Effects of Caffeine and Chlorogenic Acid on Nonalcoholic Steatohepatitis in Mice Induced by Choline-Deficient, L-Amino Acid-Defined, High-Fat Diet.

Several recent experimental studies have investigated the effects of caffeine and chlorogenic acid (CGA), representative ingredients of coffee, on nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). However, the results are conflicting, and their effects are yet to be clarified. In the present study, we examined the effects of caffeine and CGA on choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice, relatively new model mice of NASH. Seven-week-old male C57BL/6J mice were divided into the following groups: Control diet (control), CDAHFD (CDAHFD), CDAHFD supplemented with 0.05% (w/w) caffeine (caffeine), and CDAHFD supplemented with 0.1% (w/w) CGA (CGA). After seven weeks, the mice were killed and serum biochemical, histopathological, and molecular analyses were performed. Serum alanine aminotransferase (ALT) levels were significantly higher in the caffeine and CGA groups than in the CDAHFD group. On image analysis, the prevalence of Oil red O-positive areas (reflecting steatosis) was significantly higher in the caffeine group than in the CDAHFD group, and that of CD45R-positive areas (reflecting lymphocytic infiltration) in the hepatic lobule was significantly higher in the caffeine and CGA groups than in the CDAHFD group. Hepatic expression of interleukin (IL)-6 mRNA was higher in the caffeine and CGA groups than in the CDAHFD group, and the difference was statistically significant for the caffeine group. In conclusion, in the present study, caffeine and CGA significantly worsened the markers of liver cell injury, inflammation, and/or steatosis in NASH lesions in mice.

Daidzein down-regulates ubiquitin-specific protease 19 expression through estrogen receptor ß and increases skeletal muscle mass in young female mice.

Ubiquitin-specific protease 19 (USP19) is a key player in the negative regulation of muscle mass during muscle atrophy. Loss-of-function approaches demonstrate that 17ß-estradiol (E2) increases USP19 expression through estrogen receptor (ER) α and consequently decreases soleus muscle mass in young female mice under physiological conditions. Daidzein is one of the main isoflavones in soy, and activates ERß-dependent transcription. Here, we investigated the effects of daidzein on E2-increased USP19 expression and E2-decreased soleus muscle mass in young female mice. Daidzein stimulated the transcriptional activity of ERß in murine C2C12 cells and down-regulated USP19 expression. Consistently, daidzein inhibited E2-induced USP19 expression in a reporter activity using a functional half-estrogen response element (hERE) from Usp19. Daidzein inhibited E2-induced recruitment of ERα and promoted recruitment of ERß to the Usp19 hERE. Dietary daidzein down-regulated the expression of USP19 at the mRNA and protein levels and increased soleus muscle mass in female mice, but not in males. In soleus muscle from ovariectomized (OVX) female mice, dietary daidzein inhibited E2-increased USP19 mRNA expression and E2-decreased muscle mass. Furthermore, E2 induced the recruitment of ERα and ERß to the hERE, whereas daidzein inhibited E2-induced recruitment of ERα, and enhanced E2-increased recruitment of ERß, to the Usp19 hERE. These results demonstrate that dietary daidzein decreases USP19 mRNA expression through ERß and increases soleus muscle mass in young female mice, but not in male mice, under physiological conditions.

Inhibitory effects of eucalyptus and banaba leaf extracts on nonalcoholic steatohepatitis induced by a high-fructose/high-glucose diet in rats.

Nonalcoholic steatohepatitis (NASH) is a liver disease associated with metabolic syndrome. The aim of this work was to examine whether eucalyptus (Eucalyptus globulus) leaf extract (ELE) and banaba (Lagerstroemia speciosa L.) leaf extract (BLE) inhibited NASH induced by excessive ingestion of fructose in rats. Wistar rats were divided into four groups according to four distinct diets: starch diet (ST), high-fructose/high-glucose diet (FG), FG diet supplemented with ELE, or FG diet supplemented with BLE. All rats were killed after 5 weeks of treatment. Serum alanine aminotransferase and total cholesterol levels were significantly lower in the BLE group than in the FG group. Liver histopathology, including steatosis, lipogranulomas, and perisinusoidal fibrosis, was significantly attenuated in the ELE and BLE groups compared with the FG group. Levels of 2-thiobarbituric acid reactive substances (TBARS), which reflect oxidative injury to the liver, were significantly suppressed by ELE and BLE. Western blotting analysis indicated that interleukin-6 expression levels were significantly lower in the ELE and BLE groups than in the FG group. These results suggest that ELE and BLE reduced lipogenesis, oxidative stress, and inflammatory cytokine expression and thus inhibited NASH induced by excessive ingestion of fructose in rats.

ß-Carotene Increases Muscle Mass and Hypertrophy in the Soleus Muscle in Mice.

Supplements and naturally occurring nutraceuticals effective for maintenance or enhancement of skeletal muscle mass are expected to contribute to prevention of decreased mobility and increased risk of developing metabolic diseases. However, information about available food components remains widely unavailable. In the present study, we investigated the effects of dietary ß-carotene on the quantity and quality of skeletal muscle under physiological conditions. Male ddY mice (8 wk old) were orally administered ß-carotene (0.5 mg once daily) for 14 d. Dietary ß-carotene had no influence on body weight, but increased the soleus muscle/body weight ratio. The cross-sectional area (CSA) in muscle fibers of the soleus muscle was increased, indicating that administration of ß-carotene induces muscle hypertrophy. In the soleus muscle of the ß-carotene-administered mice, twitch force tended to be increased (p=0.06) and tetanic force was significantly increased, whereas specific force (force per CSA) remained unchanged. Dietary ß-carotene increased the mRNA level of insulin-like growth factor 1 (Igf-1) as its splicing variant Igf-1ea, but had no influence on the liver Igf-1 mRNA level or serum IGF-1 level. ß-Carotene promoted protein synthesis in the soleus muscle and reduced levels of ubiquitin conjugates, but had no influence on the mRNA levels of two atrogenes, Atrogin-1 and Murf1. On the other hand, ß-carotene had no influence on the processing of the autophagy marker protein light chain 3. These results indicate that in mice, administration of ß-carotene increases mass and induces functional hypertrophy in the soleus muscle, perhaps by promoting IGF-1-mediated protein synthesis and by reducing ubiquitin-mediated protein degradation.

Resveratrol inhibits hypoxia-inducible factor-1α-mediated androgen receptor signaling and represses tumor progression in castration-resistant prostate cancer.

Androgen-dependent prostate cancer inevitably progresses to incurable castration-resistant prostate cancer (CRPC) after androgen deprivation therapy. Because castration-induced hypoxia-inducible factor (HIF)-1α enhances the transcriptional activity of androgen receptor (AR) at low androgen levels mimicking the castration-resistant stage, HIF-1α is expected to be a promising target for suppression of growth of CRPC. We investigated the effect of resveratrol (3,4',5-trihydroxy-trans-stilbene) on the growth of human prostate cancer LNCaP xenografts in castrated male BALB/cSlc-nu/nu mice (5 wk old). The mice were administered a control diet or a resveratrol diet (4 g/kg diet) for 40 d. The resveratrol diet significantly suppressed tumor growth compared to the control diet. In LNCaP xenografts, dietary resveratrol decreased the protein level of HIF-1α, but not the AR coactivator ß-catenin, and reduced the mRNA levels of androgen-responsive genes. In the control group, ß-catenin was predominantly localized in the nucleus with HIF-1α in LNCaP xenografts, whereas dietary resveratrol inhibited the nuclear accumulation of ß-catenin. In hypoxic LNCaP cells at a low androgen level mimicking the castration-resistant stage, hypoxia-induced nuclear accumulation of ß-catenin was inhibited by resveratrol. Furthermore, resveratrol repressed the expression level of HIF-1α even in the presence of a proteasome inhibitor and suppressed hypoxia-enhanced AR transactivation. These results indicate that dietary resveratrol represses nuclear localization of ß-catenin by decreasing the HIF-1α expression, perhaps in a proteasome-independent manner, and inhibits ß-catenin-mediated AR signaling; this contributes to suppression of tumor growth of CRPC.

S-equol enantioselectively activates cAMP-protein kinase A signaling and reduces alloxan-induced cell death in INS-1 pancreatic ß-cells.

S-Equol is enantioselectively produced from the isoflavone daidzein by gut microflora and is absorbed by the body. An increase of pancreatic ß-cell death is directly associated with defects in insulin secretion and an increased risk of type 2 diabetes mellitus. In the present study, we demonstrate that only the S-enantiomer has suppressive effects against alloxan-induced oxidative stress in INS-1 pancreatic ß-cells. S-Equol reduced alloxan-induced cell death in a dose-dependent manner, whereas R-equol had no effects. In contrast, no significant differences were observed between the enantiomers in estrogenic activity. The cytoprotective effects of S-equol were stronger than those of its precursor daidzein and were blocked by the protein synthesis inhibitor cycloheximide. The cytoprotection was diminished when cells were incubated with a protein kinase A (PKA) inhibitor (H89), but not an estrogen receptor inhibitor. S-Equol increased intracellular cAMP levels in an enantioselective manner. S-Equol, but not R-equol, induced phosphorylation of cAMP-response element-binding protein at Ser 133, and induced cAMP-response element-mediated transcription, both of which were diminished in the presence of H89. Taken together, these results show that S-equol enantioselectively increases the survival of INS-1 cells presumably through activating PKA signaling. Thus, S-equol might have applications as an anti-type 2 diabetic agent.

Resveratrol reduces the hypoxia-induced resistance to doxorubicin in breast cancer cells.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is known to enhance the cytotoxicity of the anticancer drug doxorubicin. On the other hand, breast cancer MCF-7 cells acquire resistance to doxorubicin under hypoxic conditions. In this study, we investigated the effect of resveratrol on hypoxia-induced resistance to doxorubicin in MCF-7 cells. Resveratrol and its derivative 3,5-dihydroxy-4'-methoxy-trans-stilbene, but not 3,5-dimethoxy-4'-hydroxy-trans-stilbene, cancelled hypoxia-induced resistance to doxorubicin at a concentration of 10 µM. Carbonyl reductase 1 (CBR1) catalyzes the conversion of doxorubicin to its metabolite doxorubicinol, which is much less effective than doxorubicin. Hypoxia increased the expression of CBR1 at both mRNA and protein levels, and knockdown of CBR1 inhibited hypoxia-induced resistance to doxorubicin in MCF-7 cells. Knockdown of hypoxia-inducible factor (HIF)-1α repressed the hypoxia-induced expression of CBR1. Resveratrol repressed the expression of HIF-1α protein, but not HIF-1α mRNA, and decreased hypoxia-activated HIF-1 activity. Resveratrol repressed the hypoxia-induced expression of CBR1 at both mRNA and protein levels. Likewise, 3,5-dihydroxy-4'-methoxy-trans-stilbene decreased the hypoxia-induced expression of CBR1 protein, but not 3,5-dimethoxy-4'-hydroxy-trans-stilbene. Furthermore, resveratrol decreased the expression of HIF-1α protein even in the presence of the proteasome inhibitor MG132 in hypoxia. Theses results indicate that in MCF-7 cells, HIF-1α-increased CBR1 expression plays an important role in hypoxia-induced resistance to doxorubicin and that resveratrol and 3,5-dihydroxy-4'-methoxy-trans-stilbene decrease CBR1 expression by decreasing HIF-1α protein expression, perhaps through a proteasome-independent pathway, and consequently repress hypoxia-induced resistance to doxorubicin.

Identification of carbonyl reductase 1 as a resveratrol-binding protein by affinity chromatography using 4'-amino-3,5-dihydroxy-trans-stilbene.

The mechanisms by which resveratrol (3,4',5-trihydroxy-trans-stilbene) elicits diverse health benefits remain unclear because the intracellular target molecules of resveratrol are poorly defined. We screened resveratrol-binding proteins from lysates of MCF-7 breast cancer cells using resveratrol-affinity resin, which was constructed by immobilizing 4'-amino-3,5-dihydroxy-trans-stilbene on activated CH-Sepharose. On SDS-PAGE, two bands were detected as proteins that specifically bound to the resveratrol-affinity resin. One of these, a 30-kDa protein, was identified as human carbonyl reductase 1 (CBR1) by hybrid linear ion trap/time-of-flight mass spectrometry. Similarly, recombinant CBR1 bound to the resveratrol-affinity resin in the absence of resveratrol, but not in the presence of resveratrol. Among its activities, CBR1 catalyzes a NADPH-dependent reduction of the anticancer drug doxorubicin to the cardiotoxin doxorubicinol. The effects of doxorubicin on viability of MCF-7 cells were enhanced by resveratrol, 3,5-dihydroxy-4'-methoxy-trans-stilbene, 3,4'-dihydroxy-5-methoxy-trans-stilbene, and 4'-amino-3,5-dihydroxy-trans-stilbene at concentrations of 1 and 10 µM. Resveratrol and these derivatives inhibited CBR1 activities to a similar degree at concentrations of 100 and 200 µM. However, 3,5-dimethoxy-4'-hydroxy-trans-stilbene and m-hydroquinone had no influence on doxorubicin cytotoxicity or CBR1 activity. Resveratrol inhibited CBR1 activity through an apparent mix of competitive (Ki=55.8 µM) and noncompetitive (αKi=164 µM; α=2.98) inhibition kinetics. These results indicate that (i) resveratrol enhances the cytotoxic effects of doxorubicin on MCF-7 cells; (ii) the moiety that contains the 3,5-dihydroxyl groups of resveratrol, but not the m-hydroquinone structure alone, is required to bind CBR1; and (iii) resveratrol acts as a mixed-type inhibitor of CBR1 activity on doxorubicin.

Identification of a checkpoint modulator with synthetic lethality to p53 mutants.

The G(2) checkpoint is an indispensable pathway for cancers lacking p53 function, for delaying cell cycle progression, and for completing DNA repair. Therefore, disruption of this pathway is expected to offer selective therapy for these highly prevalent cancers. The aim of this study was to identify an inhibitor of the G(2) checkpoint including the ataxia-telangiectasia-mutated and Rad3-related checkpoint kinase 1 pathway that selectively suppresses the growth of p53-deficient cells. To obtain molecules with a novel mechanism of action, we constructed a high-throughput screening system that detected abrogation of the G(2) checkpoint in X-irradiated HT-29 cells. The screening resulted in identification of a guanidine analog, CBP-93872 that dose dependently inhibited the G(2) checkpoint induced by DNA damage. Interestingly, CBP-93872 directly suppressed the growth of p53-mutated cancer cell lines with wild-type CDKN2A by eliciting G(1) arrest, but not CDKN2A-deleted and/or wild-type p53 lines. CBP-93872 decreased phospho-cdc2 Y15 by inhibiting phosphorylation of Chk1, but did not suppress phospho-Chk2 or the kinase activities of either Chk1 or Chk2 in cellular or cell-free assays. These results suggest that a checkpoint modulator through suppression of Chk1 phosphorylation provides synthetic lethality to p53-deficient cells.

A yeast bioassay for androgenic and anti-androgenic compounds based on the NH2- and COOH-terminal interaction of androgen receptor.

Androgenic compounds induce an interaction between the NH(2)- and COOH-terminal regions (N-C interaction) of androgen receptor (AR). We describe a rapid yeast bioassay for androgenic and anti-androgenic compounds based on androgen-dependent ß-catenin-enhanced N-C interaction. The bioassay was also effective at detecting compounds that inhibit the N-C interaction in ways that do not involve binding to the ligand-binding domain.

Biodegradation of diphenylarsinic acid to arsenic acid by novel soil bacteria isolated from contaminated soil.

Microorganisms capable of degrading diphenylarsinic acid (DPAA) were enriched from contaminated soil using the soil-charcoal perfusion method. Two novel bacterial strains, L2406 and L2413, that can degrade DPAA in a mineral salt medium supplemented with DPAA as the sole carbon source were isolated. Based on comparative morphology, physiology, and comparison of the 16S rRNA gene sequences, both were presumed to be species closely related to Ensifer adhaerens. As the metabolites, phenylarsonic acid (PAA) was determined by liquid chromatography-mass spectrometry analysis as well as three unknown peaks all of whose molecular weights were estimated to be 278. The increase of m/z = 16 from DPAA in the unknowns suggests monohydroxylation of DPAA at the 2-, 3- and 4-positions. The ability of strains L2406 and L2413 to degrade DPAA was suppressed in iron insufficient conditions, e.g. less than 7.2 muM iron in the culture medium. These facts strongly suggest the following hypothesis: Monooxygenase works at the initial degradation step of DPAA degradation by the isolates; and direct hydrolysis from DPAA to PAA is not likely to occur. In addition, release of arsenic acid from PAA by strain L2406 was confirmed by liquid chromatography-inductively coupled plasma mass spectrometry. From these results, strain L2406 was considered to be capable of degrading DPAA to arsenic acid via PAA when DPAA was supplied as the sole carbon source.

Cobalamin deficiency results in an abnormal increase in L-methylmalonyl-co-enzyme-A mutase expression in rat liver and COS-7 cells.

The aim of the present study was to examine the effects of cobalamin (Cbl) on the activity and expression of L-methylmalonyl-CoA mutase (MCM) in rat liver and cultured COS-7 cells. The MCM holoenzyme activity was less than 5% of the total (holoenzyme+apoenzyme) activity in the liver although rats were fed a diet containing sufficient Cbl. When weanling rats were maintained on a Cbl-deficient diet, the holo-MCM activity became almost undetectable at the age of 10 weeks. In contrast, a marked increase in the total-MCM activity occurred under the Cbl-deficient conditions, and at the age of 20 weeks it was about 3-fold higher in the deficient rats than in the controls (108 (SD 14.5) v. 35 (SD 8.5) nmol/mg protein per min (n 5); P<0.05). Western blot analysis confirmed that the MCM protein level increased significantly in the Cbl-deficient rats. However, the MCM mRNA level, determined by real-time PCR, was rather decreased. When COS-7 cells were cultured in a medium in which 10% fetal bovine serum was the sole source of Cbl, holo-MCM activity was barely detected. The supplementation of Cbl resulted in a large increase in the holo-MCM activity in the cells, but the activity did not exceed 30% of the total-MCM activity even in the presence of Cbl at 10 micromol/l. In contrast, the total-MCM activity was significantly decreased by the Cbl supplementation, indicating that Cbl deficiency results in an increase in the MCM protein level in COS-7 cells as well as in rat liver.