPARγ2 throughout the transdifferentiation process. Our data indicated that EPA acts as a dual-function stimulator of adipogenesis that both inhibits Wnt signaling and induces PPARγ2 expression to facilitate the transdifferentiation program, and the transcriptional activation of PPARγ2 by PPARγ1 is not only the key factor for the transdifferentiation of myoblasts to adipocytes, but also the crucial evidence for successful transdifferentiation. The present findings provided insight for the first time as to how EPA induces the transdifferentiation of myoblasts to adipocytes, but also provide new clues for strategies to prevent and treat some metabolic diseases.

Insights into CYP2B6-mediated drug-drug interactions

Mounting evidence demonstrates that CYP2B6 plays a much larger role in human drug metabolism than was previously believed. The discovery of multiple important substrates of CYP2B6 as well as polymorphic differences has sparked increasing interest in the genetic and xenobiotic factors contributing to the expression and function of the enzyme. The expression of CYP2B6 is regulated primarily by the xenobiotic receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) in the liver. In addition to CYP2B6, these receptors also mediate the inductive expression of CYP3A4, and a number of important phase II enzymes and drug transporters. CYP2B6 has been demonstrated to play a role in the metabolism of 2%-10% of clinically used drugs including widely used antineoplastic agents cyclophosphamide and ifosfamide, anesthetics propofol and ketamine, synthetic opioids pethidine and methadone, and the antiretrovirals nevirapine and efavirenz, among others. Significant inter-individual variability in the expression and function of the human CYP2B6 gene exists and can result in altered clinical outcomes in patients receiving treatment with CYP2B6-substrate drugs. These variances arise from a number of sources including genetic polymorphism, and xenobiotic intervention. In this review, we will provide an overview of the key players in CYP2B6 expression and function and highlight recent advances made in assessing clinical ramifications of important CYP2B6-mediated drug-drug interactions.

A single nucleotide polymorphism in the Bax gene promoter affects transcription and influences retinal ganglion cell death.

Pro-apoptotic Bax is essential for RGC (retinal ganglion cell) death. Gene dosage experiments in mice, yielding a single wild-type Bax allele, indicated that genetic background was able to influence the cell death phenotype. DBA/2J(Bax+/-) mice exhibited complete resistance to nerve damage after 2 weeks (similar to Bax(-/-) mice), but 129B6(Bax+/-) mice exhibited significant cell loss (similar to wild-type mice). The different cell death phenotype was associated with the level of Bax expression, where 129B6 neurons had twice the level of endogenous Bax mRNA and protein as DBA/2J neurons. Sequence analysis of the Bax promoters between these strains revealed a single nucleotide polymorphism (T(129B6) to C(DBA/2J)) at position -515. A 1.5- to 2.5-fold increase in transcriptional activity was observed from the 129B6 promoter in transient transfection assays in a variety of cell types, including RGC5 cells derived from rat RGCs. Since this polymorphism occurred in a p53 half-site, we investigated the requirement of p53 for the differential transcriptional activity. Differential transcriptional activity from either 129B6 or DBA/2J Bax promoters were unaffected in p53(-/-) cells, and addition of exogenous p53 had no further effect on this difference, thus a role for p53 was excluded. Competitive electrophoretic mobility-shift assays identified two DNA-protein complexes that interacted with the polymorphic region. Those forming Complex 1 bound with higher affinity to the 129B6 polymorphic site, suggesting that these proteins probably comprised a transcriptional activator complex. These studies implicated quantitative expression of the Bax gene as playing a possible role in neuronal susceptibility to damaging stimuli.