Stimulation with Peptidoglycan induces interleukin 6 and TLR2 expression and a concomitant downregulation of expression of adiponectin receptors 1 and 2 in 3T3-L1 adipocytes.

BACKGROUND: Inflammation is a major component of obesity and diabetes, and toll-like receptors (TLRs) play critical roles in the regulation of inflammation and response to pathogen associated molecular patterns (PAMPs) and fatty acids in. Although immune cells such as macrophages are primarily responsible for recognition and clearance of pathogens, adipocytes are also closely involved in the regulation of innate immunity and inflammation. Whereas it has been demonstrated that adipocytes respond to TLR4 stimulation with lipopolysacccharide, very little is known about their response to the TLR2 agonist, peptidoglycan. METHODS: We investigated the response to peptidoglycan from Staphylococcus aureus in differentiated 3T3-L1 adipocytes. Real-time PCR analysis was used to quantify the expression of interleukin 6 (IL6), adiponectin receptors (adipoR1 and adipoR2), toll-like receptor 2 (TLR2) and 4 (TLR2 4). Media level of IL6 was determined with ELISA. RESULTS: Adipocyte stimulation peptidoglycan induces IL6 expression (P < 0.01). Both siRNA mediated suppression of TLR2 and immunoneutralization of TLR2 with a TLR2 specific antibody inhibited response to peptidoglycan (P < 0.05). We also examined the regulation of TLR2 and TLR4 mRNA in peptidoglycan treated cells. Both peptidoglycan and lipopolysaccharide (LPS) robustly induce TLR2 mRNA expression, whereas TLR4 mRNA is weakly induced by LPS only (P < 0.05). Additionally, peptidoglycan downregulates the mRNA expression of adiponectin receptors, adipoR1 and adipoR2 (P < 0.05). CONCLUSION: Obesity and type 2 diabetes are associated with increased expression of TLR2, this receptor could play a significant but previously unrecognized role in the establishment of chronic inflammation in adipose tissue in obesity.

Derivatives of Z-bisdehydrodoisynolic acid provide a new description of the binding-activity paradox and selective estrogen receptor modulator activity.

Z-Bisdehydrodoisynolic acid [(+/-)-Z-BDDA], an estrogenic carboxylic acid, is highly active in vivo yet binds poorly to estrogen receptors (ERs). Studies of Z-BDDA and its enantiomers demonstrate therapeutic potential as selective ER modulators; however, the activity vs. binding paradox has remained. One possible explanation is that the carboxylic acid group of Z-BDDA may be modified in vivo to an ester or amide. Synthesis of these derivatives showed the relative binding affinity (RBA) of the methyl ester for ERalpha and ERbeta was increased approximately 14- and 20-fold, respectively, relative to the parent compound. Yet, this increased affinity did not result in increased reporter gene expression. In contrast, the amide showed an unexpected approximately 4-fold decrease in RBA to both ERs compared with the parent. The relationship among the RBAs of ester, acid, and amide is consistent with their predicted polarity, suggesting the carboxylic acid, and not the carboxylate of BDDA, binds to ERs. Studies at pH 6.5, 7.4, and 8.0 were consistent with a simple acid-base equilibrium model, with BDDA binding as the undissociated acid and with affinity equal to or exceeding that of estradiol, consistent with high in vivo potency. Furthermore, the alcohol BDD-OH also demonstrated high affinity and increased activity in gene expression assays. In addition to suggesting a resolution to the decades-old binding/activity paradox, these studies may provide a direction for definitive in vivo metabolic and pharmacokinetic studies and provide additional insight into the chemical and metabolic determinants of BBDA's unique tissue selectivity and selective ER modulator activities.

(+)-Z-Bisdehydrodoisynolic acid ameliorates obesity and the metabolic syndrome in female ZDF rats.

OBJECTIVE: The putative selective estrogen receptor modulator (+)-Z-bisdehydrodoisynolic acid (Z-BDDA) has been found to improve cardiovascular risk in rodents. The objective of this study was to investigate the effectiveness of (+)-Z-BDDA compared with the antidiabetic drug, rosiglitazone, in treating obesity and risk factors associated with the metabolic syndrome. RESEARCH METHODS AND PROCEDURES: Female Zucker Diabetic Fatty rats were randomly assigned to three treatment groups for 29 weeks: control (C), 1.8 mg (+)-Z-BDDA/kg diet [control diet + (+)-Z-BDDA (CB)], or 100 mg rosiglitazone/kg diet [control diet + rosiglitazone (CR)]. At sacrifice, physiological, biochemical, and molecular parameters were examined. RESULTS: CB animals gained less weight and exhibited a decrease in total body lipids (p < 0.05) as compared with C or CR rats. Body weight and total body lipids were the highest in CR rats (p < 0.05). Liver weights in CB and CR rats were lower (p < 0.05) than in C rats, whereas kidney weights were lower in CB (p < 0.05) than in C and CR animals. Fasting plasma glucose was lower (p < 0.05) in the CB and CR animals when compared with C animals. C rats exhibited the highest concentration of total plasma cholesterol, and CR-treated rats exhibited the lowest concentration. Plasma triglycerides followed the same pattern as plasma cholesterol. Histomorphometry of heart vasculature revealed that CB and CR treatments produced a significant shift from small to large venules and arterioles compared with C (p < 0.05). Liver expression profiles of peroxisome proliferator-activated receptor (PPAR) alpha, PPARgamma, and PPAR-regulated genes revealed encouraging CB-induced effects. DISCUSSION: These results suggest that (+)-Z-BDDA may have applications in treating obesity and complications associated with the metabolic syndrome.

Soy isoflavones exert antidiabetic and hypolipidemic effects through the PPAR pathways in obese Zucker rats and murine RAW 264.7 cells.

The hypocholesterolemic and anti-atherosclerotic mechanism by which soy may exert a beneficial effect remains unclear. Peroxisome-proliferator activated receptors (PPAR) are promiscuous nuclear receptors that regulate the transcription of genes involved in lipid and glucose homeostasis and lipid metabolism within the cell. We hypothesize that the isoflavones improve lipid and glucose metabolism by acting as an antidiabetic PPAR agonist. Male and female obese Zucker rats (OZR) were used as a model of Type 2 diabetes, and OZR fed a high isoflavone soy protein diet displayed improvements in lipid metabolism consistent with results in humans treated with antidiabetic PPAR agonists such as the fibrates or glitazones. Liver triglyceride and cholesterol concentrations were lower in all OZR fed high-isoflavone soy protein diets than in rats fed low-isoflavone and casein diets (P < 0.05). Concurrently, PPAR-directed gene expression was evaluated in a cell culture model. An isoflavone-containing soy extract doubled PPAR-directed gene expression (P < 0.05) in RAW 264.7 cells containing either a PPARalpha or PPARgamma expression plasmid. A similar induction was observed when the soy isoflavones genistein or daidzein were used to treat cells. Both isoflavones doubled PPARalpha-directed gene expression (P < 0.05), whereas they increased PPARgamma-directed gene expression 200-400% (P < 0.05). This study suggests that soy isoflavones improve lipid metabolism, produce an antidiabetic effect, and activate PPAR receptors.

Activities of a non-classical estrogen, Z-bis-dehydrodoisynolic acid, with ERalpha and ERbeta.

(+/-)-Z-bis-Dehydrodoisynolic acid [(+/-)-Z-BDDA] is highly estrogenic in vivo, yet binds to estrogen receptor (ER) poorly. This paradox has raised the possibility of alternative ERs and/or molecular mechanisms. To address the possibility of high activities of Z-BDDA with ERbeta, we determined the activities of (+)-Z-BDDA and (-)-Z-BDDA, in cell culture and in vitro, comparing ERbeta to ERalpha. Transfectional analysis in Hela cells showed (-)-Z-BDDA is an agonist for gene activation with both ERalpha (EC(50) congruent with 0.3nM) and ERbeta (EC(50) congruent with 5nM), while little to no activity was observed with (+)-Z-BDDA. Similarly, in gene repression assays, (-)-Z-BDDA was active (EC(50) congruent with 0.2nM), but again minimal activity was exhibited by (+)-Z-BDDA. Binding to ERalpha and ERbeta in vitro used both competition and a direct binding assay. For ERalpha, the relative affinity of (-)-Z-BDDA was approximately 6% by competition and 1.7% by direct binding versus 17beta-estradiol (E2; 100%), while (+)-Z-BDDA also demonstrated binding, but with relative affinities of only 0.08% by competition and 0.3% by the direct assay. For ERbeta, the affinity of (-)-Z-BDDA was approximately 7% by competition and 1.5% by the direct assay relative to E2 (100%), while (+)-Z-BDDA had lower affinity, approximately 0.2% that of E2 by both assays. The paradox of potent in vivo activity but lower activity in receptor binding and in cell culture reporter gene assays, previously seen with ERalpha is now also associated with ERbeta. The failure of ERbeta to explain the activity-binding paradox indicates the need for additional in vivo metabolic and pharmacokinetic studies and continued consideration of alternative mechanisms.

Antioxidant effects of phyto-and synthetic-estrogens on cupric ion-induced oxidation of human low-density lipoproteins in vitro.

Oxidation of low-density lipoproteins (LDL) promotes the formation of atherosclerotic plaques. Estrogenic compounds (EC) from foods and other natural products, and synthetic estrogenic compounds (SECs) may prevent heart disease by inhibiting LDL oxidation. In the present study, we tested the antioxidant capacities of two phytoestrogens, daidzein (DAI) and genistein (GEN), and four SECs, (+)- and (-)-Z-bisdehydrodoisynolic acid (ZBDDA), and (+)- and (-)-hydroxy-allenoic acid (HAA), on isolated human LDL subjected to oxidation by cupric sulfate. The effects of these estrogenic compounds on the kinetics of conjugated diene formation in LDL undergoing oxidation were evaluated with a lag-time assay with continuous monitoring of absorbance at 234 nm. Lag-time data revealed that (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA had similarly stronger antioxidant activities than either GEN or DAI. We also found that (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA strongly inhibited the formation of Cu+-induced thiobarbituric acid reactive substances (TBARS) in LDL, and that GEN and DAI were less effective for inhibiting LDL lipid peroxidation. Finally, electrophoretic evaluation suggested that (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA protected the apolipoprotein B-100 of LDL against oxidation better than did GEN or DAI. In summary, the four SECs, (+)-HAA, (-)-HAA, (+)-ZBDDA, and (-)-ZBDDA, were more potent antioxidants than the phytoestrogens, DAI and GEN.