Sorghum (Sorghum bicolor) Extract-Induced Adipogenesis Is Independent of PPARγ Ser273 Phosphorylation in 3T3-L1 Adipocytes.

Previously we showed that the water-soluble fraction of sorghum extract (SE) improves adipogenesis in 3-isobutyl-1-methylxanthine (IBMX)/dexamethasone/insulin (MDI)/thiazolidinedione (TZD)-induced 3T3-L1 preadipocytes but downregulates genes related to peroxisome proliferator-activated receptor γ (PPARγ) and adipogenesis in both MDI- and MDI/TZD-induced 3T3-L1 adipocytes. In this study, we showed that SE treatment altered the accumulation of stained lipids in 3T3-L1 adipocytes induced by MDI/troglitazone (Tro). Immunoblot analyses indicated that SE treatment reduced adipocyte protein 2 (aP2) expression and induced peroxisome proliferator-activated receptor α (PPARα) protein expression in the presence of Tro in 3T3-L1 adipocytes. MDI/Tro treatment, but not MDI treatment, of 3T3-L1 cells induced PPARγ phosphorylation at Ser273. SE downregulated PPARγ expression in MDI-induced 3T3-L1 adipocytes and did not affect its phosphorylation at Ser273 in MDI- and MDI/Tro-induced 3T3-L1 adipocytes. Therefore, SE likely promotes adipogenesis and lipid metabolism in 3T3-L1 preadipocytes by cooperating with Tro independent of PPARγ Ser273 phosphorylation.

A New Agonist for Peroxisome Proliferation-activated Receptor γ (PPARγ), Fraglide-1 from Zhenjiang Fragrant Vinegar: Screening and Characterization Based on Cell Culture Experiments.

Zhenjiang fragrant vinegar (Kozu) is a black rice vinegar that has been used as a traditional Chinese medicine and has various health benefits, including anti-obesity effects. In the present study, using luciferase reporter assays of PPARγ promoter activity, a novel ingredient from 8-year-old Kozu, 5-hydroxy-4-phenyl-butenolide, was isolated. The newly found agonist was named as "Fraglide-1". Moreover, in subsequent experiments, it was confirmed that fraglide-1 was an PPARγ agonist and it could increase expression level of the uncoupling protein (UCP)-1. Fraglide-1 was chemically synthesized and it was verified that expression of the PPARγ was increased in dose dependent manner. Although Kozu has been consumed globally as a functional food for thousands of years, the mechanisms behind its health effects have not been characterized. The active ingredient of Kozu was successfully found and the results unraveled a longtime mystery about Kozu for its beneficial health effect.

A new synthetic matrix metalloproteinase inhibitor reduces human mesenchymal stem cell adipogenesis.

Development of adipose tissue requires the differentiation of less specialized cells, such as human mesenchymal stem cells (hMSCs), into adipocytes. Since matrix metalloproteinases (MMPs) play critical roles in the cell differentiation process, we conducted investigations to determine if a novel mercaptosulfonamide-based MMP inhibitor (MMPI), YHJ-7-52, could affect hMSC adipogenic differentiation and lipid accumulation. Enzyme inhibition assays, adipogenic differentiation experiments, and quantitative PCR methods were employed to characterize this inhibitor and determine its effect upon adipogenesis. YHJ-7-52 reduced lipid accumulation in differentiated cells by comparable amounts as a potent hydroxamate MMPI, GM6001. However, YHJ-7-82, a non-inhibitory structural analog of YHJ-7-52, in which the zinc-binding thiol group is replaced by a hydroxyl group, had no effect on adipogenesis. The two MMPIs (YHJ-7-52 and GM6001) were also as effective in reducing lipid accumulation in differentiated cells as T0070907, an antagonist of peroxisome-proliferator activated receptor gamma (PPAR-gamma), at a similar concentration. PPAR-gamma is a typical adipogenic marker and a key regulatory protein for the transition of preadiopocyte to adipocyte. Moreover, MMP inhibition was able to suppress lipid accumulation in cells co-treated with Troglitazone, a PPAR-gamma agonist. Our results indicate that MMP inhibitors may be used as molecular tools for adipogenesis and obesity treatment research.

Quercetin metabolites inhibit MMP-2 expression in A549 lung cancer cells by PPAR-γ associated mechanisms.

Our previous study demonstrated that quercetin-metabolite-enriched plasma (QP) but not quercetin itself upregulates peroxisome proliferator-activated receptor gamma (PPAR-γ) expression to induce G2/M arrest in A549 cells. In the present study, we incubated A549 cells with QP as well as quercetin-3-glucuronide (Q3G) and quercetin-3'-sulfate (Q3'S), two major metabolites of quercetin, to investigate the effects of quercetin metabolites on cell invasion and migration, the possible mechanisms and the role of PPAR-γ. We also compared the effects of QP with those of quercetin and troglitazone (TGZ), a PPAR-γ ligand. The results showed that QP significantly suppressed cell invasion and migration, as well as matrix metalloproteinases (MMPs)-2 activity and expression in a dose-dependent manner. The effects of 10% QP on those parameters were similar to those of 10µM quercetin and 20µM TGZ. However, QP and TGZ rather than quercetin itself increased the expressions of nm23-H1 and tissue inhibitor of metalloproteinase (TIMP-2). Furthermore, we demonstrated that Q3G and Q3'S also inhibited the protein expression of MMP-2. GW9662, a PPAR-γ antagonist, significantly diminished such an effect of Q3G and Q3'S. Silencing PPAR-γ expression in A549 cells also significantly diminished the suppression effect of Q3G and Q3'S on MMP-2 expression. Taken together, our study demonstrated that QP inhibited cell invasion and migration through nm23-H1/TIMP-2/MMP-2 associated mechanisms. The upregulation of PPAR-γ by quercetin metabolites such as Q3G and Q3'S could play an important role in the effects of QP.

Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons.

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB1 receptor activity. Here we investigated the possibility of a negative regulation by CB1 receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2'-chloroethylamide (ACEA) in a CB1-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL(-/-) mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB1 activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.

Identification of evodiamine as the bioactive compound in evodia (Evodia rutaecarpa Benth.) fruit extract that activates human peroxisome proliferator-activated receptor gamma (PPARγ).

The dried unripe fruit from Evodia rutaecarpa Benth., known as Wu zhu yu in China, has long been used in traditional Chinese medicine. In this research, we provide evidence that evodia fruit extract activates peroxisome proliferator-activated receptor gamma (PPARγ) and, as identified through HPLC fractionation and mass spectroscopy, the activating phytochemical is evodiamine. Evodiamine was shown to bind to and activate PPARγ. It was also shown to activate PPARγ-regulated gene expression in human hepatoma cells similar to known PPARγ ligands and that the expression was blocked by a PPARγ specific antagonist.

Small-Molecule targeting of translation initiation for cancer therapy.

Translation initiation plays a critical role in the regulation of cell growth and tumorigenesis. We report here that inhibiting translation initiation through induction of eIF2α phosphorylation by small-molecular-weight compounds restricts the availability of the eIF2.GTP.Met-tRNAi ternary complex and abrogates the proliferation of cancer cells in vitro and tumor growth in vivo. Restricting the availability of the ternary complex preferentially down-regulates the expression of growth-promoting proteins and up-regulates the expression of ER stress response genes in cancer cells as well as in tumors excised from either animal models of human cancer or cancer patients. These findings provide the first direct evidence for translational control of gene-specific expression by small molecules in vivo and indicate that translation initiation factors are bona fide targets for development of mechanism-specific anti-cancer agents.

The effect of omega-3 polyunsaturated fatty acids on the inflammatory response of the amnion.

OBJECTIVE: To investigate the effect of omega-3 PUFAs, eicosapentanoic acid (EPA) and docosohexanoic acid (DHA) on inflammatory cytokine production in the amnion. STUDY DESIGN: Amnion explants were obtained at elective caesarean sections and cultured in vitro with EPA and DHA. IL-8 and IL-6 secretion was determined by ELISA, the role of PPARγ was investigated using specific agonists and antagonists and activity of MMP assessed by gelatin zymography. RESULTS: A combination of EPA and DHA significantly reduced the concentration of IL-8 and IL-6 released into the supernatant compared to untreated controls (p<0.001). Stimulation of PPARγ with troglitazone reduced IL-8 production, and the PPARγ antagonist GW9662 partially reversed this effect. The activity of MMP-9 was also significantly reduced by treatment with EPA and DHA in combination compared to untreated control (p<0.05). CONCLUSION: The omega-3 PUFAs EPA and DHA decrease the inflammatory response of the amnion, and this may be partially mediated through PPARγ.

Anti-adipogenic diarylheptanoids from Alnus hirsuta f. sibirica on 3T3-L1 cells.

A new diarylheptanoid, (5S)-hydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-hepta-1E-en-3-one (1), was isolated along with seventeen known diarylheptanoids (2-18) from the methanol extract of Alnus hirsuta f. sibirica leaves using bioactivity-guided fractionation. Among the isolated compounds, compounds 1 and 2 and 4-12 reduced lipid accumulation dose-dependently in 3T3-L1 preadipocytes. Of the compounds active in the present assay system, the most potent compound 7, platyphyllonol-5-O-ß-d-xylopyranoside, significantly suppressed the induction of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer binding protein α (C/EBPα) protein expression, and inhibited adipocyte differentiation induced by troglitazone, a PPARγ agonist. It was demonstrated that compound 7 has anti-adipogenic activity mediated by the regulation of PPARγ dependent pathways.

Lupenone isolated from Adenophora triphylla var. japonica extract inhibits adipogenic differentiation through the downregulation of PPARγ in 3T3-L1 cells.

Adenophora triphylla var. japonica (Campanulaceae) is known to have anti-inflammatory and anti-tussive effects. Dysfunction of adipocytes and adipose tissue in obesity is related to various inflammatory cytokines or adipokines. In this study, we investigated whether lupenone isolated from A. triphylla var. japonica extract inhibits adipocyte differentiation and expression of adipogenic marker genes in 3T3-L1 preadipocytes. We demonstrated that lupenone resulted in a significant reduction in lipid accumulation and expression of adipogenic marker genes in a dose-dependent manner. In addition, lupenone decreased the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) induced by troglitazone, and we also demonstrated that lupenone suppressed the PPARγ and CCAAT-enhancer-binding protein α (C/EBPα) protein levels. These findings demonstrated that lupenone isolated from A. triphylla var. japonica extract effectively inhibited adipocyte differentiation through downregulation of related transcription factor, particularly the PPARγ gene.

1ß-Hydroxy-2-oxopomolic acid isolated from Agrimonia pilosa extract inhibits adipogenesis in 3T3-L1 cells.

In order to determine anti-adipogenic effect, this study investigated 1ß-hydroxy-2-oxopomolic acid (HOA) isolated from Agrimonia pilosa inhibits adipocyte differentiation and expression of adipogenic marker genes, such as peroxisome proliferator activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), glucose transporter 4 (GLUT4), adiponectin, adipocyte fatty acid-binding protein 2 (aP2), adipocyte determination and differentiation factor 1/sterol regulatory element binding protein 1c (ADD1/SREBP1c), resistin, and fatty acid synthase (Fas) in 3T3-L1 preadipocyte. We demonstrated that HOA induced a significant decrease in lipid accumulation and expression of adipogenic marker genes in a dose-dependent manner. In addition, HOA reduced the transcripitional activity of PPARγ induced by troglitazone, a potent diabetes agent; it also suppressed expression of PPARγ and C/EBPα protein levels. Our data suggest that HOA isolated from Agrimonia pilosa inhibits adipocyte differentiation through downregulation of various adipocytokines by blocking PPARγ and C/EBPα expression.

Effects of impressic acid from Acanthopanax koreanum on NF-κB and PPARγ activities.

Impressic acid, 3α,11α-dihydroxylup-20(29)-en-28-oic acid, is a lupane-type triterpenoid isolated from Acanthopanax koreanum, which has been used as a Korean folk medicine for rheumatism, hepatitis, diabetes, and inflammatory disorders. Recently, it was reported that impressic acid has inhibitory effects on the LPS-stimulated pro-inflammatory cytokine production in bone marrow-derived dendritic cells and on the nuclear factor of activated T-cells transcription factor activity. Thus, to investigate whether impressic acid has effects on the inhibition of nuclear factor kappa B (NF-κB) and activation of peroxisome proliferator-activated receptor gamma (PPARγ), luciferase reporter assays were used. The effects on the expression of NF-κB and PPARγ target genes were also examined by reverse transcription-polymerase chain reaction. In this study, impressic acid was found to inhibit tumor necrosis factor (TNF)-α induced NF-κB activity and to up-regulate transcriptional activity of PPARγ.

Regulation of dectin-1-mediated dendritic cell activation by peroxisome proliferator-activated receptor-gamma ligand troglitazone.

Dectin-1 is the major receptor for fungal ß-glucans. The activation of Dectin-1 leads to the up-regulation of surface molecules on dendritic cells (DCs) and cytokine secretion. Furthermore, Dectin-1 is important for the recruitment of leukocytes and the production of inflammatory mediators. Peroxisome proliferator-activated receptor-γ (PPAR-γ) and its ligands, cyclopentenone prostaglandins or thiazolidinediones, have modulatory effects on B-cell, T-cell, and DC function. In the present study, we analyzed the effects of troglitazone (TGZ), a high-affinity synthetic PPAR-γ ligand, on the Dectin-1-mediated activation of monocyte-derived human DCs. Dectin-1-mediated activation of DCs was inhibited by TGZ, as shown by down-regulation of costimulatory molecules and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation. Furthermore, TGZ inhibited the T-cell-stimulatory capacity of DCs. These effects were not due to a diminished expression of Dectin-1 or to a reduced phosphorylation of spleen tyrosine kinase; they were mediated by the inhibition of downstream signaling molecules such as mitogen-activated protein kinases and nuclear factor-κB. Furthermore, curdlan-mediated accumulation of caspase recruitment domain 9 (CARD9) in the cytosol was inhibited by TGZ. Our data demonstrate that the PPAR-γ ligand TGZ inhibits Dectin-1-mediated activation by interfering with CARD9, mitogen-activated protein kinase, and nuclear factor-κB signaling pathways. This confirms their important role as negative-feedback regulators of potentially harmful inflammatory responses.

SorLA modulates atheroprotective properties of CLA by regulating monocyte migration.

OBJECTIVE: We have previously shown that CLA induces regression of pre-established atherosclerotic lesions in apoE(-/-) mice. CLA is a known ligand of peroxisome proliferator activated receptors (PPARs) and it is postulated that CLA mediates its atheroprotective effects through activation of PPARs. Earlier work in our group identified the monocyte/macrophage cell as the primary cellular target of CLA. In this study we identified novel genes regulated by CLA during the regression of atherosclerosis and characterised a role for one of these, SorLA. SorLA is a member of the vacuolar protein sorting 10 protein (Vps10p) domain receptor family, which has structural homology with the LDLR family. METHODS AND RESULTS: Expression of SorLA was identified with its Vps10p family member Sort1 by transcriptomic analysis of murine aorta following CLA-induced regression of atherosclerosis. Decreased expression of both receptors was confirmed by real-time PCR in the aorta of CLA-supplemented mice. SorLA protein expression was predominantly localised to monocyte/macrophage cells in the vasculature by immunohistochemistry. CLA and the PPAR-γ agonists, troglitazone, and 15-deoxy-prostaglandin (PG) J(2), decreased protein and RNA expression of SorLA in THP-1 monocytes; while pre-treatment with a PPAR-γ antagonist established a PPAR-γ dependent role for CLA regulation of SorLA. CLA inhibits monocyte migration. Consistent with a role for SorLA in mediating this response, overexpression of SorLA increased migration of THP-1 monocytes to monocyte chemoattractant protein-1 with a coincident increase in UPAR expression. CONCLUSION: CLA may mediate its atheroprotective effects in part through reduced expression of SorLA and a resulting inhibition of monocyte migration in vitro.

Stimulators of AMP-activated kinase (AMPK) inhibit seawater- but not cAMP-induced oocyte maturation in a marine worm: Implications for interactions between cAMP and AMPK signaling.

Previous studies have shown that elevations in intraoocytic cAMP prevent mammalian oocytes from maturing, whereas cAMP degradation allows these oocytes to begin maturation, as evidenced by the onset of oocyte nuclear disassembly (="germinal vesicle breakdown", GVBD). Moreover, such cAMP degradation not only reduces cAMP levels but also generates AMP, which in turn can stimulate AMP-activated kinase (AMPK), a well-documented inducer of GVBD in mice. Alternatively, in some marine invertebrates, intraoocytic cAMP triggers, rather than blocks, GVBD, and whether AMPK up- or downregulates maturation in these species has not been tested. Thus, AMPK was monitored in the nemertean worm Cerebratulus during GVBD stimulated by seawater (SW) or cAMP elevators. In oocytes lacking surrounding follicle cells, AMPK activity was initially elevated in immature oocytes but subsequently reduced during SW- or cAMP-induced GVBD, given that the catalytic alpha-subunit of AMPK in maturing oocytes displayed a decreased stimulatory phosphorylation at T172 and an increased inhibitory phosphorylation at S485/491. Accordingly, AMPK-mediated phosphorylation of acetyl-CoA carboxylase, a known target of active AMPK, also declined during maturation. Moreover, treatments with either ice-cold calcium-free seawater (CaFSW) or AMPK agonists dissolved in SW maintained AMPK activity and inhibited GVBD. Conversely, adding cAMP elevators to CaFSW- or SW-solutions of AMPK activators restored GVBD while promoting S485/491 phosphorylation and AMPK deactivation. Collectively, such findings not only demonstrate for the first time that intraoocytic AMPK can block GVBD in the absence of surrounding follicle cells, but these results also provide evidence for a novel GVBD-regulating mechanism involving AMPK deactivation by cAMP-mediated S485/491 phosphorylation.

A fraction of unripe kiwi fruit extract regulates adipocyte differentiation and function in 3T3-L1 cells.

Adipocyte dysfunction is strongly associated with the development of insulin resistance and diabetes, and regulation of adipogenesis is important in prevention of diabetes. We prepared a 100% methanol fraction of methanolic extract from unripe kiwi fruit (Actinidia deliciosa), designated KMF (kiwi fruit methanol fraction). When applied to 3T3-L1 preadipocyte cells, KMF promoted adipocyte differentiation, increased glycerol-3-phosphate dehydrogenase (GPDH) activity, and increased triglyceride (TG) content. KMF markedly increased mRNA expression of peroxisome proliferator-activated receptor gamma (PPARgamma)-the master adipogenic transcription factor-and its target genes. Moreover, KMF increased mRNA expression and protein secretion of adiponectin, whereas mRNA expression and secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were decreased. Compared with troglitazone, KMF decreased the production of reactive oxygen species (ROS) and nuclear factor-kappaB (NFkappaB) activation. Glucose uptake was stimulated by KMF in differentiated 3T3-L1 adipocytes. Taken together, these results indicate that KMF may exert beneficial effects against diabetes via its ability to regulate adipocyte differentiation and function.

Mechanisms of membrane transport of poorly soluble drugs: role of micelles in oral absorption processes.

Micelles formed in the GI tract by bile acid and lecithin play an important role in oral absorption of poorly soluble drugs. In this situation, the drug molecules are present in equilibrium between the free and micellar states. In this study, the relationship between the free drug concentration and the membrane permeability of poorly soluble drugs was examined. Permeability across a Caco-2 monolayer and a dialysis membrane were measured in a side-by-side chamber system. The concentrations of sodium taurocholate (NaTC) and lecithin were varied to allow measurement of membrane permeability at different concentrations of free drugs. For troglitazone, hexylparaben, and heptylparaben, an increase in the NaTC and lecithin concentrations caused the permeability across the Caco-2 monolayer to decrease slightly, whereas the permeability across the dialysis membrane decreased markedly. In contrast, the changes in permeability of griseofulvin with an increased micelle concentration were similar for the Caco-2 monolayer and the dialysis membrane. Assuming that the permeability for the dialysis membrane reflects the free drug concentration in the medium, these results suggest that troglitazone and alkylparabens, but not griseofulvin, can partition directly from micelles to Caco-2 monolayers. This mechanism may contribute to oral absorption of drugs that are poorly soluble in water.

Technical pitfalls and improvements for high-speed screening and QSAR analysis to predict inhibitors of the human bile salt export pump (ABCB11/BSEP).

Drug-induced hepatotoxicity is one of the major problems encountered in drug discovery and development. Selection of a candidate compound for pre-clinical studies in the drug discovery process is a critical step that can determine the speed and expenditure of clinical development. Because inhibition of human adenosine triphosphate-binding cassette transporter ABCB11 (SPGP/bile salt export pump) has severe consequences, which include intrahepatic cholestasis and hepatotoxicity, resulting from exposure to toxic xenobiotics or drug interactions, in vitro screening methods are necessary for quantifying and characterizing the inhibition of ABCB11. In line with such initiatives, we developed methods for in vitro high-speed screening and quantitative structure-activity relationship (QSAR) analysis to investigate the interaction of ABCB11 with a variety of compounds. We identified one set of chemical fragmentation codes closely linked with inhibition of ABCB11. Furthermore, the high-speed screening method enables us to analyze the kinetics of ABCB11-inhibition by test compounds and to distinguish competitive and non-competitive inhibitors. Troglitazone and novobiocin were found to be competitive inhibitors to taurocholate, whereas porphyrins were non-competitive inhibitors. Kinetics-based classification of inhibitors is considered important to improve the accuracy of our QSAR analysis. The present mini-review addresses technical pitfalls and improvements for high-speed screening and QSAR analysis in the ABCB11 inhibition study.

Optimization of in vitro conditions for bovine subcutaneous and intramuscular preadipocyte differentiation.

The objective of these experiments was to develop an in vitro cell culture system for differentiation of bovine preadipocytes, which will permit examination of differences in differentiation between intramuscular (i.m.) and subcutaneous (s.c.) bovine preadipocytes. Stromal-vascular cells from bovine i.m. and s.c. adipose depots were isolated and cultured. Clonally derived s.c. preadipocytes were used to determine the ability of insulin, bovine serum lipids, octanoate, acetic acid, dexamethasone (DEX), and troglitazone (TRO) to elicit differentiation of these cells when added to serum-free medium. Addition of 10 and 20 microL/mL of a commercially available serum lipids supplement to low-glucose Dulbecco's modified Eagle's medium containing 280 nM insulin increased glycerol-3-phosphate dehydrogenase (GPDH) activity (P < 0.01). Inclusion of 1.25 to 10 microM TRO to medium containing 280 nM insulin and 20 microL/ mL serum lipids supplement also increased GPDH activity (P < 0.001) compared with 0 microM TRO. The combination of 280 nM insulin, 1 mM octanoate, and 10 mM acetic acid, with 48 h exposure to 0.25 microM DEX caused morphological differentiation in a small number of cells but did not stimulate GPDH activity (P = 0.99). When used together, 280 nM insulin, 20 microL/mL of serum lipids supplement, 40 microM TRO, and 0.25 microM DEX stimulated differentiation compared with the aforementioned treatment (P < 0.001). Omission of TRO or insulin from this medium reduced GPDH activity by 68% (P < 0.001), whereas removal of DEX tended to reduce GPDH activity (P = 0.06). Preadipocytes from s.c. (n = 3) and i.m. (n = 2) adipose tissues of 3 steers were used to determine the effects of TRO on differentiation using the established conditions. Forty to sixty microM TRO enhanced differentiation compared with 0 microM TRO (P < 0.02) in both depots. No depot differences in response to TRO were detected (P = 0.32). These data demonstrate that bovine preadipocytes are capable of differentiation in response to combinations of insulin, serum lipids, DEX, and TRO. Although TRO enhanced differentiation of bovine preadipocytes, no differential effects of TRO on the differentiation of s.c. and i.m. cells were detected.

Evaluation of a basic physiologically based pharmacokinetic model for simulating the first-time-in-animal study.

The objective of this study was to evaluate a physiologically based pharmacokinetic (PBPK) approach for predicting the plasma concentration-time curves expected after intravenous administration of candidate drugs to rodents. The predictions were based on a small number of properties that were either calculated based on the structure of the candidate drug (octanol:water partition coefficient, ionization constant(s)) or obtained from the typical high-throughput screens implemented in the early drug discovery phases (fraction unbound in plasma and hepatic intrinsic clearance). The model was tested comparing the predicted and the observed pharmacokinetics of 45 molecules. This dataset included six known drugs and 39 drug candidates from different discovery programs, so that the performance of the model could be evaluated in a real discovery case scenario. The plasma concentration-time curves were predicted with good accuracy, the pharmacokinetic parameters being on average two- to three-fold of actual values. Multivariate analysis was used for identifying the candidate properties which were likely associated to biased predictions. The application of this approach was found useful for the prioritization of the in vivo pharmacokinetics screens and the design of the first-time-in-animal studies.