Consumption of Lactobacillus acidophilus and Bifidobacterium longum does not alter phytoestrogen metabolism and plasma hormones in men: a pilot study.

OBJECTIVE: The aim of this study was to determine whether equol excretion status and plasma hormone and leptin concentrations can be influenced by consumption of a probiotic supplement. A secondary focus was to investigate whether male equol excretors have a hormone profile consistent with reduced prostate cancer risk. DESIGN: The design was a randomized, single-blinded, placebo-controlled, parallel-arm trial. SUBJECTS: Thirty-one (31) of the initially enrolled 39 subjects, 18 to 37 years old, completed all study requirements. INTERVENTION: Subjects consumed either probiotic capsules (containing Lactobacillus acidophilus and Bifidobacterium longum) or placebo capsules for 2 months. Fasting plasma concentrations of testosterone (T), dihydrotestosterone (DHT), androstanediol glucuronide (AAG), androstenedione (A), dehydroepiandrosterone sulfate (DHEAS), sex hormone-binding globulin (SHBG), and leptin were measured on days 1 and 57. Urinary excretion of genistein, glycitein, daidzein, O-desmethylangolensin (O-Dma), and equol was measured on days 4 and 61 following a 4-day soy challenge. RESULTS: Probiotic consumption did not significantly alter equol excretor status, plasma hormone, or leptin concentrations in these subjects. At baseline, there were no differences in plasma hormone concentrations between equol excretors and nonexcretors; however, the low number of equol excretors included in this study limits the strength of this finding. CONCLUSIONS: The 2-month intervention with probiotic capsules did not significantly alter equol excretion, plasma hormone, or leptin concentrations in these subjects. A secondary finding was that male equol excretors in this study did not exhibit a hormone profile consistent with reduced prostate cancer risk, although this result should be interpreted with caution.

Organization and regulation of the human rasGAP gene.

ras GTPase activating protein (rasGAP) is highly conserved among mammalian species and is required for normal cardiovascular system development. Expression of this protein exhibits both quantitative and qualitative variability among tissues. Using a combination of DNA sequencing and database analyses, we have determined that the human rasGAP gene spans 122 kb and is composed of 25 exons; the size of each intron and the intron/exon junctions also have been elucidated. With one exception, all intron/exon boundaries conform to the GT/AG rule; the splice donor site of intron 3 is GC/AG. Results of RNA ligase mediated rapid amplification of cDNA ends followed by sequence determination indicate that the transcription start point (TSP) is approximately 588 bp upstream from the translational start site and is uninterrupted by introns; this extremely long 5' untranslated region is continuous with the first coding exon. Analysis of 1 kb of sequence upstream of the TSP did not identify any of the typical promoter elements (TATA or CAAT boxes). Sequential deletions of this 1 kb region followed by secreted alkaline phosphatase reporter gene analysis revealed that transcription is supported by this region of the rasGAP gene. Because the highest efficiency is demonstrated by a 213 bp sequence just upstream from the TSP (-786 to -584), this region is identified as containing the rasGAP minimal promoter. Sequence analysis of this 213 bp sequence shows few candidate sites for transcription factor binding. A 406 bp fragment surrounding the TSP exhibits characteristics of a CpG island (68% C+G; observed/expected ratio of CpG=0.95). RapidScan analysis revealed that high levels of rasGAP transcript are present in placenta and testis, but transcript is not detectable in kidney and intestinal tract. These data suggest that rasGAP transcription is regulated by an atypical mechanism capable of producing quantitative variability among tissue types.

Vitamin A and its derivatives induce hepatic glycine N-methyltransferase and hypomethylation of DNA in rats.

Regulation of S-adenosylmethionine (SAM) and the SAM/S-adenosylhomocysteine (SAH) ratio by the key cytosolic enzyme glycine N-methyltransferase (GNMT) is essential in optimizing methyl group supply and subsequent functioning of methyltransferase enzymes. Therefore, inappropriate activation of GNMT may lead to the loss of methyl groups vital for many SAM-dependent transmethylation reactions. Previously, we demonstrated that the retinoid derivatives 13-cis- (CRA) and all-trans-retinoic acid (ATRA) mediated both the activity of GNMT and its abundance. The present study was conducted to determine whether vitamin A had a similar ability to up-regulate GNMT and to assess the biological importance of GNMT modulation by examining both the transmethylation and transsulfuration pathways after retinoid treatment. Rats were fed a control (10% casein + 0.3% L-methionine) diet and orally given retinyl palmitate (RP), CRA, ATRA or vehicle daily for 10 d. RP, CRA and ATRA elevated hepatic GNMT activity 32, 74 and 124%, respectively, compared with the control group. Moreover, the retinoid-mediated changes in GNMT activity were reflected in GNMT abundance (38, 89 and 107% increases for RP-, CRA-, and ATRA-treated rats, respectively). In addition, hepatic DNA, a substrate for SAM-dependent transmethylation, was hypomethylated (approximately 100%) after ATRA treatment compared with the control group. In contrast, the transsulfuration product glutathione was unaffected by retinoid treatment. These results provide evidence of the following: 1) vitamin A, like its retinoic acid derivatives, can induce enzymatically active GNMT; and 2) inappropriate induction of GNMT can lead to a biologically important loss of methyl groups and the subsequent impairment of essential transmethylation processes.

Activation and induction of glycine N-methyltransferase by retinoids are tissue- and gender-specific.

Glycine N-methyltransferase (GNMT) is a key protein in the liver that functions to regulate S-adenosylmethionine (SAM) and the SAM/S-adenosylhomocysteine ratio. Significant GNMT expression is also present in the kidney and pancreas. Inappropriate regulation of GNMT may have negative consequences on methyl group and folate metabolism. We have demonstrated that retinoid compounds significantly elevated hepatic GNMT activity and abundance (approximately 2-fold) in male rats. However, pancreatic GNMT activity and abundance were not altered by retinoid treatment. Likewise, retinoid administration was without effect on renal GNMT activity. Hepatic GNMT activity was also elevated in female rats treated with all-trans-retinoic acid, but to a lesser extent compared to males. Collectively, these results indicate that the modulation of methyl group metabolism by retinoids is tissue- and gender-specific, and may compromise the availability of methyl groups for SAM-dependent transmethylation reactions. In support of this, SAM-dependent synthesis of creatinine was significantly reduced 21% following all-trans-retinoic acid treatment.

Hepatic glycine N-methyltransferase is up-regulated by excess dietary methionine in rats.

Glycine N-methyltransferase (GNMT) regulates S-adenosylmethionine (SAM) levels and the ratio of SAM:S-adenosylhomocysteine (SAH). In liver, methionine availability, both from the diet and via the folate-dependent one-carbon pool, modulates GNMT activity to maintain an optimal SAM:SAH ratio. The regulation of GNMT activity is accomplished via posttranslational and allosteric mechanisms. We more closely examined GNMT regulation in various tissues as a function of excess dietary methyl groups. Sprague Dawley rats were fed either a control diet (10% casein plus 0.3% L-methionine) or the control diet supplemented with graded levels (0.5-2%) of L-methionine. Pair-fed control groups of rats were included due to the toxicity associated with high methionine consumption. As expected, the hepatic activity of GNMT was significantly elevated in a dose-dependent fashion after 10 d of feeding the diets containing excess methionine. Moreover, the abundance of hepatic GNMT protein was similarly increased. The kidney had a significant increase in GNMT as a function of dietary methionine, but to a much lesser extent than in the liver. For pancreatic tissue, neither the activity of GNMT nor the abundance of the protein was responsive to excess dietary methionine. These data suggest that additional mechanisms contribute to regulation of GNMT such that synthesis of the protein is greater than its degradation. In addition, methionine-induced regulation of GNMT is dose dependent and appears to be tissue specific, the latter suggesting that the role it plays in the kidney and pancreas may in part differ from its hepatic function.