Synergistic Phytochemicals Fail to Protect Against Ovariectomy Induced Bone Loss in Rats.

Menopause induces a loss of bone as a result of estrogen deficiency. Despite pharmaceutical options for the treatment of osteopenia and osteoporosis, many aging women use dietary supplements with estrogenic activity to prevent bone loss and other menopausal-related symptoms. Such supplements are yet to be tested for efficacy against a Food and Drug Administration (FDA) approved medication for menopausal bone loss such as zoledronic acid (ZA). The postmenopausal rat model was used to investigate the efficacy of various synergistic phytochemical blends mixed into the diet for 16 weeks. Retired-breeder, Fischer 344 rats were randomly assigned to sham or ovariectomy surgery and 4 treatment groups: ZA; genistein supplementation; and a low dose and high dose blend of genistein, resveratrol, and quercetin. Ovariectomy resulted in a loss of both trabecular and cortical bone which was prevented with ZA. The phytochemical blends tested were unable to reverse these losses. Despite the lack of effectiveness in preventing bone loss, a significant dose-response trend was observed in the phytochemical-rich diets in bone adipocyte number compared to ovariectomized control rats. Data from this study indicate that estrogenic phytochemicals are not as efficacious as ZA in preventing menopausal-related bone loss but may have beneficial effects on bone marrow adiposity in rats.

DNA cytosine hydroxymethylation levels are distinct among non-overlapping classes of peripheral blood leukocytes.

BACKGROUND: Peripheral blood leukocytes are the most commonly used surrogates to study epigenome-induced risk and epigenomic response to disease-related stress. We considered the hypothesis that the various classes of peripheral leukocytes differentially regulate the synthesis of 5-methylcytosine (5mCG) and its removal via Ten-Eleven Translocation (TET) dioxygenase catalyzed hydroxymethylation to 5-hydroxymethylcytosine (5hmCG), reflecting their responsiveness to environment. Although it is known that reductions in TET1 and/or TET2 activity lead to the over-proliferation of various leukocyte precursors in bone marrow and in development of chronic myelomonocytic leukemia and myeloproliferative neoplasms, the role of 5mCG hydroxymethylation in peripheral blood is less well studied. RESULTS: We developed simplified protocols to rapidly and reiteratively isolate non-overlapping leukocyte populations from a single small sample of fresh or frozen whole blood. Among peripheral leukocyte types we found extreme variation in the levels of transcripts encoding proteins involved in cytosine methylation (DNMT1, 3A, 3B), the turnover of 5mC by demethylation (TET1, 2, 3), and DNA repair (GADD45A, B, G) and in the global and gene-region-specific levels of DNA 5hmCG (CD4+ T cells≫CD14+ monocytes>CD16+ neutrophils>CD19+ B cells>CD56+ NK cells>Siglec8+ eosinophils>CD8+ T cells). CONCLUSIONS: Our data taken together suggest a potential hierarchy of responsiveness among classes of leukocytes with CD4+, CD8+ T cells and CD14+ monocytes being the most distinctly poised for a rapid methylome response to physiological stress and disease.

Load-specific physical activity scores are related to tibia bone architecture.

Assessment of physical activity in clinical bone studies is essential. Two bone-specific physical activity scoring methods, the Bone Loading History Questionnaire (BLHQ) and Bone-Specific Physical Activity Questionnaire (BPAQ), have shown correlations with bone density and geometry, but not architecture. The purpose of this study was to determine relationships between physical activity scoring methods and bone architecture in non-Hispanic white adolescent females (N = 24; 18-19 years of age). Bone loading scores (BLHQ [hip and spine] and past BPAQ) and energy expenditure (7-day physical activity recall) were determined from respective questionnaires. Estimates of trabecular and cortical bone architecture at the nondominant radius and tibia were assessed via magnetic resonance imaging. Total body and regional areal bone mineral density (aBMD), as well as total body fat mass and fat-free soft tissue (FFST) mass were assessed via dual energy X-ray absorptiometry. Pearson's correlations and partial correlations adjusting for height, total body fat mass, and FFST were performed. Hip BLHQ scores were correlated with midtibia cortical volume (r = .43; p = .03). Adjusted hip and spine BLHQ scores were correlated with all midtibia cortical measures (r = .50-0.58; p < .05) and distal radius apparent trabecular number (r = .46-0.53; p < .05). BPAQ scores were correlated with all midtibia cortical (r = .41-0.51; p < .05) and most aBMD (r = .47-0.53; p < .05) measures. Energy expenditure was inversely associated with femoral neck aBMD only after statistical adjustment (r = .49, p < .05). These data show that greater load-specific physical activity scores, but not energy expenditure, are indicative of greater midtibia cortical bone quality, thus supporting the utility of these instruments in musculoskeletal research.

Acute exposure to high-fat diets increases hepatic expression of genes related to cell repair and remodeling in female rats.

High-fat diets (HFD) promote the development of both obesity and fatty liver disease through the up-regulation of hepatic lipogenesis. Insulin resistance, a hallmark of both conditions, causes dysfunctional fuel partitioning and increases in lipogenesis. Recent work has demonstrated that systemic insulin resistance occurs in as little as the first 72 hours of an HFD, suggesting the potential for hepatic disruption with HFD at this time point. The current study sought to determine differences in expression of lipogenic genes between sexes in 3-month-old male and female Long-Evans rats after 72 hours of a 40% HFD or a 17% fat (chow) diet. Owing to the response of estrogen on hepatic signaling, we hypothesized that a sexual dimorphic response would occur in the expression of lipogenic enzymes, inflammatory cytokines, apoptotic, and cell repair and remodeling genes. Both sexes consumed more energy when fed an HFD compared with their low fat-fed controls. However, only the males fed the HFD had a significant increase in body fat. Regardless of sex, HFD caused down-regulation of lipogenic and inflammatory genes. Interestingly, females fed an HFD had up-regulated expression of apoptotic and cell repair-related genes compared with the males. This may suggest that females are more responsive to the acute hepatic injury effects caused by HFDs. In summary, neither male nor female rats displayed disrupted hepatic metabolic pathways after 72 hours of the HFD treatment. In addition, female rats appear to have protection from increases in fat deposition, possibly due to increased caloric expenditure; male rats fed an HFD were less active, as demonstrated by distance traveled in their home cage.

Bioactive flavonoid p-hydroxycinnamic acid stimulates osteoblastogenesis and suppresses adipogenesis in bone marrow culture.

The bioactive flavonoid p-hydroxycinnamic acid (HCA), which is an intermediate-metabolic substance in plants and fruits, is synthesized from tyrosine. The biological effect of HCA is poorly understood. Among cinnamic acid and its related compounds, HCA has a specific-anabolic effect on bone, being found to stimulate osteoblastogenesis and to inhibit osteoclastogenesis through the suppression of NF-κB signaling, thereby preventing bone loss. Bone marrow mesenchymal stem cells give rise to ostoblasts and adipocytes. HCA might therefore have effects on osteoblastogenesis and adipogenesis in bone marrow culture. This study demonstrates (1) that HCA has stimulatory effects on osteoblastogenesis and mineralization and suppressive effects on adipogenesis in mouse bone marrow culture and (2) that HCA depresses adipogenesis in mouse 3T3-L1 preadipocytes in vitro. Such effects of HCA might be involved in the differentiation of mesenchymal stem cells.

Adenovirus 36, adiposity, and bone strength in late-adolescent females.

Adenovirus 36 (Ad36) is the only adenovirus to date that has been linked with obesity in humans. Our previous studies in late-adolescent females suggest that excess weight in the form of fat mass is associated with lower cortical bone strength. The purpose of this study was to assess the relationship between Ad36-specific antibodies, adiposity, and bone strength in our sample of late-adolescent females. A cross-sectional study of 115 females aged 18 to 19 years was performed. Participants were classified according to adiposity by dual-energy X-ray absorptiometry (body fat percentage as normal-fat [ < 32% body fat; n = 93] or high-fat [ ≥ 32% body fat; n = 22]), and according to the presence of Ad36-specific neutralizing antibodies. Peripheral quantitative computed tomography measured bone parameters at the 4% (trabecular bone) and 20% (cortical bone) site, and muscle cross-sectional area (MCSA) at the 66% site, from the distal metaphyses of the radius and the tibia. Bone strength was determined from volumetric bone mineral density and bone geometry to calculate bone strength index (BSI; trabecular site) and polar strength-strain index (SSI; cortical site). After adjustment for MCSA and limb length, radial SSI was lower in Ad36+ versus Ad36- subjects from the high-fat group (p < 0.03), but not the normal-fat group. No significant differences were observed between groups in tibial SSI or BSI. These data support an association of adiposity and cortical bone strength at the radius with the presence of neutralizing antibodies to Ad36 in late-adolescent females.

Exogenous regucalcin suppresses osteoblastogenesis and stimulates adipogenesis in mouse bone marrow culture.

Regucalcin plays a pivotal role in regulating intracellular calcium homeostasis and consequently has a profound effect on multiple intracellular signal transduction pathways. The regucalcin transgenic rat displays pronounced bone loss and hyperlipidemia. Consistent with these effects exogenous regucalcin has been shown to promote osteoclastogenesis in mouse bone marrow cultures and to suppress the differentiation and mineralization of MC3T3 osteoblast precursors. Regucalcin may induce hyperlipidemia in vivo by suppressing osteoblast differentiation and stimulating adipogenesis in bone marrow mesenchymal stem cells. The present study demonstrates that exogenous regucalcin suppresses differentiation to osteoblasts and stimulates adipogenesis in mouse bone marrow cell culture ex vivo. Moreover, exogenous regucalcin was found to enhance adipogenesis stimulated by insulin which is involved in the extracellular signal-related kinase pathway in 3T3-L1 adipocytes in vitro.

What is new for an old molecule? Systematic review and recommendations on the use of resveratrol.

BACKGROUND: Resveratrol is a natural compound suggested to have beneficial health effects. However, people are consuming resveratrol for this reason without having the adequate scientific evidence for its effects in humans. Therefore, scientific valid recommendations concerning the human intake of resveratrol based on available published scientific data are necessary. Such recommendations were formulated after the Resveratrol 2010 conference, held in September 2010 in Helsingør, Denmark. METHODOLOGY: Literature search in databases as PUBMED and ISI Web of Science in combination with manual search was used to answer the following five questions: (1)Can resveratrol be recommended in the prevention or treatment of human diseases?; (2)Are there observed "side effects" caused by the intake of resveratrol in humans?; (3)What is the relevant dose of resveratrol?; (4)What valid data are available regarding an effect in various species of experimental animals?; (5)Which relevant (overall) mechanisms of action of resveratrol have been documented? CONCLUSIONS/SIGNIFICANCE: The overall conclusion is that the published evidence is not sufficiently strong to justify a recommendation for the administration of resveratrol to humans, beyond the dose which can be obtained from dietary sources. On the other hand, animal data are promising in prevention of various cancer types, coronary heart diseases and diabetes which strongly indicate the need for human clinical trials. Finally, we suggest directions for future research in resveratrol regarding its mechanism of action and its safety and toxicology in human subjects.

Synergism between resveratrol and other phytochemicals: implications for obesity and osteoporosis.

Resveratrol, a phytoalexin, has gained much attention recently due to its effects on sirtuins. While the anti-cancer properties of resveratrol have been extensively investigated, the anti-adipogenic and osteogenic effects of resveratrol are also gaining considerable interest. The finding that resveratrol supplementation mimics caloric restriction prompted researchers to study the effects of resveratrol on lipid metabolism. Mesenchymal stem cells are the precursors for both adipocytes and osteoblasts. In the aging population, differentiation to adipocytes dominates over the differentiation to osteoblasts in bone marrow, contributing to the increased tendency for fractures to occur in the elderly. Thus, an inverse relationship exists between adipocytes and osteoblasts in the bone marrow. Resveratrol acts on several molecular targets in adipocytes and osteoblasts leading to a decrease in adipocyte number and size and an increase in osteogenesis. Furthermore, resveratrol in combination with genistein and quercetin synergistically decreased adipogenesis in murine and human adipocytes. A recent in vivo study showed that phytochemicals including resveratrol in combination with vitamin D prevented weight gain and bone loss in a postmenopausal rat model. Therefore, combinations of resveratrol with other phytochemicals may lead to potential novel potent therapies for both obesity and osteoporosis.

Emerging role of apelin as a therapeutic target in cancer: a patent review.

Since tumors cannot grow or spread without forming new blood vessels, inhibiting angiogenesis is an excellent approach for the treatment of cancer. Further, inhibitors of angiogenesis have mild side effects since they act on endothelial cells, which eliminate the possibility of developing resistance or tolerance in tumor cells, unlike that seen with chemotherapy drugs. The anti-vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab acts by preventing new blood vessel formation in solid tumors and is approved by FDA to treat colorectal, lung, breast, glioblastoma and kidney cancers. The registration of this drug and its ongoing success in the clinic has validated the targeting of angiogenesis as an important approach to the treatment of solid tumors. Apelin is a novel angiogenic factor and recent studies indicate that apelin promotes angiogenesis, lymphangiogenesis and tumor growth in vivo and the angiogenic potential of apelin is similar to that of VEGF. Also, apelin expression is upregulated and has been shown to be associated with clinical outcome in certain human cancers. Thus, inhibition of apelin activity might lead to a new class of anti-angiogenesis drugs which should be more efficacious than those currently on the market due to their ability to be both anti-angiogenic as well as anti-lymphangiogenic. There are very few patents on the angiogenic effects of apelin and this review article focuses on these patented claims related to inhibiting apelin signaling and sheds more light on how blocking apelin signaling might open doors to a new class of angiogenic inhibitors.

Central (ICV) leptin injection increases bone formation, bone mineral density, muscle mass, serum IGF-1, and the expression of osteogenic genes in leptin-deficient ob/ob mice.

Both central and peripheral leptin administrations reduce body weight, food intake, and adiposity in ob/ob mice. In this study we compared effects of intracerebroventricular (ICV) and subcutaneous (SC) administration of leptin on bone metabolism in the appendicular and axial skeleton and adipose tissue gene expression and determined the effects of ICV leptin on bone marrow gene expression in ob/ob mice. In experiment 1, leptin (1.5 or 0.38 µg/d) or control was continuously injected ICV for 12 days. Gene expression analysis of femoral bone marrow stromal cells showed that expression of genes associated with osteogenesis was increased after ICV injection, whereas those associated with osteoclastogenesis, adipogenesis, and adipocyte lipid storage were decreased. In experiment 2, leptin was injected continuously ICV (0.0 or 1.5 µg/d) or SC (0.0 or 10 µg/d) for 12 days. In both experiments, regardless of mode of administration, leptin decreased body weight, food intake, and body fat and increased muscle mass, bone mineral density, bone mineral content, bone area, marrow adipocyte number, and mineral apposition rate. Serum insulin was decreased, whereas serum osteocalcin, insulin-like growth factor 1, osteoprotegerin, pyridinoline, and receptor activator of nuclear factor κB ligand concentrations were increased. In experiment 2, expression of genes in adipose tissue associated with apoptosis, lipid mobilization, insulin sensitivity, and thermogenesis was increased, whereas expression of genes associated with cell differentiation and maturation was decreased regardless of mode of administration. Thus ICV injection of leptin promotes expression of pro-osteogenic factors in bone marrow, leading to enhanced bone formation in ob/ob mice.

Central leptin versus ghrelin: effects on bone marrow adiposity and gene expression.

This study compared the central effects of ghrelin and leptin on body and bone marrow adiposity and gene expression in adipose tissue and bone marrow. Male Sprague-Dawley rats were injected intracerebroventricular (ICV) twice daily with control, 66 ng ghrelin (G66), 330 ng ghrelin (G330), or 5 µg leptin (L5) for 5 days. Food intake (FI) and body weight (BW) were measured daily. Gene expression in adipose tissue and bone marrow was assessed using RT-PCR. Leptin reduced FI (P < 0.05) and BW (P < 0.05), whereas ghrelin increased BW (P < 0.05) without affecting FI. Leptin decreased fat pad weights, whereas ghrelin (G330) increased fat pad weights (P < 0.05). In epididymal adipose tissue, leptin increased expression of lipolysis marker ADRB2 and thermogenesis marker MFN2 and decreased expression of adipogenic markers, FASN, SLC2A4, and SCD1, whereas ghrelin increased expression of FASN and SCD1. Leptin decreased bone marrow adipocyte size and number; however, ghrelin had no effect on these parameters. In whole bone marrow, leptin decreased expression of FASN and SCD1 and increased expression of DLK1, whereas ghrelin (G330) decreased expression of COL1A1. Thus, leptin induces similar changes in bone marrow and adipose tissue gene expression, reflecting the decreased adiposity in both compartments.

Octanoate and decanoate induce apoptosis in 3T3-L1 adipocytes.

The effect of octanoate and decanoate, respectively, eight- and 10-carbon medium-chain fatty acids (MCFAs), on apoptotic signaling in 3T3-L1 adipocytes was investigated. 3T3-L1 adipocytes were treated with various concentrations of octanoate or decanoate. Cell viability, apoptosis, and expression of apoptosis-related proteins were investigated. Results indicated that both octanoate and decanoate decreased viability, increased apoptosis, and increased reactive oxygen species production. Immunoblotting analysis showed an increase in the levels of cytoplasmic cytochrome c and cleaved poly(ADP-ribose) polymerase by octanoate and decanoate. Concomitantly, we observed that pro-caspase-3 was decreased, resulting in the induced accumulation of the cleaved form of caspase-3 by both octanoate and decanoate. In addition, both octanoate and decanoate increased the expression of pro-apoptotic Bax with an accompanied decrease of anti-apoptotic Bcl-2. These results show that octanoate and decanoate mediate adipocyte apoptosis via a caspase-dependent mitochondrial pathway in 3T3-L1 adipocytes. MCFAs thus decrease adipocyte number by initiating the apoptotic process in 3T3-L1 adipocytes.

Anti-obesity effects of xanthohumol plus guggulsterone in 3T3-L1 adipocytes.

Xanthohumol (XN) and guggulsterone (GS) have each been shown to inhibit adipogenesis and induce apoptosis in adipocytes. In the present study effects of the combination of XN + GS on 3T3-L1 adipocyte apoptosis and adipogenesis were investigated. Mature adipocytes were treated with XN and GS individually and in combination. XN and GS individually decreased cell viability, but XN + GS caused an enhanced decrease in viability and potentiated induction of apoptosis. Likewise, XN + GS caused a potentiated increase in caspase-3/7 activation, whereas neither of the compounds showed any effect individually. In addition, western blot analysis revealed that XN + GS increased Bax expression and decreased Bcl-2 expression, whereas individual compounds did not show any significant effect. XN and GS both decreased lipid accumulation. Individually, XN at 1.5 microM and GS at 3.12 microM decreased lipid accumulation by 26 +/- 4.5% (P < .001) each, whereas XN1.5 + GS3.12 decreased lipid accumulation by 78.2 +/- 1.8% (P < .001). Moreover, expression of the adipocyte-specific proteins was down-regulated with XN1.5 + GS3.12, but no effect was observed with the individual compounds. Finally, XN + GS caused an enhanced stimulation of lipolysis. Thus, combination of XN and GS is more potent in exerting anti-obesity effects than additive effects of the individual compounds.

Genistein inhibits differentiation of primary human adipocytes.

Genistein, a major soy isoflavone, has been reported to exhibit antiadipogenic and proapoptotic potential in vivo and in vitro. It is also a phytoestrogen which has high affinity to estrogen receptor beta. In this study, we determined the effect of genistein on adipogenesis and estrogen receptor (ER) alpha and beta expression during differentiation in primary human preadipocytes. Genistein inhibited lipid accumulation in a dose-dependent manner at concentrations of 6.25 microM and higher, with 50 microM genistein inhibiting lipid accumulation almost completely. Low concentrations of genistein (3.25 microM) increased cell viability and higher concentrations (25 and 50 microM) decreased it by 16.48+/-1.35% (P<.0001) and 50.68+/-1.34% (P<.0001). Oil Red O staining was used to confirm the effects on lipid accumulation. The inhibition of lipid accumulation was associated with inhibition of glycerol-3-phosphate dehydrogenase activity and down-regulation of expression of adipocyte-specific genes, including peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, glycerol-3-phosphate dehydrogenase, adipocyte fatty acid binding protein, fatty acid synthase, sterol regulatory element-binding protein 1, perilipin, leptin, lipoprotein lipase and hormone-sensitive lipase. These effects of genistein during the differentiation period were associated with down-regulation of ERalpha and ERbeta expression. This study adds to the elucidation of the molecular pathways involved in the inhibition of adipogenesis by phytoestrogens.

ICV vs. VMH injection of leptin: comparative effects on hypothalamic gene expression.

Leptin regulates feeding behavior and body weight by binding to its receptors localized in specific areas of the hypothalamus. Leptin injected twice daily for 4 days either into the right ventromedial hypothalamus (VMH) or into the right lateral cerebral ventricle (ICV) and using Real-Time Taqman RT-PCR, mRNA expression levels of selected genes in the arcuate nucleus-median eminence (ARC-ME) complex were quantitatively measured. Expression of selected genes from the ipsi- vs. contralateral VMH areas in rats injected with leptin into the VMH was also compared. VMH injections of leptin increased ARC-ME mRNAs of proopiomelanocortin (POMC), 27.3% (p<0.05); gamma-aminobutyric acid A receptor (GABRD), 89.3% (p<0.01); and thyrotropin-releasing hormone (TRH), 57.7% (p<0.01); and decreased janus kinase 2 (JAK2), 44.4% (p<0.001); suppressor of cytokine signaling 3 (SOCS3), 86.6% (p<0.001); signal transducer and activator of transcription 3 (STAT3), 46.8% (p<0.01); tyrosine hydroxylase (TH), 51.1% (p<0.001); prostaglandin E synthase (PTGES), 96.5% (p<0.001); tumor necrosis factor-alpha (TNF-alpha), 47% (p<0.01); and secretin, 55.4% (p<0.001). Only GABRD, 76.6% (p<0.01) and SCT, 64.9% (p<0.01) were up-regulated in the hypothalamic ARC-ME of rats with ICV leptin injections. VMH injections of leptin induced identical reductions in expression levels of CART, SOCS3, PTGES, and TNF-alpha in both VMH areas; except TH mRNA, whose expression was lowered ipsilaterally. Food intake, body and fat pad weights and serum insulin and leptin were also decreased in rats given leptin through VMH. This study suggests that leptin either unilateral exposure through VMH or bilateral exposure through ICV injections induces divergent ARC-ME gene profiles.

Adipose tissue gene expression profiles in ob/ob mice treated with leptin.

Leptin plays a critical role in regulating body weight, lipid metabolism, apoptosis and microvasculature of adipose tissue. To explore multiple signaling pathways of leptin action on adipose tissue, real-time PCR utilizing TaqMan low-density arrays was performed to compare mRNA expression in adipose tissue of ob/ob mice treated with vehicle or leptin (2.5 microg/d or 10 microg/d) for 14 days via subcutaneous osmotic minipumps. Of the 24 target genes selected for characterization, many were differentially expressed between control ob/ob mice and leptin-treated ob/ob mice. Increases in mRNA expression were found for hormone sensitive lipase (HSL), uncoupling protein 2 (UCP2), adrenergic receptor 3 (ADR3), mitofusin 2 (Mfn2), sirtuin 3 (Sirt3), transcription factor sterol regulatory element binding factor 1 (SREBF1), Bcl-2, Bax, Caspase 3, tumor necrosis factor alpha (TNFalpha), adiponectin and angiopoietin 2 (Ang-2). Decreases in expression were found for stearoyl-coenzyme A desaturase 1 (SCD1), fatty acid synthase (FAS), and retinol binding protein 4 (RBP4). There were no changes in expression of transcription factors involved in adipocyte differentiation (C/EBPalpha, PPARalpha, and PPARgamma). These results confirm that alterations in the expression of specific adipose tissue genes including those associated with the promotion of lipid mobilization, energy dissipation, and apoptosis may mediate leptin-induced fat loss in ob/ob mice.

Enhanced effects of xanthohumol plus honokiol on apoptosis in 3T3-L1 adipocytes.

OBJECTIVE: To study the effects of xanthohumol (XN), a flavonoid found in hops (Humulus lupulus) and honokiol (HK), a lignan isolated from Magnolia officinalis, alone and in combination, on apoptotic signaling in 3T3-L1 adipocytes. METHODS AND PROCEDURES: 3T3-L1 mature adipocytes were incubated with various concentrations of XN and HK alone and in combination. Viability and apoptosis were quantified using an MTS-based cell viability assay and single-stranded DNA assay, respectively. Expression of apoptosis related proteins including cleaved poly(ADP-ribose) polymerase (PARP), cytochrome c, Bcl-2, caspase-3/7, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Akt was analyzed by western blotting. RESULTS: Combinations of XN and HK significantly decreased viability and induced apoptosis in a dose-dependent manner and more than the additive responses to XN and HK alone. Western blot analysis showed an increase in cleaved PARP and cytochrome c release and decrease in expression of Bcl-2 protein by XN plus HK, whereas XN and HK individually had no effect. Furthermore, the combination of XN and HK activated PTEN and inactivated Akt by decreasing levels of phosphorylated PTEN and phosphorylated Akt. DISCUSSION: We demonstrated that although XN and HK showed little or no effect as individual compounds, in combination (XN plus HK) they showed enhanced activity in inducing apoptosis via the cytochrome c/caspase-3/PARP and PTEN/Akt pathways in 3T3-L1 adipocytes.

A putative role for apelin in the etiology of obesity.

Apelin, the endogenous ligand of the G protein-coupled APJ receptor has been shown to promote tumor angiogenesis. However, the effect of apelin on inducing angiogenesis in adipose tissue has not been investigated. In this review, we propose a putative role for apelin in promoting angiogenesis in adipose tissue. We further propose that targeting adipose tissue vasculature by blocking apelin signaling with anti-apelin antibodies will lead not only to inhibition of angiogenesis in adipose tissue but also to decreased adiposity.

ICV leptin effects on spontaneous physical activity and feeding behavior in rats.

Although leptin causes negative energy balance by reducing food intake and increasing energy expenditure, the effect of leptin on spontaneous physical activity (SPA) is not clearly established. To test the hypothesis that leptin enhances SPA in rats, male Sprague-Dawley rats were injected intracerebroventricularly (ICV) with either 10mug of leptin or artificial cerebrospinal fluid (aCSF) before dark onset (12:00h) once daily for 5 successive days. The rats were individually housed in behavioral monitoring cages to measure feeding behavior and SPA throughout the study. Both groups had a diurnal pattern of SPA being low during the light period and high during the dark period. Specifically, there were two peaks of SPA during the dark period, with the first peak taking place around the dark onset and the second occurring approximately 6h towards the light onset. Leptin treatment resulted in a significant increase in SPA whether or not it was expressed in terms of light-dark, daily or diurnal basis. Increased SPA was consistently observed throughout the entire 5-day study in spite of the fact that the rats were consistently eating less and losing body weight. With reduction in weight of fat pads and increase in apoptosis of fat pads but no change in body temperature, leptin decreased size, duration and number of meals without altering eating rate, thereby increasing satiety. Our data show that increased activity is a key determinant in negative energy balance induced by leptin, which cannot be accounted for solely by the leptin-induced food intake reduction.