Resveratrol potentiates effects of simvastatin on inhibition of rat ovarian theca-interstitial cells steroidogenesis.

BACKGROUND: Polycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, hyperplastic theca compartment and increased androgen production due to, at least in part, excessive expression of several key genes involved in steroidogenesis. Previously, our group has demonstrated that simvastatin, competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a rate-limiting step of the mevalonate pathway, reduces rat-theca interstitial cell steroidogenesis by inhibiting Cyp17a1 gene expression, the key enzyme of the androgen biosynthesis pathway. Recently, we demonstrated that resveratrol, a bioflavonoid abundant in red grapes, decreases rat theca-interstitial cell steroidogenesis and this suppressive effect is mediated through mechanisms independent of the mevalonate pathway. The present study evaluated the effect of combining simvastatin and resveratrol treatments on rat theca-interstitial cell steroidogenesis. METHODS: Rat theca-interstitial cells isolated from 30 day-old female rats were cultured for up to 48 h with or without simvastatin (1 µM) and/or resveratrol (3-10 µM). Steroidogenic enzymes gene expression was evaluated by quantitative real time PCR and steroid levels were measured by liquid chromatography-mass spectrometry. Comparisons between groups were performed using ANOVA and Tukey test. RESULTS: Resveratrol potentiated inhibitory effects of simvastatin on androstenedione and androsterone production in theca-interstitial cells. This suppressive effect correlated with profound inhibition in Cyp17a1 mRNA expression in the presence of a combination of resveratrol and simvastatin. CONCLUSIONS: The present findings indicate that resveratrol potentiates the simvastatin-induced inhibitory effect on theca-interstitial cell androgen production, raising the possibility of development of novel treatments of PCOS.

Letrozole increases ovarian growth and Cyp17a1 gene expression in the rat ovary.

OBJECTIVE: To evaluate the effects of letrozole on ovarian size and steroidogenesis in vivo, as well as on proliferation and steroidogenesis of theca-interstitial cells alone and in coculture with granulosa cells using an in vitro model. DESIGN: In vivo and in vitro studies. SETTING: Research laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): In vivo effects of letrozole were studied in intact rats receiving either letrozole (90-day continuous-release SC pellets, 400 µg/d) or placebo pellets (control group). In in vitro experiments, theca cells were cultured alone or in coculture with granulosa cells in the absence or presence of letrozole. MAIN OUTCOME MEASURE(S): Deoxyribonucleic acid synthesis was determined by thymidine incorporation assay; steroidogenesis by mass spectrometry; and steroidogenic enzyme messenger RNA (mRNA) expression by polymerase chain reaction. RESULT(S): In vivo, letrozole induced an increase in ovarian size compared with the control group and also induced a profound increase of androgen, LH levels, and Cyp17a1 mRNA expression. Conversely, a decrease in Star, Cyp11a1, and Hsd3b1 transcripts was observed in letrozole-exposed rats. In vitro, letrozole did not alter either theca cell proliferation or Cyp17a1 mRNA expression. Similarly, letrozole did not affect Cyp17a1 transcripts in granulosa-theca cocultures. CONCLUSION(S): These findings suggest that letrozole exerts potent, but indirect, effect on growth of rat ovary and dramatically increases androgen levels and Cyp17a1 mRNA expression, the key enzyme regulating the androgen biosynthesis pathway. The present findings reveal novel mechanisms of action of letrozole in the rat ovary.

Effects of resveratrol on growth and function of rat ovarian granulosa cells.

OBJECTIVE: To evaluate the effects of resveratrol on growth and function of granulosa cells. Previously, we demonstrated that resveratrol exerts profound proapoptotic effects on theca-interstitial cells. DESIGN: In vitro study. SETTING: Research laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Granulosa cells were cultured in the absence or presence of resveratrol. MAIN OUTCOME MEASURE(S): DNA synthesis was determined by thymidine incorporation assay, apoptosis by activity of caspases 3/7, cell morphology by immunocytochemistry, steroidogenesis by mass spectrometry, antimüllerian hormone (AMH), and vascular endothelial growth factor (VEGF) expression by polymerase chain reaction and Western blot. RESULT(S): Resveratrol induced a biphasic effect on DNA synthesis, whereby a lower concentration stimulated thymidine incorporation and higher concentrations inhibited it. Additionally, resveratrol slightly increased the cell number and modestly decreased the activity of caspases 3/7 with no effect on cell morphology or progesterone production. However, resveratrol decreased aromatization and VEGF expression, whereas AMH expression remained unaltered. CONCLUSION(S): Resveratrol, by exerting cytostatic but not cytotoxic effects, together with antiangiogenic actions mediated by decreased VEGF in granulosa cells, may alter the ratio of theca-to-granulosa cells and decrease vascular permeability, and therefore may be of potential therapeutic use in conditions associated with highly vascularized theca-interstitial hyperplasia and abnormal angiogenesis, such as those seen in women with polycystic ovary syndrome.

Resveratrol reduces steroidogenesis in rat ovarian theca-interstitial cells: the role of inhibition of Akt/PKB signaling pathway.

Polycystic ovary syndrome is characterized by theca-interstitial hyperplasia and increased expression of steroidogenic genes, leading to excessive androgen production. Resveratrol, a natural polyphenol, promotes apoptosis and reduces rat theca-interstitial cell growth, in part by inhibiting the mevalonate pathway and decreasing the availability of substrates of isoprenylation [farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP)]. This study evaluated the effect of resveratrol on rat theca-interstitial cell steroidogenesis. Because resveratrol may activate sirtuins, this study also investigated whether steroidogenesis was affected by sirtuin inhibitors (nicotinamide, sirtinol). Theca-interstitial cells were cultured with or without resveratrol (1-10 µm), GGPP (30 µm), FPP (30 µm), nicotinamide (1 mm), and/or sirtinol (10 µm). Resveratrol did not affect progesterone levels but reduced androgen production in a concentration-dependent fashion (androstenedione by up to 78% and androsterone by up to 76%). This inhibitory effect correlated with a decrease in mRNA expression of genes regulating androgen production, especially Cyp17a1 (by up to 73%). GGPP and FPP had no effect on androgen levels and Cyp17a1 mRNA levels and did not alter the effects induced by resveratrol. Similarly, sirtuin inhibitors did not reverse resveratrol-induced inhibition of steroidogenesis. However, resveratrol decreased activity of serine-threonine kinase/protein kinase B pathway, a cell-signaling pathway involved in ovarian steroidogenesis. The present findings indicate that resveratrol reduces androgen production primarily by inhibiting Cyp17a1 mRNA expression, and this inhibition may be mediated, in part, by blocking the activity of the serine-threonine kinase/protein kinase B pathway. These findings may be of clinical relevance to conditions associated with excessive production of androgens by theca cells, such as polycystic ovary syndrome.

Simvastatin reduces steroidogenesis by inhibiting Cyp17a1 gene expression in rat ovarian theca-interstitial cells.

Polycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, theca-interstitial hyperplasia, and increased androgen production by theca cells. Previously, our group has demonstrated that statins (competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, a rate-limiting step of the mevalonate pathway) reduce proliferation of theca-interstitial cells in vitro and decrease serum androgen levels in women with PCOS. The present study evaluated the effect of simvastatin on rat ovarian theca-interstitial cell steroidogenesis. Because actions of statins may be due to reduced cholesterol availability and/or isoprenylation of proteins, the present study also investigated whether steroidogenesis was affected by cell- and mitochondrion-permeable 22-hydroxycholesterol, isoprenylation substrates (farnesyl-pyrophosphate [FPP] and geranylgeranyl-pyrophosphate [GGPP]), as well as selective inhibitors of farnesyltransferase (FTI) and geranylgeranyltransferase (GGTI). Theca-interstitial cells were cultured for 12, 24, and 48 h with or without simvastatin, GGPP, FPP, FTI, GGTI, and/or 22-hydroxycholesterol. Simvastatin decreased androgen levels in a time- and concentration-dependent fashion. This inhibitory effect correlated with a decrease in mRNA levels of Cyp17a1, the gene encoding the key enzyme regulating androgen biosynthesis. After 48 h, GGPP alone and FPP alone had no effect on Cyp17a1 mRNA expression; however, the inhibitory action of simvastatin was partly abrogated by both GGPP and FPP. The present findings indicate that statin-induced reduction of androgen levels is likely due, at least in part, to the inhibition of isoprenylation, resulting in decreased expression of CYP17A1.

Resveratrol inhibits the mevalonate pathway and potentiates the antiproliferative effects of simvastatin in rat theca-interstitial cells.

OBJECTIVE: To examine the mechanisms of action of resveratrol and its interaction with simvastatin on growth and the mevalonate pathway in rat theca-interstitial cells. DESIGN: In vitro study. SETTING: Research laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Theca-interstitial cells were cultured in the absence or presence of resveratrol, simvastatin, mevalonic acid, farnesyl pyrophosphate, and/or geranylgeranyl pyrophosphate. MAIN OUTCOME MEASURE(S): DNA synthesis was assessed by thymidine incorporation assay; 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) expression and activity were evaluated with the use of quantitative real-time polymerase chain reaction, Western blot analysis, and HMGCR activity assay. Cholesterol synthesis was determined by the conversion of [(14)C]-acetate to [(14)C]-cholesterol. RESULT(S): Resveratrol potentiated the simvastatin-induced inhibition on cell proliferation in a concentration-dependent manner. Inhibitory effects of resveratrol were partly abrogated by the addition of mevalonic acid, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. Resveratrol reduced HMGCR expression and activity, and decreased cholesterol synthesis. In contrast, simvastatin inhibited HMGCR activity with a compensatory increase in HMGCR expression. Resveratrol counteracted this effect of simvastatin on HMGCR expression but augmented the simvastatin-induced inhibition on HMGCR activity and cholesterol synthesis. CONCLUSION(S): Resveratrol inhibits the mevalonate pathway via distinctly different mechanisms than statins. These observations demonstrate a novel mechanism of action of resveratrol and underscore the potential translational/clinical relevance of resveratrol interactions with simvastatin.

Simvastatin induces apoptosis and alters cytoskeleton in endometrial stromal cells.

CONTEXT: Statins are competitive inhibitors of 3-hydroxy-3methylglutaryl-coenzyme A reductase, with antimitotic, antioxidant, antiinflammatory, and immunomodulatory properties. Recent studies have shown that statins reduce the growth of human endometrial stromal (HES) cells and protect from the development of endometriosis in animal models. OBJECTIVES: The present study was conducted to evaluate the effects of simvastatin on apoptosis and cytoskeleton of HES cells. DESIGN AND SETTING: In vitro experiments were performed in the university research laboratory. PATIENTS: HES cells were obtained from endometrial biopsies collected from nine subjects in the proliferative phase of their menstrual cycle. MAIN OUTCOME MEASURES: The effect of simvastatin (10 and 30 mum) and/or geranylgeranyl pyrophosphate (GGPP, 30 mum) on caspase 3 and 7 activity, DNA fragmentation, and HES cell morphology was evaluated. RESULTS: Simvastatin induced significant time- and concentration-dependent apoptotic effects on HES cells as determined by increased activity of executioner caspases and DNA fragmentation. Simvastatin also caused profound alterations in HES cell morphology and F-actin cytoskeleton. This effect was abrogated by geranylgeranyl pyrophosphate, an important product of the mevalonate pathway. CONCLUSIONS: Simvastatin induces apoptosis and disruption of the cytoskeleton of HES cells by reducing isoprenylation in cultures of human endometrial stroma. The present findings may lead to the development of novel treatments for endometriosis involving statins.

Effects of resveratrol on proliferation and apoptosis in rat ovarian theca-interstitial cells.

Polycystic ovary syndrome (PCOS) is characterized by ovarian dysfunction and associated with ovarian theca-interstitial (T-I) cell hyperplasia, hyperinsulinemia, systemic inflammation and oxidative stress. This in vitro study tested whether rat T-I cell growth with or without insulin can be altered by resveratrol, a natural polyphenol with anti-carcinogenic, anti-inflammatory, anti-proliferative and antioxidant properties. Rat T-I cells were cultured with and without resveratrol and/or insulin, and the effects on DNA synthesis, number of viable cells and markers of apoptosis were evaluated. Resveratrol alone induced a potent concentration-dependent inhibition of cell growth by inhibiting DNA synthesis, decreasing the number of viable cells and increasing the activity of executioner caspases 3 and 7; these effects of resveratrol counteracted the pro-proliferative and anti-apoptotic effects of insulin. Immunofluorescence analysis of cells incubated with resveratrol showed concentration- and time-dependent morphological changes consistent with apoptosis. The present findings indicate that resveratrol promotes apoptosis to reduce rat T-I cell growth in vitro as well as inhibiting insulin-induced rat T-I cell growth. This suggests a possibility that resveratrol and/or mechanisms mediating its effect may be relevant to the development of novel treatments for PCOS, which is characterized by both excessive ovarian mesenchyma growth and hyperinsulinemia.

Epigenetic regulation of hepatic endoplasmic reticulum stress pathways in the ethanol-fed cystathionine beta synthase-deficient mouse.

UNLABELLED: We tested the hypothesis that the pathogenesis of alcoholic liver injury is mediated by epigenetic changes in regulatory genes that result from the induction of aberrant methionine metabolism by ethanol feeding. Five-month-old cystathionine beta synthase heterozygous and wild-type C57BL/6J littermate mice were fed liquid control or ethanol diets by intragastric infusion for 4 weeks. Both ethanol-fed groups showed typical histopathology of alcoholic steatohepatitis, with reduction in liver S-adenosylmethionine (SAM), elevation in liver S-adenosylhomocysteine (SAH), and reduction in the SAM/SAH ratio with interactions of ethanol and genotype effects. Hepatic endoplasmic reticulum stress signals including glucose-regulated protein-78 (GRP78), activating transcription factor 4, growth arrest and DNA damage-inducible gene 153 (GADD153), caspase 12, and transcription factor sterol response element binding protein-1c (SREBP-1c) were up-regulated in ethanol-fed mice with genotype interactions and negative correlations with the SAM/SAH ratio. Immunohistochemical staining showed reduction in trimethylated histone H3 lysine-9 (3meH3K9) protein levels in centrilobular regions in both ethanol groups, with no changes in trimethylated histone H3 lysine-4 levels. The chromatin immunoprecipitation assay revealed a decrease in levels of suppressor chromatin marker 3meH3K9 in the promoter regions of GRP78, SREBP-1c, and GADD153 in ethanol-treated heterozygous cystathionine beta synthase mice. The messenger RNA expression of the histone H3K9 methyltransferase EHMT2 (G9a) was selectively decreased in ethanol-fed mice. CONCLUSION: The pathogenesis of alcoholic steatohepatitis is mediated in part through the effects of altered methionine metabolism on epigenetic regulation of pathways of endoplasmic reticulum stress relating to apoptosis and lipogenesis.

Role of isoprenylation in simvastatin-induced inhibition of ovarian theca-interstitial growth in the rat.

Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate-limiting step of the mevalonate pathway. The pleiotropic effects of statins may be due to inhibition of cholesterol synthesis, as well as decreased availability of several biologically important intermediate components of the mevalonate pathway, including two substrates for isoprenylation (farnesyl pyrophosphate [FPP] and geranylgeranyl pyrophosphate [GGPP]). Recently, we demonstrated statin-induced inhibition of ovarian theca-interstitial cell proliferation in vitro, as well as reduction of testosterone levels in women with polycystic ovary syndrome (PCOS). This study evaluates the relative contribution of inhibition of isoprenylation and/or cholesterol availability to the modulation of theca-interstitial proliferation. Rat theca-interstitial cells were cultured in chemically defined media with or without simvastatin, FPP, GGPP, squalene, and/or two membrane-permeable forms of cholesterol (25-hydroxycholesterol and 22-hydroxycholesterol). Simvastatin inhibited DNA synthesis and the count of viable cells. The effects of simvastatin were partly abrogated by FPP and GGPP but not by squalene or cholesterol. Inhibition of farnesyl transferase and geranylgeranyl transferase reduced cell proliferation. The present findings indicate that simvastatin inhibits proliferation of theca-interstitial cells, at least in part, by reduction of isoprenylation. These observations provide likely mechanisms explaining clinically observed improvement of ovarian hyperandrogenism in women with PCOS.

Interactive effects of COMT Val108/158Met and MTHFR C677T on executive function in schizophrenia.

Schizophrenia is characterized by heritable deficits in executive function. Two common, functional polymorphisms, catechol-O-methyltransferase (COMT) Val108/158Met and methylenetetrahydrofolate reductase (MTHFR) C677T, have separately been associated with executive function performance in schizophrenia. Given the closely related biochemistry of MTHFR and COMT, it is plausible that the T and Val alleles act synergistically to impair executive function. This investigation of 185 outpatients with schizophrenia examined the interactive effects of these two polymorphisms on Wisconsin Card Sorting Task (WCST) performance. Two WCST measures consistently associated with schizophrenia, perseverative errors and inability to generate categories, were contrasted among compound COMT-MTHFR genotype groups. Individuals homozygous for the COMT Val allele who also carried at least one copy of the MTHFR T allele exhibited a significantly higher percentage of perseverative errors than patients in the other genotype groups. While the T allele also exerted a negative effect on category generation, COMT genotype did not contribute to category performance. It is plausible that cumulative effects of the MTHFR T and COMT Val alleles on intracellular methylation profiles and prefrontal dopamine transmission underlie their interactive effect on perseverative errors.

Contribution of methylenetetrahydrofolate reductase (MTHFR) polymorphisms to negative symptoms in schizophrenia.

BACKGROUND: Folate deficiency may contribute to negative symptoms in schizophrenia, but the underlying mechanism remains uncertain. We examined whether the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C functional polymorphisms contribute to negative symptoms. METHODS: Outpatients with schizophrenia (n = 200) were evaluated with the Positive and Negative Syndrome Scale (PANSS). Subjects also provided a blood sample for MTHFR genotype and serum chemistries. Comparisons of PANSS symptoms, folate, and homocysteine status were conducted based on genotype. RESULTS: The 677T allele load was associated with negative symptom severity. Contrary to our expectations, the T allele was also found to be protective against positive symptoms. The A1298C polymorphism did not contribute to negative symptoms, and only weakly to positive symptoms. The specific effects of the C677T polymorphism were confirmed with haplotype analysis. Among patients homozygous for the 667T allele, serum folate levels correlated with negative symptom severity. CONCLUSIONS: Increased MTHFR 677T allele load confers risk for negative symptoms in schizophrenia, while reducing severity of positive symptoms. Further, the biochemical interaction of low serum folate with 677T-variant MTHFR may induce downstream effects salient to the expression of negative symptoms.

Relations of glutamate carboxypeptidase II (GCPII) polymorphisms to folate and homocysteine concentrations and to scores of cognition, anxiety, and depression in a homogeneous Norwegian population: the Hordaland Homocysteine Study.

BACKGROUND: Glutamate carboxypeptidase II (GCPII) encodes for intestinal folate hydrolase and brain N-acetylated alpha-linked acidic dipeptidase. Previous studies provided conflicting results on the effect of the GCPII 1561C-->T polymorphism on folate and total homocysteine (tHcy) concentrations. OBJECTIVE: We aimed to determine the potential effects of 2 polymorphisms of GCPII on plasma folate and tHcy concentrations, cognition, anxiety, and depression in a large aging cohort of Norwegians enrolled in the Hordaland Homocysteine Study. DESIGN: DNA samples were genotyped for the GCPII 1561C-->T and 484A-->G polymorphisms, and the results were linked to plasma folate and tHcy concentrations and to scores for cognition, anxiety, and depression. RESULTS: The 2 polymorphisms were in linkage disequilibrium and were associated with concentrations of tHcy. After adjustment for covariates, persons in the CT or combined CT and TT groups of the 1561C-->T polymorphism had higher plasma folate concentrations and lower tHcy concentrations than did those in the CC group. Subjects with the TT genotype had lower Symbol Digit Modalities Test (SDMT) scores than did subjects with the CC genotype. Compared with abstainers, moderate alcohol drinkers had higher plasma folate concentrations and higher scores on the Mini Mental State Examination. However, women abstainers with the CT genotype had lower SDMT scores than did abstainers with the CC genotype or moderate drinkers with the CT genotype. CONCLUSIONS: The 1561C-->T polymorphism is associated with higher plasma folate and lower tHcy concentrations and with lower SDMT cognitive scores in women who abstain from alcohol.

S-adenosylmethionine attenuates hepatic lipid synthesis in micropigs fed ethanol with a folate-deficient diet.

BACKGROUND: To demonstrate a causative role of abnormal methionine metabolism in the pathogenesis of alcoholic steatosis, we measured the effects on hepatic lipid synthesis of supplementing ethanol and folate-deficient diets with S-adenosylmethionine (SAM), a metabolite that regulates methionine metabolism. METHODS: Yucatan micropigs were fed folate-deficient diets as control, with ethanol at 40% of kcal, and with ethanol supplemented with SAM at 0.4 g/1,000 kcal for 14 weeks. Histopathology, triglyceride levels and transcripts, and protein levels of the regulatory signals of hepatic lipid synthesis were measured in terminal omental adipose and liver samples. RESULTS: Feeding ethanol at 40% of kcal with folate-deficient diets for 14 weeks increased and supplemental SAM maintained control levels of liver and plasma triglyceride. Serum adiponectin, liver transcripts of adiponectin receptor-1 (AdipoR1), and phosphorylated adenosine monophosphate kinase-beta (p-AMPKbeta) were each reduced by ethanol feeding and were sustained at normal levels by SAM supplementation of the ethanol diets. Ethanol feeding activated and SAM supplementation maintained control levels of ER stress-induced transcription factor sterol regulatory element-binding protein-1c (SREBP-1c) and its targeted transcripts of lipid synthesizing enzymes acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and glycerol-3-phosphate acyltransferase (GPAT). CONCLUSIONS: Ethanol feeding with a folate-deficient diet stimulates hepatic lipid synthesis by down-regulating adiponectin-mediated pathways of p-AMPK to increase the expression of nSREBP-1c and its targeted lipogenic enzymes. Preventing abnormal hepatic methionine metabolism by supplementing ethanol diets with SAM reduces liver triglyceride levels by up-regulation of adiponectin-mediated pathways to decrease fatty acid and triglyceride synthesis. This study demonstrates that ethanol-induced hepatic lipid synthesis is mediated in part by abnormal methionine metabolism, and strengthens the concept that altered methionine metabolism plays an integral role in the pathogenesis of steatosis.

Effects of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism on executive function in schizophrenia.

BACKGROUND: The methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism has been associated with both overall schizophrenia risk and severity of negative symptoms. This study examined whether schizophrenia patients homozygous for the risk allele (T/T) exhibit greater impairment in executive function, and determined the extent to which MTHFR's effects on negative symptoms underlie this relationship. METHODS: 200 outpatients with chronic schizophrenia were evaluated with the Verbal Fluency Test (VFT), Wisconsin Card Sort Test (WCST), and California Verbal Learning Test (CVLT). Performance was stratified by MTHFR C667T genotype. Path analysis determined the extent to which MTHFR effects on negative symptoms mediated the relationship between genotype and cognitive measures. RESULTS: T/T subjects exhibited significantly greater deficits on the VFT and had more difficulty achieving the first category on the WCST. Genotype groups did not differ in CVLT performance. C677T effects on negative symptoms contributed to, but did not fully account for, genotype effects on VFT. Negative symptoms did not mediate WCST performance. CONCLUSIONS: MTHFR C677T genotype contributes to certain executive function deficits in schizophrenia. These deficits remained significant when taking into account mediating effects of negative symptoms. Although the intermediate mechanisms for C677T effects remain uncertain, these results suggest that MTHFR-related cognitive impairment and negative symptoms reflect differing neural substrates.

Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism.

Autism is a behaviorally defined neurodevelopmental disorder usually diagnosed in early childhood that is characterized by impairment in reciprocal communication and speech, repetitive behaviors, and social withdrawal. Although both genetic and environmental factors are thought to be involved, none have been reproducibly identified. The metabolic phenotype of an individual reflects the influence of endogenous and exogenous factors on genotype. As such, it provides a window through which the interactive impact of genes and environment may be viewed and relevant susceptibility factors identified. Although abnormal methionine metabolism has been associated with other neurologic disorders, these pathways and related polymorphisms have not been evaluated in autistic children. Plasma levels of metabolites in methionine transmethylation and transsulfuration pathways were measured in 80 autistic and 73 control children. In addition, common polymorphic variants known to modulate these metabolic pathways were evaluated in 360 autistic children and 205 controls. The metabolic results indicated that plasma methionine and the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), an indicator of methylation capacity, were significantly decreased in the autistic children relative to age-matched controls. In addition, plasma levels of cysteine, glutathione, and the ratio of reduced to oxidized glutathione, an indication of antioxidant capacity and redox homeostasis, were significantly decreased. Differences in allele frequency and/or significant gene-gene interactions were found for relevant genes encoding the reduced folate carrier (RFC 80G > A), transcobalamin II (TCN2 776G > C), catechol-O-methyltransferase (COMT 472G > A), methylenetetrahydrofolate reductase (MTHFR 677C > T and 1298A > C), and glutathione-S-transferase (GST M1). We propose that an increased vulnerability to oxidative stress (endogenous or environmental) may contribute to the development and clinical manifestations of autism.

Abnormal transsulfuration and glutathione metabolism in the micropig model of alcoholic liver disease.

BACKGROUND: Alcoholic liver disease is associated with abnormalities of methionine metabolic enzymes that may contribute to glutathione depletion. Previously, we found that feeding micropigs a combination of ethanol with a folate-deficient diet resulted in the greatest decreases in S-adenosylmethionine and glutathione and increases in liver S-adenosylhomocysteine and oxidized disulfide glutathione. METHODS: To study the mechanisms of glutathione depletion, we analyzed the transcripts and activities of enzymes involved in its synthesis and metabolism in liver and plasma specimens that were available from the same micropigs that receive folate-sufficient or folate-depleted diets with or without 40% of energy as ethanol for 14 weeks. RESULTS: Ethanol feeding, folate deficiency, or their combination decreased liver and plasma glutathione and the activities of hepatic copper-zinc superoxide dismutase and glutathione peroxidase and increased the activity of manganese superoxide dismutase and glutathione reductase. Hepatic levels of cysteine and taurine were unchanged while plasma cysteine was increased in the combined diet group. Cystathionine beta-synthase transcripts and activity were unaffected by ethanol feeding, while the activities of other transsulfuration enzymes involved in glutathione synthesis were increased. Glutathione transferase transcripts were increased 4-fold and its mean activity was increased by 34% in the combined ethanol and folate-deficient diet group, similar in magnitude to the observed 36% reduction in hepatic glutathione. CONCLUSIONS: Chronic ethanol feeding and folate deficiency acted individually or synergistically to affect methionine metabolism in the micropig by depleting glutathione pools and altering transcript expressions and activities of enzymes involved in its synthesis, utilization, and regeneration. The data suggest that the observed decrease in hepatic glutathione during ethanol feeding reflects its increased utilization to meet increased antioxidant demands, rather than reduction in its synthesis.

Chronic ethanol feeding and folate deficiency activate hepatic endoplasmic reticulum stress pathway in micropigs.

Previously, we showed that feeding micropigs ethanol with a folate-deficient diet promoted the development of hepatic injury while increasing hepatic levels of homocysteine and S-adenosylhomocysteine (SAH) and reducing the level of S-adenosylmethionine (SAM) and the SAM-to-SAH ratio. Our present goals were to evaluate mechanisms for hepatic injury using liver specimens from the same micropigs. The effects of ethanol feeding or folate-deficient diets, singly or in combination, on cytochrome P-450 2E1 (CYP2E1) and signal pathways for apoptosis and steatosis were analyzed using microarray, real-time PCR, and immunoblotting techniques. Apoptosis was increased maximally by the combination of ethanol feeding and folate deficiency and was correlated positively to liver homocysteine and SAH. Liver CYP2E1 and the endoplasmic reticulum stress signals glucose-regulated protein 78 (GRP78), caspase 12, and sterol regulatory element binding protein-1c (SREBP-1c) were each activated in pigs fed folate-deficient or ethanol diets singly or in combination. Liver mRNA levels of CYP2E1, GRP78, and SREBP-1c, and protein levels of CYP2E1, GRP78, nuclear SREBP, and activated caspase 12 each correlated positively to liver levels of SAH and/or homocysteine and negatively to the SAM-to-SAH ratio. The transcripts of the lipogenic enzymes fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase were elevated in the ethanol-fed groups, and each was positively correlated to liver homocysteine levels. The induction of abnormal hepatic methionine metabolism through the combination of ethanol feeding with folate deficiency is associated with the activation of CYP2E1 and enhances endoplasmic reticulum stress signals that promote steatosis and apoptosis.