Rhododendrol, a reductive metabolite of raspberry ketone, suppresses the differentiation of 3T3­L1 cells into adipocytes.

Obesity is a serious medical condition worldwide, and a major risk factor for type 2 diabetes, metabolic syndrome, cancer and cardiovascular disease. In addition to changes in dietary habits and physical activity, consuming supplements to maintain good health and prevent obesity is important in modern society. Raspberry ketone (RK) is a natural phenolic ketone found in the European red raspberry (Rubus idaeus L.) and is hypothesized to prevent obesity when administered orally. The present study found that RK was reduced to rhododendrol (ROH) in human liver microsomes and cytosol. The present study investigated whether the metabolite ROH had anti­adipogenic effects using mouse 3T3­L1 cells. The effects of ROH or RK on lipid accumulation during differentiation of 3T3­L1 pre­adipocyte into adipocyte were determined using Oil Red O staining. CCAAT enhancer­binding protein α (C/EBPα) and peroxisome proliferator­activated receptor γ (PPARγ) mRNA and protein expression were examined using reverse transcription­quantitative PCR and western blotting analysis, respectively. The present study revealed that ROH suppressed lipid accumulation in the cells, similar to RK. In addition, ROH suppressed the mRNA expression levels of C/EBPα and PPARγ in 3T3­L1 adipocytes. Furthermore, ROH suppressed PPARγ protein expression in 3T3­L1 adipocytes. These findings suggested that ROH is an active metabolite with an anti­adipogenic effect, which may contribute to the anti­obesity effect of orally administered RK. The present study indicated that it is important to understand the biological activity of the metabolites of orally administered compounds.

Carbonic anhydrase 3 increases during liver adipogenesis even in pre-obesity, and its inhibitors reduce liver adipose accumulation.

The abnormal lipid metabolism in the liver that occurs after high caloric intake is the main cause of nonalcoholic fatty liver disease (NAFLD). Differences between samples from healthy livers and livers from individuals with NAFLD indicate that changes in liver function occur during disease progression. Here, we examined changes in protein expression in a fatty liver model in the early stages of obesity to identify potential alterations in function. The proteins expressed in the liver tissue of pre-obese rats were separated via SDS/PAGE and stained with Coomassie brilliant blue-G250. Peptide mass fingerprinting indicated an increase in the expression of carbonic anhydrase 3 (CA3) relative to controls. Western blotting analysis confirmed the increase in CA3 expression, even in an early fat-accumulation state in which excessive weight gain had not yet occurred. In human hepatoma HepG2 cells, fat accumulation induced with oleic acid also resulted in increased CA3 expression. When the cells were in a state of fat accumulation, treating them with the CA3 inhibitors acetazolamide (ACTZ) or 6-ethoxyzolamide (ETZ) suppressed fat accumulation, but only ETZ somewhat reduced the fat-induced upregulation of CA3 expression. Expression of CA3 was therefore upregulated in response to the consumption of a high-fat diet, even in the absence of an increase in body weight. The suppression of CA3 activity by ACTZ or ETZ reduced fat accumulation in hepatocytes, suggesting that CA3 is involved in the development of fatty liver.

[Occupational Exposure of Pharmacists to Drugs during the Preparation of Powder Drugs in Dispensing Pharmacies].

Occupational exposure of pharmacists to drugs during powder drug preparation in dispensing pharmacies was investigated. First, we determined frequently prescribed tipepidine hibenzate and ambroxol hydrochloride suspended in the air of the dispensing room. The median concentration of the drugs in the air was 0.01 µg/m3 and 0.02 µg/m3, respectively; these values indicate that the air in the dispensing room was contaminated with powder drug. To estimate drug exposure during powder drug preparation, drug dust was collected near the nose of workers. Analysis of the active ingredients of the drugs used in the preparation revealed that eight drugs, including bethanechol, l-carbocisteine, and zonisamide, were detected in the range of 1.5-1220 µg/m3. Assuming that the respiratory volume of an adult was 0.008 m3/min, it was estimated that 0.4-36 µg of the ingredients were exposed per prescription by multiplying concentration, respiratory volume and sampling time (3-5 min). Furthermore, the effect of wearing a medical mask on the drug powder exposure was evaluated using a self-made apparatus. When the amount of drug powder collected on filters that is either covered with or without a medical mask was compared, the covered filter exhibited reduced drug powder accumulation to less than 10% the amount collected on the uncovered filter. The present data suggested that a medical mask might decrease the drug dust allergies in dispensing pharmacists.

Possible mechanism of superoxide formation through redox cycling of plumbagin in pig heart.

The purpose of this study is to elucidate the possible mechanism of superoxide formation through redox cycling of plumbagin (PLG) in pig heart. Of four 1,4-naphthoquinones tested in this study, PLG was most efficiently reduced in the cytosolic fraction of pig heart. On the other hand, lawsone (LAS) was little reduced. Thus, whether or not PLG and LAS induce the formation of superoxide anion radical in pig heart cytosol was examined, by using the methods of cytochrome c reduction and chemiluminescence. PLG significantly induced the formation of superoxide anion radical, even though LAS had no ability to mediate superoxide formation. PLG was a significant inhibitor for the stereoselective reduction of 4-benzoylpyridine (4-BP) catalyzed by tetrameric carbonyl reductase (TCBR) in pig heart cytosol. Furthermore, PLG was confirmed to competitively inhibit the 4-BP reduction, and the optimal pH for the PLG reduction was around 6.0 similar to that for the 4-BP reduction. These results suggest that PLG mediates superoxide formation through its redox cycling involved in the two-electron reduction catalyzed by TCBR, and induces oxidative stress in pig heart.

Thrombomodulin suppresses invasiveness of HT1080 tumor cells by reducing plasminogen activation on the cell surface through activation of thrombin-activatable fibrinolysis inhibitor.

Cell malignancy is negatively correlated with the expression of thrombomodulin (TM), a thrombin receptor expressed on the surface of various cells, including tumor cells. TM accelerates thrombin-activatable fibrinolysis inhibitor (TAFI) activation catalyzed by thrombin. The active form of TAFI (TAFIa) contributes to inhibition of plasmin formation through its carboxypeptidase B (CPB)-like activity. Here, we examined whether TM- and tumor cell-dependent TAFI activation participates in controlling pericellular fibrinolysis and cell invasion. Human fibrosarcoma HT1080 cells retained the ability to activate both prothrombin and plasminogen, but did not express TM. HT1080 cells mediated activation of TAFI in plasma in the presence of soluble-type TM (sTM) through cell-dependent prothrombin activation. HT1080 cells stably expressing TM (TM-HT1080) mediated plasma TAFI activation without added sTM, but HT1080 (wild-type) and Mock-transfected HT1080 cells (Mock) did not. Production of TAFIa suppressed cell-mediated plasminogen activation. Matrigel invasion by wild-type and Mock cells was enhanced two-fold by diluted plasma (10%), whereas the plasma-induced invasion of TM-HT1080 cells (65% of wild-type invasion) was lower than those of wild-type and Mock cells. Cell invasion by TM-HT1080 was partially enhanced by addition of a TAFIa/CPB inhibitor. These results suggest that TM suppresses pericellular fibrinolysis and plasma-induced tumor cell invasion, and that it is mediated, at least in part, by plasma TAFI activation.

Verotoxin-1 stimulation of macrophage-like THP-1 cells up-regulates tissue factor expression through activation of c-Yes tyrosine kinase: Possible signal transduction in tissue factor up-regulation.

Verotoxin (VT)-producing Escherichia coli (E. coli) O157:H7 infections are frequently complicated by thrombotic angiopathy, hemolytic uremic syndrome (HUS) and neurological symptoms. The present data demonstrate that VT-1 (Shiga toxin) stimulation of macrophage-like THP-1 cells up-regulates the activity, antigen and mRNA levels of tissue factor (TF), a key cofactor of the coagulation-inflammation-thrombosis circuit. This up-regulation is accompanied by phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase), IkappaB kinase beta (IKKbeta) and extracellular signal-regulated kinase 2 (ERK2). Changes in TF mRNA levels were in parallel with the activation of NF-kappaB/Rel and Egr-1 activation, but not with AP-1. Inhibition of PI3-kinase attenuated VT-1-induced phosphorylation of IKKbeta and ERK2, and the up-regulation of TF mRNA levels. VT-1 stimulation rapidly activated c-Yes tyrosine kinase, a member of the Src family. Treatment of the cells with c-Yes antisense oligos attenuated the VT-1-induced phosphorylation of PI3-kinase, IKKbeta and ERK2, activations of NF-kappaB/Rel and Egr-1, and up-regulation of TF mRNA levels. These results suggest that VT-1-induced macrophage stimulation activates c-Yes, which then up-regulates TF expression through activation of the IKKbeta/proteasome/NF-kappaB/Rel and MEK/ERK2/Egr-1 pathways via activation of PI3-kinase. Induction of macrophage TF expression by VT-1 may play an important role in the acceleration of the coagulation-inflammation-thrombosis circuit during infections by VT-producing E. coli.