Dectin-3-targeted antifungal liposomes efficiently bind and kill diverse fungal pathogens.

DectiSomes are anti-infective drug-loaded liposomes targeted to pathogenic cells by pathogen receptors including the Dectins. We have previously used C-type lectin (CTL) pathogen receptors Dectin-1, Dectin-2, and DC-SIGN to target DectiSomes to the extracellular oligoglycans surrounding diverse pathogenic fungi and kill them. Dectin-3 (also known as MCL, CLEC4D) is a CTL pathogen receptor whose known cognate ligands are partly distinct from other CTLs. We expressed and purified a truncated Dectin-3 polypeptide (DEC3) comprised of its carbohydrate recognition domain and stalk region. We prepared amphotericin B (AmB)-loaded pegylated liposomes (AmB-LLs) and coated them with this isoform of Dectin-3 (DEC3-AmB-LLs), and we prepared control liposomes coated with bovine serum albumin (BSA-AmB-LLs). DEC3-AmB-LLs bound to the exopolysaccharide matrices of Candida albicans, Rhizopus delemar (formerly known as R. oryzae), and Cryptococcus neoformans from one to several orders of magnitude more strongly than untargeted AmB-LLs or BSA-AmB-LLs. The data from our quantitative fluorescent binding assays were standardized using a CellProfiler program, AreaPipe, that was developed for this purpose. Consistent with enhanced binding, DEC3-AmB-LLs inhibited and/or killed C. albicans and R. delemar more efficiently than control liposomes and significantly reduced the effective dose of AmB. In conclusion, Dectin-3 targeting has the potential to advance our goal of building pan-antifungal DectiSomes.

DC-SIGN targets amphotericin B-loaded liposomes to diverse pathogenic fungi.

BACKGROUND: Life-threatening invasive fungal infections are treated with antifungal drugs such as Amphotericin B (AmB) loaded liposomes. Our goal herein was to show that targeting liposomal AmB to fungal cells with the C-type lectin pathogen recognition receptor DC-SIGN improves antifungal activity. DC-SIGN binds variously crosslinked mannose-rich and fucosylated glycans and lipomannans that are expressed by helminth, protist, fungal, bacterial and viral pathogens including three of the most life-threatening fungi, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. Ligand recognition by human DC-SIGN is provided by a carbohydrate recognition domain (CRD) linked to the membrane transit and signaling sequences. Different combinations of the eight neck repeats (NR1 to NR8) expressed in different protein isoforms may alter the orientation of the CRD to enhance its binding to different glycans. RESULTS: We prepared two recombinant isoforms combining the CRD with NR1 and NR2 in isoform DCS12 and with NR7 and NR8 in isoform DCS78 and coupled them to a lipid carrier. These constructs were inserted into the membrane of pegylated AmB loaded liposomes AmB-LLs to produce DCS12-AmB-LLs and DCS78-AmB-LLs. Relative to AmB-LLs and Bovine Serum Albumin coated BSA-AmB-LLs, DCS12-AmB-LLs and DCS78-AmB-LLs bound more efficiently to the exopolysaccharide matrices produced by A. fumigatus, C. albicans and C. neoformans in vitro, with DCS12-AmB-LLs performing better than DCS78-AmB-LLs. DCS12-AmB-LLs inhibited and/or killed all three species in vitro significantly better than AmB-LLs or BSA-AmB-LLs. In mouse models of invasive candidiasis and pulmonary aspergillosis, one low dose of DCS12-AmB-LLs significantly reduced the fungal burden in the kidneys and lungs, respectively, several-fold relative to AmB-LLs. CONCLUSIONS: DC-SIGN's CRD specifically targeted antifungal liposomes to three highly evolutionarily diverse pathogenic fungi and enhanced the antifungal efficacy of liposomal AmB both in vitro and in vivo. Targeting significantly reduced the effective dose of antifungal drug, which may reduce drug toxicity, be effective in overcoming dose dependent drug resistance, and more effectively kill persister cells. In addition to fungi, DC-SIGN targeting of liposomal packaged anti-infectives have the potential to alter treatment paradigms for a wide variety of pathogens from different kingdoms including protozoans, helminths, bacteria, and viruses which express its cognate ligands.

Antifungal Liposomes Directed by Dectin-2 Offer a Promising Therapeutic Option for Pulmonary Aspergillosis.

Invasive fungal diseases cause millions of deaths each year. There are currently approximately 300,000 acute cases of aspergillosis, most of which result from a pulmonary infection of immunocompromised patients by the common soil organism and opportunistic pathogen Aspergillus fumigatus Patients are treated with antifungal drugs, such as amphotericin B (AmB). However, AmB has serious limitations due to human organ toxicity. AmB is slightly less toxic if loaded in liposomes, such as AmBisome or AmB-loaded liposomes (AmB-LLs). Even with antifungal therapy, recurrent infections are common, and 1-year fatality rates may exceed 50%. We have previously shown that coating AmB-LLs with the extracellular oligomannan-binding domain of the C-type lectin receptor Dectin-2 (DEC2-AmB-LLs) effectively targets DEC2-AmB-LLs to cell walls, exopolysaccharide matrices, and biofilms of fungal pathogens in vitroIn vitro, DEC2-AmB-LLs reduce the effective dose of AmB for 95% inhibition and killing of A. fumigatus 10-fold compared to that of untargeted AmB-LLs. Herein we tested the antifungal activity of DEC2-AmB-LLs relative to that of untargeted AmB-LLs in immunosuppressed mice with pulmonary aspergillosis. Remarkably, DEC2-AmB-LLs bound 30-fold more efficiently to A. fumigatus at sites of infection in the lungs. Furthermore, Dectin-2-targeted liposomes delivering AmB at a dose of 0.2 mg/kg of body weight significantly reduced the fungal burden in lungs compared to results with untargeted AmB-LLs at 0.2 mg/kg and micellar voriconazole at 20 mg/kg and prolonged mouse survival. By dramatically increasing the efficacy of antifungal drugs at low doses, targeted liposomes have the potential to create a new clinical paradigm to treat diverse fungal diseases.IMPORTANCE Invasive aspergillosis (IA) generally results from a pulmonary infection of immunocompromised patients by the common soil organism and opportunistic pathogen Aspergillus fumigatus The susceptible population has expanded rapidly due to the increased number of cancer patients with immunocompromising chemotherapy and transplant patients taking immunosuppressants. Patients are treated with antifungals, such as liposomal amphotericin B, with per-patient costs exceeding $50,000 in the United States. However, AmB has serious side effects due to host toxicity, which limits its usage and contributes to the lack of fungal clearance in patients at safe doses. Fifty percent of IA patients die within a year. Herein, we employed liposomal amphotericin B coated with the innate immune receptor Dectin-2 to direct antifungals specifically to the fungal pathogen. Using two mouse models of pulmonary aspergillosis, we demonstrate that Dectin-2-targeted delivery of amphotericin B to A. fumigatus resulted in remarkably higher efficacy than that of the untargeted antifungal formulations.

Patients with obstructive sleep apnea have suppressed levels of soluble cytokine receptors involved in neurodegenerative disease, but normal levels with airways therapy.

PURPOSE: Obstructive sleep apnea (OSA) results in systemic intermittent hypoxia. By one model, hypoxic stress signaling in OSA patients alters the levels of inflammatory soluble cytokines TNF and IL6, damages the blood brain barrier, and activates microglial targeting of neuronal cell death to increase the risk of neurodegenerative disorders and other diseases. However, it is not yet clear if OSA significantly alters the levels of the soluble isoforms of TNF receptors TNFR1 and TNFR2 and IL6 receptor (IL6R) and co-receptor gp130, which have the potential to modulate TNF and IL6 signaling. METHODS: Picogram per milliliter levels of the soluble isoforms of these four cytokine receptors were estimated in OSA patients, in OSA patients receiving airways therapy, and in healthy control subjects. Triplicate samples were examined using Bio-Plex fluorescent bead microfluidic technology. The statistical significance of cytokine data was estimated using the nonparametric Wilcoxon rank-sum test. The clustering of these high-dimensional data was visualized using t-distributed stochastic neighbor embedding (t-SNE). RESULTS: OSA patients had significant twofold to sevenfold reductions in the soluble serum isoforms of all four cytokine receptors, gp130, IL6R, TNFR1, and TNFR2, as compared with control individuals (p = 1.8 × 10-13 to 4 × 10-8). Relative to untreated OSA patients, airways therapy of OSA patients had significantly higher levels of gp130 (p = 2.8 × 10-13), IL6R (p = 1.1 × 10-9), TNFR1 (p = 2.5 × 10-10), and TNFR2 (p = 5.7 × 10-9), levels indistinguishable from controls (p = 0.29 to 0.95). The data for most airway-treated patients clustered with healthy controls, but the data for a few airway-treated patients clustered with apneic patients. CONCLUSIONS: Patients with OSA have aberrantly low levels of four soluble cytokine receptors associated with neurodegenerative disease, gp130, IL6R, TNFR1, and TNFR2. Most OSA patients receiving airways therapy have receptor levels indistinguishable from healthy controls, suggesting a chronic intermittent hypoxia may be one of the factors contributing to low receptor levels in untreated OSA patients.

Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy.

Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2's mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases.IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

Binding and Synergizing Motif within Coleopteran Cadherin Enhances Cry3Bb Toxicity on the Colorado Potato Beetle and the Lesser Mealworm.

Cry3Bb toxin from Bacillus thuringiensis is an important insecticidal protein due to its potency against coleopteran pests, especially rootworms. Cadherin, a protein in the insect midgut epithelium, is a receptor of Cry toxins; in some insect species toxin-binding domains of cadherins-synergized Cry toxicity. Previously, we reported that the DvCad1-CR8-10 fragment of Diabrotica virgifera virgifera cadherin-like protein (GenBank Accession #EF531715) enhanced Cry3Bb toxicity to the Colorado Potato Beetle (CPB), Leptinotarsadecimlineata (L. decimlineata). We report that individual CR domains of the DvCad1-CR8-10 fragment were found to have strong binding affinities to α-chymotrypsin-treated Cry3Bb. The dissociation constant (Kd) of Cry3Bb binding to the CR8, CR9, and CR10 domain was 4.9 nM, 28.2 nM, and 4.6 nM, respectively. CR8 and CR10, but not CR9, enhanced Cry3Bb toxicity against L. decimlineata and the lesser mealworm Alphitobius diaperinus neonates. In-frame deletions of the DvCad1-CR10 open reading frame defined a high-affinity binding and synergistic site to a motif in residues I1226-D1278. A 26 amino acid peptide from the high affinity Cry3Bb-binding region of CR10 functioned as a Cry3Bb synergist against coleopteran larvae.

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.

Differential protection of Cry1Fa toxin against Spodoptera frugiperda larval gut proteases by cadherin orthologs correlates with increased synergism.

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin.

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.

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.

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.

Withaferin A induces apoptosis and inhibits adipogenesis in 3T3-L1 adipocytes.

Withaferin A (WA), a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera (also called ashwagandha) has been reported to have anti-tumor, anti-angiogenesis, and pro-apoptotic activity. We investigated the effects of WA on viability, apoptosis and adipogenesis in 3T3-L1 adipocytes. Pre- and post-confluent preadipocytes and mature adipocytes were treated with WA (1-25 microM) up to 24 hrs. Viability and apoptosis were measured by CellTiter-Blue Cell Viability Assay and single strand DNA ELISA Assay, respectively. WA decreased viability and induced apoptosis in all stages of cells. Induction of apoptosis by WA in mature adipocytes was mediated by increased ERK1/2 phosphorylation and altered Bax and Bcl2 protein expression. The effect of WA on adipogenesis was examined by AdipoRed Assay after treating with WA (0.1-1 microM) during the differentiation period. WA decreased lipid accumulation in a dose-dependent manner and decreased the expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha and adipocyte fatty acid binding protein. The effects on apoptosis and lipid accumulation were also confirmed with Hoechst staining and Oil Red O staining, respectively. These results show that WA acts on adipocytes to reduce cell viability and adipogenesis and also induce apoptosis.

Resveratrol potentiates genistein's antiadipogenic and proapoptotic effects in 3T3-L1 adipocytes.

Genistein (G) and resveratrol (R) individually inhibit adipogenesis in 3T3-L1 adipocytes and induce apoptosis in cancer cells. We investigated whether the combination of G and R resulted in enhanced effects on adipogenesis, lipolysis, and apoptosis in 3T3-L1 cells. Preadipocytes and mature adipocytes were treated with G and R individually at 50 and 100 micromol/L (G100; R100) and in combination. Both in preadipocytes and mature adipocytes, G and R individually decreased cell viability dose-dependently, but G100 + R100 further decreased viability by 59 +/- 0.97% (P < 0.001) and 69.7 +/- 1.2% (P < 0.001) after 48 h compared with G100 and R100, respectively. G100 + R100 induced apoptosis 242 +/- 8.7% (P < 0.001) more than the control after 48 h, whereas G100 and R100 individually increased apoptosis only 46 +/- 9.2 and 46 +/- 7.9%, respectively. G and R did not modulate mitogen-activated protein kinase expression by themselves, but G100 + R100 increased Jun-N-terminal kinase phosphorylation by 38.8 +/- 4.4% (P < 0.001) and decreased extracellular signal-regulating kinase phosphorylation by 48 +/- 3.4% (P < 0.001). Individually, G and R at 25 micromol/L (G25; R25) decreased lipid accumulation by 30 +/- 1.7% and 20.07 +/- 4.27%, respectively (P < 0.001). However, G25 + R25 decreased lipid accumulation by 77.9 +/- 3.4% (P < 0.001). Lipolysis assay revealed that neither G25 nor R25 induced lipolysis, whereas G25 + R25 significantly increased lipolysis by 25.5 +/- 4.6%. The adipocyte-specific proteins PPARgamma and CCAAT/enhancer binding protein-alpha were downregulated after treatment with G + R, but no effect was observed with individual compounds. These results indicate that G and R in combination produce enhanced effects on inhibiting adipogenesis, inducing apoptosis, and promoting lipolysis in 3T3-L1 adipocytes. Thus, the combination of G and R is more potent in exerting antiobesity effects than the individual compounds.

Gene expression in arcuate nucleus-median eminence of rats treated with leptin or ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) and leptin are cytokine-like% hormones and act on their corresponding receptors in the hypothalamic arcuate nucleus (ARC). The present study was designed to assess effects of intracerebroventricular (ICV) injection of leptin and CNTF on gene expression in micropunched hypothalamic arcuate nucleus-median eminence (ARC-ME) complex samples from rats. Male Sprague Dawley rats were implanted with lateral cerebroventricular cannulas for administration of control, 10 microg/d leptin or 5 microg/d CNTF for four days. Real-time Taqmantrade mark RT-PCR was used to quantitatively compare the mRNA levels of selected genes in the ARC-ME complex. Leptin and CNTF increased ARC-ME mRNA levels of signal transducer and activator of transcription 3 (STAT3) by 64.5 and 124.7% (p<0.01), suppressor of cytokine signaling 3 (SOCS3) by 258.9 and 1063.9% (p<0.01), cocaine and amphetamine regulated transcript (CART) by 102.7 and 123.1% (p<0.01), and proopiomelanocortin (POMC2) by 374.1 and 264.9% (p<0.01), respectively. Leptin increased growth hormone releasing hormone (GHRH) by 309.9% (p<0.01), while CNTF increased janus kinase 2 (JAK2) mRNA by 31.7% (p<0.01) and decreased gonadotropin releasing hormone 1 (GNRH1) by 59.7% (p<0.01), mitogen activated protein kinase 1 (MAPK1) by 19.4% (p<0.05) and tyrosine hydroxylase (TH) by 74.5% (p<0.05). Significant reduction in daily food intake and body weights by both the treatments was observed. Also, decrease in weights of fat pads was concomitant with lowered serum insulin and leptin levels. Our findings show that leptin and CNTF engage both convergent and divergent pathways involved in feeding, cellular signaling, inflammation, and other related regulatory systems.