Rhabdomyolysis is a severe adverse effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins). This myopathy is strongly enhanced by the combination with statins and fibrates, another hypolipidaemic agent. We have evaluated the initial step of statin-induced apoptosis by the detection of membrane flip-flop using flow cytometric analysis. L6 rat myoblasts were treated with various statins (atorvastatin (3 microM), cerivastatin (3 microM), fluvastatin (3 microM), pravastatin (3 mM), or simvastatin (3 microM)) for 2, 4 or 6 h followed by reacting with FITC-conjugated annexin V for the detection of initial apoptosis signal (flip-flop). Various statin-treated myoblasts were significantly stained with FITC-annexin V at 6 h, whereas they were not detected at 2 h. Moreover, immunoblot analysis indicated that when the cells were treated with cerivastatin (3 microM), membrane-associated Ras protein was activated and detached until 6 h, resulting in cell death through the consequent activation of caspase-8. On the other hand, since cytosolic Ras activation did not activate, there is still an unknown mechanism in statin-related Ras depletion. In conclusion, statin-induced apoptosis in muscular tissue was directly initiated by the farnesyl-anchored Ras protein depletion from cell membrane with subsequent apoptosis.

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BackgroundWe investigated the prevalence of hallux valgus (HV) and examined its association with various factors in a cross-sectional study of Japanese female university students.MethodsA questionnaire survey of foot symptoms, lifestyle, and body mass index (BMI) was administered to 343 women who provided informed consent at a women’s university. Footprints were obtained and bone density was measured. Associations of HV with various factors were analyzed by logistic regression analysis.ResultsBig toe pain was reported in 26.5% of the women. HV (HV angle, ≥15°) was present in the left foot in 22.4%, the right foot in 20.7%, and unilaterally or bilaterally in 29.7% of women. Mild HV (HV angle, ≥15° to <20°) was noted in the left foot and right foot in 13.4% and 13.1% of women, respectively; no severe HV (HV angle, ≥40°) was observed. HV was associated with big toe pain (adjusted OR: 3.56, 95% CI: 2.01–6.32), history of HV in the mother or maternal grandmother (adjusted OR: 2.45, 95% CI: 1.19–5.02), and history of HV in other family members (adjusted OR: 3.09, 95% CI: 1.35–7.06). Moderate HV was associated with big toe pain (adjusted OR: 4.58, 95% CI: 2.17–9.66) and history of HV in the mother or maternal grandmother (adjusted OR: 3.36, 95% CI: 1.40–8.07). The proportion of women with big toe pain increased significantly with HV severity.ConclusionsHV was present in about 30% of female university students. Young women with big toe pain or a family history of HV should be evaluated for HV.

We investigated the inhibitory effect of caffeic acid phenethyl ester (CAPE) on the differentiation of 3T3-L1 mouse fibroblasts to adipocytes. 3T3-L1 cells were differentiated for adipocytes given high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 mM dexamethasone (DEX), 500 mM isobutylmethylxanthine (IBMX), and 5 mg/ml insulin for 7 days. After differentiation, cells were stained with Oil-Red-O to detect oil droplets in adipocytes. Additionally, the cells were lysed and measured for triglyceride contents. Total RNA was isolated from differentiated cells on day 0, 4 and 7. Then, RNA was analyzed using reverse transcription (RT)-polymerase chain reaction (PCR). CAPE dose-dependently suppressed oil droplet accumulation and reduced the droplet size. These findings showed that CAPE at concentrations of 25 to 50 mM could significantly inhibit triglyceride deposition (pϽ0.05). Treatment of 3T3-L1 with CAPE reduced the mRNA levels of peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer-binding protein (C/EBPalpha). Fatty acid synthase (Fas) and adipocyte-specific fatty acid binding protein (aP2) are known to be associated with lipid metabolism in adipocytes, and both Fas mRNA and aP2 mRNA were significantly suppressed by CAPE treatment. These findings suggested that CAPE suppresses 3T3-L1 differentiation to adipocytes through inhibition of PPARgamma, C/EBPalpha, Fas and aP2 expression.Key words caffeic acid phenethyl ester; adipocyte differentiation; fatty acid synthase; peroxisome proliferator-activated receptor gamma; CCAAT/enhancer-binding protein alpha; adipocyte-specific fatty acid binding protein 2 Biol. Pharm. Bull. 33(9) 1484-1488 (2010) © 2010 Pharmaceutical Society of Japan * To whom correspondence should be addressed. e-mail: manju129@mukogawa-u.ac.jp Oil-Red-O Staining After differentiation, cells were stained with Oil-Red-O to detect oil droplets in adipocytes. Cells were washed 3 times with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 2 min and then stained with 3.3 mg/ml Oil-Red-O in 75% isopropanol for 30 min. Cells were washed with PBS 3 times and observed under a microscope (Olympus CKX41, Japan). Stained oil droplets in the cells were dissolved containing 4% (v/v) Nonidet-P40 in isopropanol with gentle agitation for 5 min. Supernatant was measured with a spectrophotometer at 500 nm.Triglyceride Determination Differentiated cells were lysed with Lysis buffer (50 mM Tris, 0.15 M NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Tween-20, pH 7.5 with HCl) and measured for triglyceride content on day 4 and 7. The triglyceride content in the cell lysates were quantified using the Triglyceride E test WAKO. The concentration was corrected using DNA as an internal standard. DNA was quantified using a Quant-iT TM dsDNA HS Assay kit. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) To detect mRNA expression levels, total RNA was extracted from differentiated cells on day 0, 4 and 7 with an RNeas...

Obesity is a condition in which excess body fat accumulates due to lipids producing adipocytes and an increased number of differentiated mature cells. Recently, new findings have shown that macrophages infiltrate into adipose tissues and produce various pro-inflammatory cytokines in obese subjects. The inflammatory changes induced by the cross-talk between adipocytes and macrophages are critical for the pathophysiology of obesity and thus of metabolic syndrome. Caffeic acid phenethyl ester (CAPE) is known to have many functions, including antibacterial, anticancer and anti-inflammatory properties, but there is no evidence of its effect on the inflammatory responses in hypertrophic adipocytes through stimulation by macrophages. We investigated the effect of CAPE on macrophages and hypertrophic adipocytes in this study. CAPE significantly suppressed the levels of lipopolysaccharide (LPS)-induced interleukin (IL)-1-beta, tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1 from a macrophage cell line, RAW264.7. Supernatants of stimulated RAW264.7 macrophages drastically increased mRNA levels of pro-inflammatory cytokines such as IL-6, MCP-1 and TNF-alpha in 3T3-L1 hypertrophic adipocytes. CAPE also significantly and dose-dependently reduced the gene expression of these cytokines. Our findings indicate that CAPE has inhibitory effects on the production of pro-inflammatory cytokines from LPS-stimulated RAW264.7 macrophages. In addition, CAPE suppressed gene expressions of cytokines under inflammatory conditions of hypertrophic adipocytes, suggesting that it may have the potential to suppress inflammation by macrophage infiltration into adipose tissue in obese patients.Key words caffeic acid phenethyl ester; hypertrophic adipocyte; macrophage; pro-inflammatory cytokine Obesity is a major risk factor for metabolic syndrome, which includes a number of disorders, such as hypertension, heart diseases and diabetes.1,2) Obesity is a condition in which excess body fat accumulates due to lipids producing hypertrophic adipocytes and an increased number of differentiated mature cells. This process is regulated by genetic and environmental factors, such as nutrient intake.3,4) It is known that adipocytes in obese individuals show cell hypertrophy and chronic inflammation. The inflammatory changes induced by the cross-talk between adipocytes and macrophages are critical for the pathophysiology of obesity and thus of metabolic syndrome. 5,6) Recent studies have demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, 7,8) which produce various inflammatory proteins. The inflammation process is associated with the activation of macrophages and the release of pro-inflammatory cytokines, such as interleukin (IL)-1-beta, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-6. 9,10) MCP-1, known to play a pivotal role in macrophage trafficking and activation, is produced by macrophages and adipocytes. Several reports suggest t...

The generation of oxidative stress, characterized by enhanced reactive oxygen species (ROS) formation, has been found in obesity. ROS production was increased during the differentiation of 3T3-L1 cells into adipocytes. We previously reported that caffeic acid phenethyl ester (CAPE) suppresses 3T3-L1 differentiation to adipocytes through the inhibition of peroxisome proliferator-activated receptor γ. In this study, the preventive effect of CAPE on oxidative stress in 3T3-L1 cells was observed. The results were as follows: (1) ROS production during 3T3-L1 cell differentiation to adipocytes was significantly (p < 0.05) suppressed by CAPE treatment in a concentration-dependent manner, (2) with CAPE treatment, the extracellular superoxide dismutase mRNA expression level significantly increased, but the NOX4 mRNA expression level did not change, and (3) CAPE treatment significantly increased superoxide dismutase (SOD) activity in 3T3-L1 cells. From these results, we suggest that the increased oxidative stress in 3T3-L1 differentiation to adipocytes is attenuated by CAPE treatment. This attenuation may be partly caused by increased SOD production.

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Natural killer (NK) cells have many functional activities, including cytotoxicity and the capacity to produce cytokines and chemokines. NK cell activity is regulated partly by eicosanoids, which are produced from arachidonic acid (ARA) and eicosapentaenoic (EPA) acid. In this study, we investigated the effects of long-term therapy with ARA or docosahexaenoic acid (DHA) on the cytotoxic effects of the NK cells of young rats, which were fed on a nonfish oil diet for two generations. Control oil, ARA (240 mg/kg BW/day) or DHA (240 mg/kg BW/day) were orally administrated to the rats for 13 weeks before determining the cytotoxic activity of NK cells from the spleen against YAC-1 mouse lymphoma cell line, as well as the plasma levels of docosanoids or eicosanoids and inflammatory cytokines. Long-term ARA administration significantly suppressed the cytotoxic activity of NK cells. Moreover, ARA administration significantly increased the plasma levels of ARA, prostaglandin (PG) E2, and PGD2. However, DHA administration did not produce any different effects compared with those in the control rats. Furthermore, the inflammatory cytokine levels were not affected by the administration of ARA or DHA. These results suggest that long-term ARA administration has an inhibitory effect on the tumor cytotoxicity of NK cells in rat spleen lymphocytes owing to the enhanced synthesis of PGE2 and PGD2 from ARA because of the elevated plasma ARA levels in young rats.