Sex hormone imbalances and adipose tissue dysfunction impacting on metabolic syndrome; a paradigm for the discovery of novel adipokines.

Sex hormone imbalance is causally related with visceral adipose tissue (AT) dysfunction and visceral obesity - an etiological component of metabolic syndrome (MetS), associated with high risk of both cardiovascular disease (CVD) and type 2 diabetes. In general, premenopausal women appear to be protected from CVD and the dramatic decline in sex steroid hormone occurring during menopausal transitions or other sex-related disorders influence the regional distribution, function, and metabolism of AT and increase the risk of CVD. Visceral AT dysfunction, manifesting as abnormality of fatty acid metabolism, increased oxidative stress, endothelial dysfunction, and excessive production of adipokines have been proposed in the pathogenesis of MetS. However, direct evidence of molecular mechanisms of depot-specific AT alterations, and dysfunction causally related to MetS is limited in studies on postmenopausal women due to difficulty in collecting discrete AT specimens at different ages and repeated sampling from different fat depots. This can be overcome using animal models that can mimic the cluster of pathology leading to MetS and help establish the molecular basis of links between loss of gonadal function on various AT depots and their contribution to MetS. Our group used sex hormone imbalance FSH receptor knock out (FORKO) female mice to recapitulate different aspects of the MetS and addressed the mechanism of visceral obesity related to MetS and discover two novel sex steroid hormone-regulated deep mesenteric estrogen-dependent adipose (MEDAs) genes. Taken together, such recent studies raise hopes for pharmacologic intervention strategies targeting sex steroid hormone signaling in AT to provide protection against AT dysfunction.

Novel genes of visceral adiposity: identification of mouse and human mesenteric estrogen-dependent adipose (MEDA)-4 gene and its adipogenic function.

Visceral adiposity represents a high risk factor for type 2 diabetes, metabolic syndrome, and cardiovascular disease as well as various cancers. While studying sex hormone imbalance-induced early obesity and late onset of insulin resistance in FSH receptor knock out female mice, we identified a novel mesenteric estrogen-dependent adipose gene (MEDA-4) selectively up-regulated in a depot-specific manner in mesenteric adipose tissue. Meda-4 cloned from both mouse and human adipose tissue codes for a 34-kDa cytosolic protein with 91% homology. Mouse Meda-4 mRNA is expressed highest in visceral adipose tissue and localizes predominantly in the adipocyte fraction. Human MEDA-4 is also more abundant in omental fat than sc depot in obese patients. In 3T3-L1 cells endogenous Meda-4 expression increases early during differentiation, and its overexpression promotes differentiation of preadipocytes into adipocytes and enhances glucose uptake. Conversely, short hairpin RNA-mediated knockdown of Meda-4 reduces both adipogenic and glucose uptake potential. In promoting adipogenesis, Meda-4 up-regulates transcription factor peroxisome proliferator-activated receptor-γ2. Meda-4 promotes lipid accumulation in adipocytes, regulating adipocyte fatty acid-binding protein 2, CD36, lipoprotein lipase, hormone-sensitive lipase, acyl-Coenzyme A oxidase-1, perilipin-1, and fatty acid synthase expression. 17ß-Estradiol reduced Meda-4 expression in mesenteric adipose tissue of ovariectomized mice and in 3T3-L1 adipocytes. Thus our study identifies Meda-4 as a novel adipogenic gene, capable of promoting differentiation of preadipocytes into adipocytes, increasing lipid content and glucose uptake in adipocytes. Therefore it might play an important role in adipose tissue expansion in normal and aberrant hormonal conditions and pathophysiological states.

Bone marrow transplantation restores follicular maturation and steroid hormones production in a mouse model for primary ovarian failure.

Recent studies suggest that bone marrow stem cells (BMSCs) are promising grafts to treat a variety of diseases, including reproductive dysfunction. Primary ovarian failure is characterized by amenorrhea and infertility in a normal karyotype female, with an elevated serum level of follicle-stimulating hormone (FSH) and a decrease level of estrogen caused by a mutation in FSH receptor (FSHR) gene. Currently, there is no effective treatment for this condition. The phenotype of FSHR (-/-) mouse, FORKO (follitropin receptor knockout), is a suitable model to study ovarian failure in humans. Female FORKO mice have elevated FSH, decreased estrogen levels, are sterile because of the absence of folliculogenesis, and display thin uteri and small nonfunctional ovaries. In this study, we determined the effects of BMSC transplantation on reproductive physiology in this animal model. Twenty four hours post BMSC transplantation, treated animals showed detectable estroidogeneic changes in daily vaginal smear. Significant increase in total body weight and reproductive organs was observed in treated animals. Hemotoxylin and eosin (H&E) evaluation of the ovaries demonstrated significant increase in both the maturation and the total number of the follicles in treated animals. The FSH dropped to 40-50% and estrogen increased 4-5.5 times in the serum of treated animals compared to controls. The FSHR mRNA was detected in the ovaries of treated animals. Our results show that intravenously injected BMSCs were able to reach the ovaries of FORKO mice, differentiate and express FHSR gene, make FSHR responsive to FSH, resume estrogen hormone production, and restore folliculogenesis.

Novel hormone-regulated genes in visceral adipose tissue: cloning and identification of proinflammatory cytokine-like mouse and human MEDA-7: implications for obesity, insulin resistance and the metabolic syndrome.

AIMS/HYPOTHESIS: We sought to characterise novel genes dysregulated by sex hormonal imbalances that induce obesity and metabolic disorder in a setting of oestrogen deficiency and androgen dominance in follicle-stimulating hormone receptor (For [also known as Fshr]) knockout female mice. METHODS: Transcriptome analysis of mesenteric adipose tissue (MAT) of mutants revealed novel genes. One novel gene named Meda-7 was selected for study. Meda-7 was cloned from mouse and human adipose tissue; its expression, hormonal regulation and function were characterised. RESULTS: Mouse Meda-7 is richly expressed in deep visceral adipose tissue and encodes a 22 kDa secreted protein with 71% homology to human mesenteric oestrogen-dependent adipose gene- 7 (MEDA-7) protein. Both have six conserved cysteines like many cytokines. In obese patients, MEDA-7 is more abundant in omental than subcutaneous fat. Meda-7 is downregulated in For-knockout female MAT at 5 months (obese state) followed by steep upregulation at 9 months (prediabetic condition) when mutants progress towards the metabolic syndrome. Meda-7 is expressed predominantly in the stromal-vascular cell fraction. In this fraction,M1-proinflammatorymacrophages are rich in Meda-7. Meda-7 dysregulation in 5-month-old For-knockout MAT is restored by oestrogen, but treatment has no effect in older mutants. Overabundance of MEDA-7 in HEK-293 cells enhances cell proliferation via p42/44 mitogen-activated protein kinases. Secreted MEDA-7 attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes, while downregulating glucose transporter-4 and upregulating both monocyte chemotactic protein-1 and suppressor of cytokine signalling-3. Downstream activity of the insulin signalling mediator, phospho-AKT, is also downregulated. CONCLUSIONS/INTERPRETATION: MEDA-7 is a hormone-regulated adipokine/proinflammatory cytokine that is implicated in causing chronic inflammation, affecting cellular expansion and blunting insulin response in adipocytes.

Role of insulin in Cr(VI)-mediated genotoxicity in Neurospora crassa.

AIMS: Chromium (III) is an insulinomimetic agent whose biological and/or environmental availability is frequently in the form of Cr(VI), which is known to be toxic. Wall-less mutant of Neurospora crassa (FGSC stock no. 4761) is known to possess insulin receptor in its cell membrane and hence is a good model for Cr toxicity studies. This study explores the toxicity of Cr(VI) and the possible consequences on simultaneous exposure to insulin in N. crassa. METHODS AND RESULTS: Comet assay of N. crassa cells treated with 100 µmol l⁻¹ Cr(VI) showed up to 50% reduction in comet tail lengths when incubated simultaneously with 0.4 U insulin. Fluorescence measurement in Cr(VI)-treated cells using DCFH-DA showed six- to eightfold increase in free radical generation, which was reduced to fourfold by 0.4 U insulin. Annexin-V/PI Flow cytometry analysis indicated necrotic cell death up to 28.7 ± 3.6% and 68.6 ± 2.5% on Cr(VI) exposure at concentrations 100 and 500 µmol l⁻¹ which was reduced by 68.3 ± 3.2% and 48.9 ± 3.6%, respectively, upon addition of insulin. CONCLUSION: Insulin-mediated protection from DNA damage by Cr(VI) is because of scavenging of free radicals liberated during exposure to Cr(VI). SIGNIFICANCE AND IMPACT OF THE STUDY: Overall, Cr(VI) toxicity depends upon available insulin, indicating that Cr(VI) toxicity may be a serious issue in insulin-deficient individuals with diabetes.

Anti-proliferative and pro-apoptotic actions of a novel human and mouse ovarian tumor-associated gene OTAG-12: downregulation, alternative splicing and drug sensitization.

In studying the age dependence and chronology of ovarian tumors in follicle stimulating hormone receptor knockout mice, we identified a novel ovarian tumor associated gene-12 (OTAG-12), which is progressively downregulated and maps to Chr. 8B3.3. OTAG-12 protein overexpression in mouse ovarian and mammary tumor cells suggested powerful anti-proliferative effects. In human epithelial ovarian cancers (OCs) and OC cell lines, OTAG-12 mRNA expression is downregulated in comparison with normal ovaries. Cloning and identification revealed that human OTAG-12 mapping to gene-rich Chr. 19p13.12 is expressed in three spliced forms: hOTAG-12a, hOTAG-12b and hOTAG-12c, of which b is predominant in the normal ovary. Functionally active hOTAG-12b is a simple protein with no disulfide bonds and a nuclear localization signal is present in all variants. Transfection of OTAG-12 variants in OC and tumorigenic HEK293 cells confirmed nuclear localization. hOTAG-12b overexpression in OC and HEK293 cells effectively suppressed cell growth, anchorage-dependent and independent colony formation followed by apoptosis, whereas hOTAG-12a and hOTAG-12c had no such effects. Deletion mutants identified the critical importance of carboxyl terminus for hOTAG-12b function. Doxycycline-inducible growth inhibition of HEK293 cells by hOTAG-12a was associated with effects on G2 cell cycle arrest and apoptosis induction. hOTAG-12b expression rendered tumorigenic cells more sensitive to four apoptotic stimuli including etoposide-a topoisomerase-II inhibitor. Doxycycline-induced hOTAG-12b expression blocked xenograft tumor growth in nude mice, whereas hOTAG-12a was ineffective. Although p53-pathway-dependent apoptotic agents could upregulate endogenous hOTAG-12b and p53 in UCI-101/107 OC cells, hOTAG-12b could also induce apoptosis in p53-null and platinum-resistant SKOV3 OC cells and Doxycycline-induced hOTAG-12b did not alter p53. Further study showed that hOTAG-12b increases mRNAs of pro-apoptotic genes such as BAD, GADD45α and CIEDB, while inhibiting anti-apoptotic NAIP and Akt1 expression, suggesting that hOTAG-12b-induced apoptosis might be p53-independent. These results indicate that hOTAG-12b is a putative ovarian tumor suppressor gene warranting further studies.

Chronology and complexities of ovarian tumorigenesis in FORKO mice: age-dependent gene alterations and progressive dysregulation of Major Histocompatibility Complex (MHC) Class I and II profiles.

Among gynecologic malignancies ovarian cancer is the deadliest and most difficult to detect at early stages. As ovarian tumors have long latency and are relatively more frequent in postmenopausal women, revealing chronological changes in model systems might help in the discovery of novel molecular targets and diagnostic biomarkers for disease detection and management. Follitropin receptor knockout (FORKO) mice with early and sustained sex steroid hormone disharmony develop various age-dependent ovarian abnormalities including increased incidence ovarian tumors in complete absence of ovulation. These mutants show various tumor cell types including those related to ovarian surface epithelium around 12-15 months of age. To explore why the FORKO mice develop ovarian tumors later in life, we assessed global gene expression changes during the pre-tumor period (at 8 months). Age-matched wild-type and FORKO mice were compared to gain a comprehensive view of genes that are misregulated, even before overt tumors appear in mutants. Applying a conservative 2-fold change to detect changes, our study identified 476 genes (338 upregulated and 138 downregulated) to be altered between 8-month-old FORKO and wild-type ovaries. Using Ingenuity Pathway Analysis (IPA), we found highly significant alterations in five functional networks in pre-tumor stage FORKO ovaries. Notably, the top network to change in 8-month-old FORKO ovaries was associated with functions implicated in immune system development and function. We selected 9 immune related genes that are reportedly altered in Epithelial Ovarian Cancer (EOC) in women and confirmed their expression and chronology of changes in FORKO ovaries before and after tumor development. Our data indicate that immune surveillance mechanisms are compromised with in a 4-month window of tumorigenic alterations. In addition, expression of previously unrecognized genes misregulated in the dysfunctional FORKO ovaries suggests mechanisms not yet appreciated to date. We propose that a better understanding of genes that change before overt tumors develop could provide useful insights into ovarian carcinogenesis and open the door to additional new targets for treating ovarian cancers.

Hypoxia preconditioning by cobalt chloride enhances endurance performance and protects skeletal muscles from exercise-induced oxidative damage in rats.

AIM: Training under hypoxia has several advantages over normoxic training in terms of enhancing the physical performance. Therefore, we tested the protective effect of hypoxia preconditioning by hypoxia mimetic cobalt chloride against exercise-induced oxidative damage in the skeletal muscles and improvement of physical performance. METHOD: Male Sprague-Dawley rats were randomly divided into four groups (n=8), namely control, cobalt-supplemented, training and cobalt with training. The red gastrocnemius muscle was examined for all measurements, viz. free radical generation, lipid peroxidation, muscle damage and antioxidative capacity. RESULTS: Hypoxic preconditioning with cobalt along with training significantly increased physical performance (33%, P<0.01) in rats compared with training-only rats. Cobalt supplementation activated cellular oxygen sensing system in rat skeletal muscle. It also protected against training-induced oxidative damage as observed by an increase in the GSH/GSSG ratio (36%, P<0.001; 28%, P<0.01 respectively) and reduced lipid peroxidation (15%, P<0.01; 31%, P<0.01 respectively) in both trained and untrained rats compared with their respective controls. Cobalt supplementation along with training enhanced the expression of antioxidant proteins haem oxygenase-1 (HO-1; 1.2-fold, P<0.05) and metallothionein (MT; 4.8-fold, P<0.001) compared with training only. A marked reduction was observed in exercise-induced muscle fibre damage as indicated by decreased necrotic muscle fibre, decreased lipofuscin content of muscle and plasma creatine kinase level (16%, P<0.01) in rats preconditioned with cobalt. CONCLUSION: Our study provides strong evidence that hypoxic preconditioning with cobalt chloride enhances physical performance and protects muscle from exercise-induced oxidative damage via GSH, HO-1 and MT-mediated antioxidative capacity.

Catalase can protect spermatozoa of FSH receptor knock-out mice against oxidant-induced DNA damage in vitro.

The aetiology of sperm DNA damage is likely multi-factorial with abnormal compaction of nuclear DNA, abortive apoptosis and oxidative stress implicated as potential causes of DNA damage. The objective of this study was to evaluate DNA damage in spermatozoa from wild-type (WT) and FSH receptor knock-out (FORKO) mice, compare the relative susceptibility of spermatozoa from these animals to oxidative DNA damage, and examine the protective effect of the antioxidant catalase on sperm DNA damage. Epididymal spermatozoa from FORKO mice (n = 5) and WT controls (n = 5) were extracted and incubated with or without catalase. Sperm DNA damage was assessed immediately after epididymal extraction (time 0 control) and following 2-h incubation at 37 °C. DNA damage was measured by the sperm chromatin structure assay and the results expressed as the %DNA fragmentation index or %DFI. Freshly retrieved epididymal spermatozoa from WT mice had a significantly lower mean (±SD) %DFI than that of FORKO mice (2.7 ± 1.8 vs. 6.4 ± 2.9%, p < 0.05). Prolonged (2-h) incubation of FORKO mice spermatozoa resulted in a significant increase in %DFI compared with the time 0 control (17.9 ± 9.2% vs. 6.4 ± 2.9%, respectively, p < 0.05) and the addition of catalase protected these spermatozoa from DNA damage (9.8 ± 4.1 vs. 17.9 ± 9.2%, respectively, p < 0.05). However, incubation of WT mice spermatozoa did not increase %DFI significantly (5.8 ± 5.0 vs. 2.7 ± 1.8, respectively, p > 0.05) and the addition of catalase (vs. no catalase) did not result in a significant reduction in %DFI (5.8 ± 5.0 vs. 7.7 ± 6.5%, respectively, p > 0.05). These data indicate that catalase may protect sperm nuclear DNA from oxidative stress in vitro. The data also demonstrate the differential susceptibility of WT and FORKO mice spermatozoa to oxidative stress.

Toward gene therapy of premature ovarian failure: intraovarian injection of adenovirus expressing human FSH receptor restores folliculogenesis in FSHR(-/-) FORKO mice.

A homozygous missense mutation, C566T, in the follicle stimulation hormone receptor (FSHR) gene has been linked to premature ovarian failure. The disease leads to infertility in a normal karyotype female with an elevated follicle stimulating hormone (FSH) and decreased serum estrogen level. Female mice carrying mutated FSHR gene, called follitropin receptor knockout (FORKO), display similar phenotype and are sterile because of a folliculogenesis block at a primary stage. We investigated the effects of bilateral intra-ovarian injection of an adenovirus expressing a normal copy of human FSHR on the reproductive system of 6-10 weeks female FORKO mice. Ad-LacZ was injected directly into each ovary of the control group. Animals were sacrificed at 2, 4, 8 and 12 weeks post-injection and tissues collected for evaluation. Treated mice showed estrogenic changes in daily vaginal smear whereas control animals remained fixated in the diestrus stage. Histological evaluation showed on average 26 +/- 4 follicles/ovary in treated group with 8 +/- 2 follicles at the antral stage compared with only 5 +/- 2 with zero follicles at antral stage in Ad-LacZ control mice. There was no significant change in serum level of progesterone, however, estrogen level increased 2-3-fold (P < 0.02) and FSH decreased by up to 50% (P < 0.04) in treated animals. FSHR mRNA was detected in the ovaries of the treated group. In conclusion, intra-ovarian injection of an adenovirus expressing human FSHR gene is able to restore FSH responsiveness and reinitiate ovarian folliculogenesis as well as resume estrogen production in female FORKO mice. Ad-LacZ injections indicate the absence of systemic viral dissemination or germ line transmission of adenovirus DNA to offspring.

Initiation of the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma) in the rat ovary and the role of FSH.

PPARgamma is highly expressed in granulosa cells by 23 days post-partum (pp) and is down-regulated in response to the LH surge. We tested the hypothesis that high levels of FSH during the neonatal period trigger the expression of PPARgamma. To determine when PPARgamma expression is initiated, ovaries were collected from neonatal rats. Messenger RNA for PPARgamma was undetectable on day 1, low from days 5-14, and increased by day 19 pp (p < 0.05). PPARgamma was detected in select granulosa cells in primary/early secondary follicles. Messenger RNA for the FSH receptor was detected as early as day 1 and remained steady throughout day 19 pp. The FSH receptor was detected by immunoblot analysis in ovaries collected 1, 2, and 5-9 days pp. In a subsequent experiment, neonatal rats were treated with acyline (GnRH antagonist) which significantly reduced FSH (p < 0.05) but not levels of mRNA for PPARgamma. The role of FSH in the induction of PPARgamma expression was further assessed in ovarian tissue from FORKO mice. Both mRNA and protein for PPARgamma were identified in ovarian tissue from FORKO mice. In summary, the FSH/FSH receptor system is present in granulosa cells prior to the onset of expression of PPARgamma. Reducing FSH during the neonatal period, or the ability to respond to FSH, did not decrease expression of mRNA for PPARgamma. These data indicate that FSH is not a primary factor initiating the expression of PPARgamma and that other agents play a role in activating its expression in the ovary.

Profilin oligomerization and its effect on poly (L-proline) binding and phosphorylation.

Profilin is a cytoskeletal protein that interacts specifically with actin, phosphoinositides and poly (l-proline). Experimental results and in silico studies revealed that profilin exists as dimer and tetramer. Profilin oligomers possess weak affinity to poly (l-proline) due to unavailability of binding sites in dimers and tetramers. Phosphorylation studies indicate that profilin dimers are not phosphorylated while teramers are preferentially phosphorylated over monomers. In silico studies revealed that PKC phosphorylation site, S137 is buried in dimer while it is accessible in tetramer.

Modulation of ovarian structure and abdominal obesity in curcumin- and flutamide-treated aging FSH-R haploinsufficient mice.

We have previously shown that follicle-stimulating hormone receptor haploinsufficient mice undergo early reproductive senescence with alterations in ovarian structures. The objective of this study was to treat aging (7-8 months) +/- follicle-stimulating hormone receptor mice that are destined for reproductive failure with 2 selected antiandrogens, curcumin and flutamide, to counteract deleterious effects of mild hyperandrogenemia on the ovary and metabolism. Both compounds significantly downregulated the expression of ovarian androgen receptor protein and simultaneously reduced cyclooxygenase 2 protein in the ovary. Immunolocalization of bone morphogenetic protein-15 in the ovary was enhanced considerably by curcumin and partially by flutamide in treated mice. Improved structural changes were evident in zona pellucida of curcumin-treated ovaries. Flutamide reduced p450c-17 (cyp-17 protein) enzyme expression in thecal/interstitial cells, whereas increased expression of 3beta-hydroxysteroid dehydrogenase in thecal cells and granulosa-lutein cells of big follicles was apparent in curcumin-treated ovaries. Reduction in abdominal adiposity was greater in flutamide-treated mice. Taken together, our study allows the following conclusions: changes in ovarian histology and oocyte components as well as adipose tissue indicate the potential for reversing ovarian decline and metabolism because of mild hyperandrogenemia that occurs with aging in follicle-stimulating hormone receptor haploinsufficienct mice.

Potentiation of vascular oxidative stress and nitric oxide-mediated endothelial dysfunction by high-fat diet in a mouse model of estrogen deficiency and hyperandrogenemia.

Estrogen deficiency is associated with increased cardiovascular risk due, in part, to hypertension, endothelial dysfunction, obesity, and hypercholesterolemia. Underlying mechanisms for this remain unclear. Here, we investigated whether high-fat intake aggravates vascular dysfunction through oxidative stress and inflammation, which could predispose to cardiovascular injury in conditions of estrogen deficiency, such as menopause. We studied female homozygous follitropin receptor knock out (FORKO) mice, which have hormonal features of clinical menopause and hypertension and wild-type (WT) and heterozygote mice (HTZ), fed a standard or high-fat diet for 4 months. Vascular function and structure were evaluated in arterial segments by pressurized myography. Acetylcholine (ACh)-induced vasodilation was reduced in FORKO vs. WT mice (P < .001). N(varpi)-nitro-l-arginine-methyl-ester inhibited Ach-induced relaxation in all groups on normal chow and in WT and HTZ on high-fat diet (FD) but had no effect in fat-fed FORKO mice. Antioxidant cocktail (superoxide dismutase, catalase, Tempol) increased ACh responses only in high-fat diet FORKO mice (P < .05). Vascular media-to-lumen ratio was increased and reactive oxygen species (ROS) generation, nitrotyrosine formation, and plasma nitrite levels were augmented in fat-fed FORKO vs. FORKO on normal chow. High-fat diet did not influence vascular inflammatory responses in any group. Our data demonstrate that FORKO mice have altered nitric oxide-sensitive vasorelaxation and vascular remodeling, which are aggravated by high-fat diet. Underlying mechanisms for this may involve decreased NO formation and increased generation of ROS and nitrotyrosine. These findings suggest that high-fat intake potentiates vascular damage in estrogen-deficient states, an effect involving increased oxidative stress.

Cardiac hypertrophy is associated with altered thioredoxin and ASK-1 signaling in a mouse model of menopause.

Oxidative stress is implicated in menopause-associated hypertension and cardiovascular disease. The role of antioxidants in this process is unclear. We questioned whether the downregulation of thioredoxin (TRX) is associated with oxidative stress and the development of hypertension and target-organ damage (cardiac hypertrophy) in a menopause model. TRX is an endogenous antioxidant that also interacts with signaling molecules, such as apoptosis signal-regulated kinase 1 (ASK-1), independently of its antioxidant function. Aged female wild-type (WT) and follitropin receptor knockout (FORKO) mice (20-24 wk), with hormonal imbalances, were studied. Mice were infused with ANG II (400 ng x kg(-1) x min(-1); 14 days). Systolic blood pressure was increased by ANG II in WT (166+/-8 vs. 121+/-5 mmHg) and FORKO (176+/-7 vs. 115+/-5 mmHg; P<0.0001; n=9/group) mice. In ANG II-infused FORKO mice, cardiac mass was increased by 42% (P<0.001). This was associated with increased collagen content and augmented ERK1/2 phosphorylation (2-fold). Cardiac TRX expression and activity were decreased by ANG II in FORKO but not in WT (P<0.01) mice. ASK-1 expression, cleaved caspase III content, and Bax/Bcl-2 content were increased in ANG II-infused FORKO (P<0.05). ANG II had no effect on cardiac NAD(P)H oxidase activity or on O(2)(*-) levels in WT or FORKO. Cardiac ANG II type 1 receptor expression was similar in FORKO and WT. These findings indicate that in female FORKO, ANG II-induced cardiac hypertrophy and fibrosis are associated with the TRX downregulation and upregulation of ASK-1/caspase signaling. Our data suggest that in a model of menopause, protective actions of TRX may be blunted, which could contribute to cardiac remodeling independently of oxidative stress and hypertension.

Aqueous extract of Rhodiola imbricata rhizome inhibits proliferation of an erythroleukemic cell line K-562 by inducing apoptosis and cell cycle arrest at G2/M phase.

Rhodiola imbricata is a medicinal plant having immunostimulating properties. The anti-proliferative effects of Rhodiola aqueous extract (RAE), were studied in human erythroleukemic cell line K-562 using MTT cell proliferation assay. The proliferation of K-562 was significantly decreased after 72h incubation with RAE at 100 and 200microg/ml. However, almost no suppressive effects could be detected in normal human peripheral blood lymphocytes or mouse macrophage cell line RAW-264.7. RAE was also found to induce intracellular reactive oxygen species (ROS) in K-562 cells at 200microg/ml when incubated overnight. The increased ROS generation may cause apoptosis, which was observed in AnnexinV-FITC and propidium iodide (PI) staining of cells treated with RAE for 72h in K-562 cells. Moreover, RAE arrested cell cycle progression in G2/M phase in early and late period of exposure. The anti-cancer activity of RAE was also confirmed by increased NK cell cytotoxicity. These observations suggest that aqueous extract of R. imbricata rhizome has very potent anti-cancer activities, which might be useful in leukemia cancer treatment.

A review of high throughput technology for the screening of natural products.

High throughput screening is commonly defined as automatic testing of potential drug candidates at a rate in excess of 10,000 compounds per week. The aim of high throughput drug discovery is to test large compound collections for potentially active compounds ('hits') in order to allow further development of compounds for pre-clinical testing ('leads'). High throughput technology has emerged over the last few years as an important tool for drug discovery and lead optimisation. In this approach, the molecular diversity and range of biological properties displayed by secondary metabolites constitutes a challenge to combinatorial strategies for natural products synthesis and derivatization. This article reviews the approach of High throughput technique for the screening of natural products for drug discovery.

Impairment of the natriuretic peptide system in follitropin receptor knockout mice and reversal by estradiol: implications for obesity-associated hypertension in menopause.

Estrogen is considered a major regulator of adipose tissue in females. Estrogen increases circulating levels of atrial natriuretic peptide (ANP), a hormone with renal and cardiovascular effects. The aim of this study was to determine the status of the natriuretic peptide system in female follitropin-receptor knockout (FORKO) mice that could be associated with obesity and hypertension observed in these mutants. Furthermore, estradiol treatment was used to reverse alterations observed. FORKO and wild-type (WT) mice received daily injections of estradiol for 4 d. On the fifth day, blood was collected for determination of plasma ANP levels, and selected tissues were collected for determination of ANP, natriuretic peptide receptor type-A (NPR-A) and type-C (NPR-C) gene expression by RT-PCR and binding of [(125)I]ANP by autoradiography. At 5 months of age, FORKO mice were heavier and had more adipose tissue than WT mice. FORKO mice had lower plasma ANP levels and atrial ANP gene expression and higher renal and adipocyte NPR-C gene expression than WT mice. Estradiol treatment reduced weight gain and increased atrial ANP synthesis as well as decreased ANP clearance NPR-C receptors, resulting in elevation of circulating ANP level. In conclusion, this study shows that FORKO females have an impaired natriuretic peptide system, which may contribute to the susceptibility of FORKO mice to developing age-related hypertension previously shown in these animals. This study establishes a relation between estrogen, adipose tissue, and ANP, which may have important implications in menopausal women.

NR1 and GluR2 expression mediates excitotoxicity in chronic hypobaric hypoxia.

Hypobaric hypoxia has been reported to cause memory dysfunction. The possible molecular mechanism involved, however, remains to be explored. The role that glutamate and its receptors play in causing excitotoxicity in ischemia and neurodegenerative diseases indicates the possible occurrence of a similar phenomenon in hypobaric hypoxia. The present study aimed to elucidate the molecular events occurring at glutamatergic synapses in hypobaric hypoxia using Sprague-Dawley rats as a model system. The animals were exposed to an altitude of 7,600 m for different durations. Hypobaric hypoxia was found to cause oxidative stress, chromatin condensation, and neurodegeneration. A temporal change in the expression of the ionotropic receptors of glutamate was also observed. Expression of the N-methyl-D-aspartate (NMDA) receptor increased, and expression of glutamate receptor subunit 2 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor decreased. We also observed increased activity of glutamate dehydrogenase, indicating greater synthesis and release of glutamate after 3 and 7 days of exposure. Administration of a selective NMDA antagonist during exposure was found to ameliorate neuronal degeneration, providing evidence for the occurrence of excitotoxicity in hypobaric hypoxia. Our study indicates that excitotoxicity occurs in hypobaric hypoxia. This study also indicates the appropriate period for drug administration during exposure to hypobaric hypoxia and establishes ionotropic receptors of glutamate as potential therapeutic targets for ameliorating high-altitude-induced cognitive dysfunction.

Changes in adiponectin and inflammatory genes in response to hormonal imbalances in female mice and exacerbation of depot selective visceral adiposity by high-fat diet: implications for insulin resistance.

Early obesity and late onset of insulin resistance associated with hormonal imbalances occur in FSH receptor-deficient follitropin receptor knockout female mice. This study tests the hypothesis that chronic high-fat diet aggravates obesogenic changes in a depot-specific manner and explores some molecular links of hormone imbalances with insulin resistance. In SV 129 mice, hormonal imbalances seem obligatory for exacerbation of diet-induced obesity. Visceral adiposity, glucose intolerance, and lipid disturbances in 9-month follitropin receptor knockout females were associated with decrease in adiponectin signaling. High-molecular-weight plasma adiponectin and adipose tissue adiponectin mRNA were decreased. Adiponectin receptors R1 and R2 mRNA was selectively altered in mesenteric fat but not periuterine fat. R2 decreased in the liver and R1 was higher in muscle. Whereas hepatic adenosine monophosphate T-activated protein kinase activity was down-regulated, both phosphoenolpyruvate carboxykinase and glucose-6-phosphatase enzymes were up-regulated. Longitudinally, diminishing sex hormone signaling in adipose tissue was associated with progressive down-regulation of adiponectin activity and gradual impaired glucose tolerance. Chronic high-fat diet in SV129 wild-type mice did not produce overt obesity but induced visceral fat depot changes accompanied by liver lipid accumulation, high cholesterol, and up-regulation of inflammation gene mRNAs. Thus, TNF-alpha, C-C motif chemokine receptor-2, and C-C motif chemokine ligand-2 were selectively elevated in mesenteric fat without altering glucose tolerance and adiponectin signaling. Our study highlights adiponectin signaling and regulation to be involved in hormone imbalance-induced insulin resistance and demonstrates selective visceral adipose depot alterations by chronic high-fat diet and induction of inflammatory genes.