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Biomedical subjects

W O Wilkison

Publications and source records attributed to W O Wilkison.

At least 19 recordsLinked to original sources
PubMed2001

Identification of a subtype selective human PPARalpha agonist through parallel-array synthesis.

Using solid-phase, parallel-array synthesis, a series of urea-substituted thioisobutyric acids was synthesized and assayed for activity on the human PPAR subtypes. GW7647 (3) was identified as a potent human PPARalpha agonist with approximately 200-fold selectivity over PPARgamma and PPARdelta, and potent lipid-lowering activity in animal models of dyslipidemia. GW7647 (3) will be a valuable chemical tool for studying the biology of PPARalpha in human cells and animal models of disease.

Animals↗
PubMed2001

Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous.

The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor gamma ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age +/- SEM, 38.9 +/- 3.1) with mild to moderate obesity (mean body mass index +/- SEM, 27.8 +/- 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 +/- 93-fold (mean +/- SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 +/- 197.5 vs 1118.5 +/- 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation.

Adipocytes↗
PubMed2001

Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells.

Mouse melanocortin receptors, MC1-R, MC3-R, MC4-R, and MC5-R, when expressed in HEK293 cells and stimulated with either alpha-melanocyte-stimulating hormone (alpha-MSH) or desacetyl-alpha-MSH, mediate increases in intracellular free calcium concentration ([Ca(2+)](i)) with EC(50) values between 0.3 and 4.3 nM. The increase in [Ca(2+)](i) is cholera toxin sensitive and pertussis toxin insensitive. The mechanism involves calcium mobilization from intracellular stores without a transient rise in inositol trisphosphate. Mouse agouti protein (55 nM) is a competitive antagonist of alpha-MSH (6-fold) and desacetyl-alpha-MSH (8-fold), coupling the mMC1-R to increased [Ca(2+)](i). Agouti protein (55 nM) significantly increased the EC(50) for alpha-MSH (3-fold), and 550 nM agouti protein significantly increased the EC(50) for desacetyl-alpha-MSH (4-fold), coupling the mMC4-R to a rise in [Ca(2+)](i). However, agouti protein antagonism of the MC4-R may not be competitive since there was a trend for the maximum response to also increase. There was no significant antagonism of the MC3-R and MC5-R by agouti protein (55 nM). Understanding the physiological relevance of the transduction of a calcium signal by melanocortin peptides may be important for future development of therapeutic targeting of the melanocortin receptors.

Agouti Signaling Protein↗
PubMed2001

Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: biochemical, cellular, and molecular analysis.

While adipocyte differentiation has been studied extensively in murine cultures, the lack of a readily available preadipocyte model has hindered equivalent studies in man. We describe methods for the isolation and culture of primary human stromal cells from surgical adipose tissue specimens. In vitro, the stromal cells rapidly differentiate in response to a combination of adipogenic agents. Among these, glucocorticoids and thiazolidinediones act together to induce the formation of lipid vacuoles within the cells. These morphologic changes accompany the increased expression of 2 characteristic adipocyte proteins, the cytoplasmic enzyme glycerol phosphate dehydrogenase (GPDH) and the secreted cytokine leptin. Likewise, stromal cell differentiation results in elevated mRNA levels for the fatty acid binding protein aP2 and the adipogenic regulatory transcription factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) in addition to leptin. The in vitro differentiated stromal cells exhibit a lipolytic response to beta-adrenergic agonists, comparable to that reported with primary human adipocytes. These studies demonstrate the validity of human adipose tissue-derived stromal cells as a reliable in vitro model for investigations of adipocyte metabolism in humans.

Adipose Tissue↗
PubMed2001

Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells.

Human adipose tissue represents an abundant reservoir of stromal cells with potential utility for tissue engineering. The current study demonstrates the ability of human adipose tissue-derived stromal cells to display some of the hallmarks of osteoblast differentiation in vitro. Following treatment with ascorbate, beta-glycerophosphate, dexamethasone, and 1,25 dihydroxy vitamin D(3), adipose tissue-derived stromal cells mineralize their extracellular matrix based on detection of calcium phosphate deposits using Alizarin Red and von Kossa histochemical stains. Fourier transform infrared analysis demonstrates the apatitic nature of these crystals. Mineralization is accompanied by increased expression or activity of the osteoblast-associated proteins osteocalcin and alkaline phosphatase. These and other osteoblast-associated gene markers are detected based on polymerase chain reaction. In contrast, the adipocyte gene markers--leptin, lipoprotein lipase, and peroxisome proliferator activated receptor gamma2--are reduced under mineralization conditions, consistent with the reciprocal relationship postulated to exist between adipocytes and osteoblasts. The current work supports the presence of a multipotent stromal cell population within human extramedullary adipose tissue. These findings have potential implications for human bone tissue bioengineering.

Adipose Tissue↗
PubMed2001

Effects of fenofibrate on lipid parameters in obese rhesus monkeys.

Fenofibrate is a member of the fibrate class of hypolipidemic agents used clinically to treat hypertriglyceridemia and mixed hyperlipidemia. The fibrates were developed primarily on the basis of their cholesterol and triglyceride lowering in rodents. Fibrates have historically been ineffective at lowering triglycerides in experimentally-induced dyslipidemia in nonhuman primate models. The spontaneously obese rhesus monkey is a well-recognized animal model for the study of human obesity and type 2 diabetes, and many of these monkeys exhibit naturally occurring lipid abnormalities, including elevated triglycerides and low HDL cholesterol (HDL-C), similar to patients with type 2 diabetes. To explore whether the obese rhesus model was predictive of the lipid lowering effects of fibrates, we evaluated fenofibrate in six hypertriglyceridemic, hyperinsulinemic, nondiabetic animals in a 20-week, dose-escalating study. The study consisted of a 4-week baseline period, two treatment periods of 10 mg/kg twice daily (b.i.d) for 4 weeks and 30 mg/kg b.i.d. for 8 weeks, and a 4-week washout period. Fenofibrate (30 mg/kg b.i.d) decreased serum triglycerides 55% and LDL-C 27%, whereas HDL-C increased 35%. Apolipoproteins B-100 and C-III levels were also reduced 70% and 29%, respectively. Food intake, body weight, and plasma glucose were not affected throughout the study. Interestingly, plasma insulin levels decreased 40% during the 30 mg/kg treatment period, suggesting improvement in insulin sensitivity. These results support the use of obese rhesus monkey as an excellent animal model for studying the effects of novel hypolipidemic agents, particularly agents that impact serum triglycerides and HDL-C.

Amino Acid Sequence↗
PubMed2000

Transcriptional regulation of the adipocyte fatty acid synthase gene by agouti: interaction with insulin.

Mice carrying dominant mutations at the agouti locus exhibit ectopic expression of agouti gene transcripts, obesity, and type II diabetes through unknown mechanisms. To gain insight into the role of agouti protein in modulating adiposity, we investigated regulation of a key lipogenic gene, fatty acid synthase (FAS) by agouti alone and in combination with insulin. Both agouti and insulin increase FAS activity in 3T3-L1 and in human adipocytes. Agouti and insulin independently and additively increase FAS activity in 3T3-L1 adipocytes. We further investigated the mechanism responsible for the agouti-induced FAS expression in these cells and demonstrated that both insulin (3-fold increase) and agouti (2-fold) increased FAS gene expression at the transcriptional level. Furthermore, insulin and agouti together exerted additive effects (5-fold increase) on FAS gene transcription. Transfection assays of FAS promoter-luciferase fusion gene constructs into 3T3-L1 adipocytes indicated that the agouti response element(s) is (are) located in the -435 to -415 region (-435/-415) of the FAS promoter. Nuclear proteins binding to this novel sequence are adipocyte specific. Thus the agouti response sequences mapped to a region upstream of the insulin-responsive element (which we previously reported to be located at -67/-52), consistent with additive effects of these two factors on FAS gene transcription.

3T3 Cells↗
PubMed2000

Role of intracellular calcium in human adipocyte differentiation.

Intracellular calcium ([Ca(2+)](i)) modulates adipocyte lipid metabolism and inhibits the early stages of murine adipogenesis. Consequently, we evaluated effects of increasing [Ca(2+)](i) in early and late stages of human adipocyte differentiation. Increasing [Ca(2+)](i) with either thapsigargin or A23187 at 0-1 h of differentiation markedly suppressed differentiation, with a 40-70% decrease in triglyceride accumulation and glycerol-3 phosphate dehydrogenase (GPDH) activity (P < 0.005). However, a 1-h pulse of either agent at 47-48 h only modestly inhibited differentiation. Sustained, mild stimulation of Ca(2+) influx with either agouti protein or 10 mM KCl-induced depolarization during 0-48 h of differentiation inhibited triglyceride accumulation and GPDH activity by 20-70% (P < 0.05) and markedly suppressed peroxisome proliferator-activated receptor gamma (PPARgamma) expression. These effects were reversed by Ca(2+) channel antagonism. In contrast, Ca(2+) pulses late in differentiation (71-72 h or 48-72 h) markedly increased these markers of differentiation. Thus increasing [Ca(2+)](i) appears to exert a biphasic regulatory role in human adipocyte differentiation, inhibiting the early stages while promoting the late stage of differentiation and lipid filling.

Adipocytes↗
PubMed2000

Adipose-derived stromal cells--their utility and potential in bone formation.

Many organs contain connective tissue or stromal cells and these cells play important roles in growth, development and tissue repair. Subcutaneous adipose tissue represents an accessible reservoir for the isolation of human stromal cells. Ex vivo, the adipose tissue-derived human stromal cells can be expanded more than 100-fold. These primary cultures respond to adipogenic agonists by accumulating lipid and expressing adipocyte specific proteins, including leptin and the peroxisome proliferator-activated receptor gamma (PPARgamma). In contrast, when the adipose tissue-derived stromal cells are exposed to osteogenic factors, they display osteoblastic gene markers and mineralize their extracellular matrix. This work demonstrates that subcutaneous adipose tissue is a readily available source of multipotential stromal cells. It is possible that these cells will be used clinically to treat a broad range of orthopedic, rheumatologic and periodontal disorders.

Adipocytes↗
PubMed1999

An agouti mutation lacking the basic domain induces yellow pigmentation but not obesity in transgenic mice.

Chronic antagonism of melanocortin receptors by the paracrine-acting agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type agouti. Functional analysis of agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the agouti basic domain, the entire 29-aa region was deleted from the agouti cDNA, and the resulting mutation (agoutiDeltabasic) was expressed in transgenic mice under the control of the beta-actin promoter (BAPaDeltabasic). Three independent lines of BAPaDeltabasic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the agoutiDeltabasic protein was functional in vivo. However, none of the BAPaDeltabasic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of agoutiDeltabasic expression in relevant tissues exceeded the level of agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the agoutiDeltabasic protein, rather than insufficient RNA synthesis, accounts for the phenotype of the BAPaDeltabasic transgenic mice. These findings implicate a functional role for the agouti basic domain in vivo, possibly influencing the biogenesis of secreted agouti protein or modulating protein-protein interactions that contribute to effective antagonism of melanocortin receptors.

Agouti Signaling Protein↗
PubMed1999

The agouti gene product stimulates pancreatic [beta]-cell Ca2+ signaling and insulin release.

Ubiquitous expression of the mouse agouti gene results in obesity and hyperinsulinemia. Human agouti is expressed in adipose tissue, and we found recombinant agouti protein to stimulate lipogenesis in adipocytes in a Ca(2+)-dependent fashion. However, adipocyte-specific agouti transgenic mice only became obese in the presence of hyperinsulinemia. Because intracellular Ca(2+) concentration ([Ca(2+)](i)) is a primary signal for insulin release, and we have shown agouti protein to increase [Ca(2+)](i) in several cell types, we examined the effects of agouti on [Ca(2+)](i) and insulin release. We demonstrated the expression of agouti in human pancreas and generated recombinant agouti to study its effects on Ca(2+) signaling and insulin release. Agouti (100 nM) stimulated Ca(2+) influx, [Ca(2+)](i) increase, and a marked stimulation of insulin release in two beta-cell lines (RIN-5F and HIT-T15; P < 0. 05). Agouti exerted comparable effects in isolated human pancreatic islets and beta-cells, with a 5-fold increase in Ca(2+) influx (P < 0.001) and a 2.2-fold increase in insulin release (P < 0.01). These data suggest a potential role for agouti in the development of hyperinsulinemia in humans.

Agouti Signaling Protein↗
PubMed1999

Role of the sulfonylurea receptor in regulating human adipocyte metabolism.

A regulatory role for intracellular Ca2+ ([Ca2+]i) in adipocyte lipogenesis, lipolysis and triglyceride accumulation has been demonstrated. Compounds acting on the pancreatic sulfonylurea receptor (SUR) to increase (e.g., glibenclamide) or decrease (e.g., diazoxide) [Ca2+]i cause corresponding increases and decreases in weight gain. However, these weight gain and loss effects have been attributed to insulin release rather than to the primary effects of these compounds on the adipocyte SUR and its associated K(ATP) channel. Accordingly, we have evaluated the direct role of the human adipocyte SUR in regulating adipocyte metabolism. We used RT-PCR with primers for a highly conserved region of SUR1 to demonstrate that human adipocytes express SUR1. The PCR product was confirmed by sequence analysis and used as a probe to demonstrate adipocyte SUR1 expression by Northern blot analysis. Adipocytes exhibited glibenclamide dose-responsive (0-20 microM) increases in [Ca2+]i (P<0.05). Similarly, glibenclamide (10 microM) caused a 67% increase in adipocyte fatty acid synthase activity (P<0.001), a 48% increase in glycerol-3-phosphate dehydrogenase activity (P<0.01) and a 68% inhibition in lipolysis (P<0.01), whereas diazoxide (10 microM) completely prevented each of these effects. These data demonstrate that human adipocytes express a SUR that regulates [Ca2+]i and, consequently, exerts coordinate control over lipogenesis and lipolysis. Accordingly, the adipocyte SUR1 may represent an important target for the development of therapeutic interventions in obesity.

ATP-Binding Cassette Transporters↗
PubMed1999

Agouti antagonism of melanocortin-4 receptor: greater effect with desacetyl-alpha-melanocyte-stimulating hormone (MSH) than with alpha-MSH.

Desacetyl-alpha-MSH is more abundant than alpha-MSH in the brain, the fetus, human blood, and amniotic fluid, but there is little information on its ability to interact with melanocortin receptors. The aim of this study is to compare and contrast the ability of desacetyl-alpha-MSH and alpha-MSH to couple melanocortin receptors stably expressed in HEK293 cells, to the protein kinase A (PKA) signaling pathway. Desacetyl-alpha-MSH activated mouse MC1, MC3, MC4 and MC5 receptors with EC50s = 0.13, 0.96, 0.53, and 0.84 nM, and alpha-MSH activated these receptors with EC50s = 0.17, 0.88, 1.05, and 1.34 nM, respectively. Mouse agouti protein competitively antagonized alpha-MSH and desacetyl-alpha-MSH coupling to the MC1-R similarly. In contrast, mouse agouti protein antagonized desacetyl-alpha-MSH much more effectively and potently than alpha-MSH coupling the MC4-R to the PKA signaling pathway. Furthermore, mouse agouti protein (10 nM) significantly reduced (1.4-fold) the maximum response of mMC4-R to desacetyl-alpha-MSH and 100 nM mouse agouti significantly increased (4.8-fold) the EC50. Minimal antagonism of alpha-MSH coupling mMC4-R to the PKA signaling pathway was observed with 10 nM mouse agouti, whereas both 50 and 100 nM mouse agouti appeared to reduce the maximum reponse (1.1- and 1.3-fold, respectively) and increase the EC50 (2.5- and 3.4-fold respectively). Mouse agouti protein did not significantly antagonize either alpha-MSH or desacetyl-alpha-MSH coupling mouse MC3 and MC5 receptors. Understanding the similarities and differences in activation of melanocortin receptors by desacetyl-alpha-MSH and alpha-MSH will contribute to delineating the functional roles for these endogenous melanocortin peptides.

Adenylyl Cyclases↗
PubMed1999

HP-228 (Trega Biosciences Inc).

HP-228 is a cytokine regulating agent from Trega Biosciences (formerly Houghten Pharmaceuticals). It is in phase II trials for the management of post-surgical pain and inflammation [195065]. The drug was indicated for the treatment of diabetes, obesity and chemotherapy-induced toxicity, but Trega has decided to discontinue the development of HP-228 for these indications [259339].

Journal Article↗
PubMed1998

Melanocortin receptor binding determinants in the agouti protein.

The agouti protein plays an important role in the development of diabetes and obesity in rodents and has been shown to be a potent antagonist of melanocortin receptors. For this reason alanine-scanning mutagenesis was performed on the agouti protein carboxyl terminus to locate residues important for melanocortin receptor binding inhibition. When agouti residues Arg116 and Phe118 are changed to alanine, very large decreases in agouti affinity for melanocortin receptor 1, 3, and 4 result. Mutation of Phe117 to alanine causes a similar increase in agouti KI app at melanocortin receptor 4. Substitution of agouti residue Asp108 with alanine results in large increases in KI app for all three melanocortin receptors examined. All of these residues are conserved in the agouti-related transcript, ART, whose expression is up-regulated in animal models of obesity. The three-dimensional structure of the agouti carboxyl terminus was modeled, and residues which decrease receptor binding by a factor of > or = 15 when mutated to alanine localize to one side of the structure. These agouti variants with altered receptor selectivity may be useful in determining the role of melanocortin receptors in diabetes and obesity.

Agouti Signaling Protein↗
PubMed1998

Effects of a potent melanocortin agonist on the diabetic/obese phenotype in yellow mice.

OBJECTIVE: To test the hypothesis that a melanocortin agonist can reverse obesity and insulin resistance in mice overexpressing the agouti protein. EXPERIMENTAL MODEL: Mice overexpressing the agouti protein either by transgene introduction (beta-actin promotor) or by mutation (Ay). DESIGN: NDPMSH was tested for pharmacokinetic suitability. NDPMSH at various doses was administered subcutaneously twice a day for 2-3 weeks. MEASUREMENTS: Fur pigmentation, various fatness parameters (core temperature, fat pad weight and body weight), blood glucose and hormones, fatty acid synthase measurement. RESULTS: NDPMSH caused fur pigmentation and core temperature changes, but failed to affect any metabolic parameters in agouti-dependent manner. CONCLUSION: NDPMSH, as a representation melanocortin agonist, does not compete with agouti in reversing agouti-dependent metabolic effects. This suggests that 1) agouti works via a receptor other than a melanocortin receptor to mediate its metabolic effects, 2) agouti-dependent metabolic effects are mediated through melanocortin receptors but not via antagonism of these receptors, or 3) NDPMSH is pharmacodynamically an inappropriate molecule for these types of studies.

Agouti Signaling Protein↗
PubMed1998

Thiazolidinedione exposure increases the expression of uncoupling protein 1 in cultured human preadipocytes.

Thiazolidinediones (TZDs) are a novel class of insulin-sensitizing agents used in the treatment of NIDDM and are potent agonists for the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma). The thiazolidinedione BRL 49653 has been shown to promote the differentiation of the HIB-1B brown preadipocyte cell line and to increase rat interscapular brown adipose tissue (BAT) mass. Given the importance of brown fat in the control of energy metabolism in rodents, this may represent an important therapeutic effect of this class of compound. To date, however, no studies examining the effects of TZDs on human brown fat have been reported. In the present study, we have measured uncoupling protein 1 (UCP-1) mRNA, a specific marker for BAT, in isolated adipocytes and subcultured preadipocytes prepared from different adult human adipose tissue depots. Consistent with previous studies of adult human whole adipose tissue, UCP-1 mRNA was detectable in isolated human adipocytes prepared from all depots studied with a rank order of perirenal, omental, and subcutaneous. BRL 49653 treatment of subcultured human pre-adipocytes prepared from all depots resulted in increased levels of UCP-1 mRNA, compared with those of the vehicle-treated cells. When exposed to BRL 49653 for 5 days, preadipocytes from the human perirenal depot accumulated lipid, and a proportion of cells showed clear mitochondrial staining for UCP-1 protein by confocal microscopy. Thus, cells of the brown fat lineage were detectable in all human adipose depots studied, and cultured human pre-adipocytes, particularly from the perirenal depot, showed a marked increase in UCP-1 expression in response to thiazolidinediones. Given the role of brown adipocytes in the enhancement of energy expenditure, promotion of brown fat adipogenesis by thiazolidinediones could contribute to the beneficial effects of these drugs on insulin resistance in humans.

Adipocytes↗