Deborah A. Winegar

Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway

Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7α-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXRα and LXRβ, two orphan members of the nuclear receptor superfamily, are activated by 24(S),25-epoxycholesterol and 24(S)-hydroxycholesterol at physiologic concentrations. In addition, we have identified an LXR response element in the promoter region of the rat cholesterol 7α-hydroxylase gene. Our data provide evidence for a new hormonal signaling pathway that activates transcription in response to oxysterols and suggest that LXRs play a critical role in the regulation of cholesterol homeostasis.

A selective peroxisome proliferator-activated receptor δ agonist promotes reverse cholesterol transport

The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of the fibrate drugs and the antidiabetic effects of the glitazone drugs in humans are due to activation of the α (NR1C1) and γ (NR1C3) subtypes, respectively. By contrast, the therapeutic potential of the δ (NR1C2) subtype is unknown, due in part to the lack of selective ligands. We have used combinatorial chemistry and structure-based drug design to develop a potent and subtype-selective PPARδ agonist, GW501516. In macrophages, fibroblasts, and intestinal cells, GW501516 increases expression of the reverse cholesterol transporter ATP-binding cassette A1 and induces apolipoprotein A1-specific cholesterol efflux. When dosed to insulin-resistant middle-aged obese rhesus monkeys, GW501516 causes a dramatic dose-dependent rise in serum high density lipoprotein cholesterol while lowering the levels of small-dense low density lipoprotein, fasting triglycerides, and fasting insulin. Our results suggest that PPARδ agonists may be effective drugs to increase reverse cholesterol transport and decrease cardiovascular disease associated with the metabolic syndrome X.

Identification of a subtype selective human PPARα 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.

HIV protease inhibitors stimulate hepatic triglyceride synthesis

Abstract—Hyperlipidemia may complicate the use of HIV protease inhibitors (PIs) in AIDS therapy. To determine the cause of hyperlipidemia, the effect of PIs on lipid metabolism was examined with HepG2 liver cells and AKR/J mice. In HepG2 cells, the PIs ABT-378, nelfinavir, ritonavir, and saquinavir stimulated triglyceride synthesis; ritonavir increased cholesterol synthesis; and amprenavir and indinavir had no effect. Moreover, nelfinavir increased mRNA expression of diacylglycerol acyltransferase and fatty acid synthase. The retinoid X receptor agonist LG100268, but not the antagonist LG100754, further increased PI-stimulated triglyceride synthesis and mRNA expression of fatty acid synthase in vitro. In fed mice, nelfinavir or ritonavir did not affect serum glucose and cholesterol, whereas triglyceride and fatty acids increased 57% to 108%. In fasted mice, ritonavir increased serum glucose by 29%, cholesterol by 40%, and triglyceride by 99%, whereas nelfinavir had no effect, suggesting these PIs have different effects on metabolism. Consistent with the in vitro results, nelfinavir and ritonavir increased triglyceride 2- to 3-fold in fasted mice injected with Triton WR-1339, an inhibitor of triglyceride clearance. We propose that PI-associated hyperlipidemia is due to increased hepatic triglyceride synthesis and suggest that retinoids or meal restriction influences the effects of select PIs on lipid metabolism.

Early Outcomes of Bariatric Surgery in Patients with Metabolic Syndrome: An Analysis of the Bariatric Outcomes Longitudinal Database

BACKGROUND Metabolic syndrome (MetS) complicating obesity is endemic in the United States. STUDY DESIGN Bariatric Outcomes Longitudinal Database, the national database for the American Society for Metabolic and Bariatric Surgery Bariatric Surgery Center of Excellence Program, was queried to identify patients undergoing bariatric surgery from June 2007 through November 2010. MetS was defined as the presence of hypertension, diabetes, and dyslipidemia at presentation for bariatric surgery. Ninety-day and 1-year outcomes were assessed to determine early outcomes in bariatric surgery patients with MetS. RESULTS Among 186,576 research-consented patients, 23,106 (12%) were diagnosed with MetS. Patients with MetS were more likely to be male (35% vs 20%; p < 0.0001), older (mean age 54 vs 44 years; p < 0.0001), and Caucasian (81% vs 74%; p < 0.0001). Of the 23,106 MetS patients, more underwent gastric bypass (RYGB) (62%) compared with gastric banding (32%), sleeve gastrectomy (4.5%), and biliopancreactic diversion with duodenal switch (BPD/DS)(1.5%). MetS patients had an increase in serious complications (2.4% vs 1.0%; p < 0.0001), readmissions (6.2% vs 4.7%; p < 0.0001), and mortality (0.3% vs 0.1%; p < 0.0001) within 90 days of operation. After adjusting for sex, age, and body mass index, RYGB patients with MetS had an increased risk of 90-day serious complications compared to RYGB patients without MetS (odds ratio 1.43; 95% CI, 1.27 to 1.61; p < 0.0001). The 12-month remission rate of diabetes was least for gastric banding (28%) compared with the other procedures (RYGB 62%, sleeve gastrectomy 52%, BPD/DS 74%). CONCLUSIONS Patients with MetS undergoing bariatric surgery showed dramatic improvement in diabetes 1-year after surgery; however, an adverse 90-day outcome was more common.

Production of monoclonal antibodies using recombinant baculovirus displaying gp64-fusion proteins.

Generation of protein immunogens is often a rate-limiting step in the production of monoclonal antibodies (Mabs). Expressing domains of proteins as fusions to the baculovirus surface glycoprotein gp64 displays foreign proteins on the surface of the virion. Antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the gp64 nucleotide sequence. This method allows immunization with whole virus, eliminating the need for purification of target antigens. Affinity-matured Mabs to the human nuclear receptors LXRbeta and FXR have been produced using baculovirus particles displaying gp64/nuclear receptor fusion proteins as the immunizing agent. Immunizations were performed directly with pelleted virus using the Repetitive Immunization Multiple Sites (RIMMS) immunization strategy for rapid Mab production. All Mabs were identified using insect cells infected with the immunizing virus. Characterization of these antibodies shows them to be class-switched and specific for LXRbeta or FXR. Additionally, high affinity antibodies that recognize gp64 and neutralize baculovirus infection of insect cells were isolated. Use of the recombinant baculovirus gp64 display system makes possible the production of Mabs once a partial DNA sequence is known. This allows the generation of antibodies prior to the isolation of purified protein, in turn providing antibodies to facilitate purification, characterization and immunolocalization of proteins.

Venous thromboembolism after bariatric surgery performed by Bariatric Surgery Center of Excellence Participants: analysis of the Bariatric Outcomes Longitudinal Database

BACKGROUND Venous thromboembolism (VTE) is an uncommon complication of bariatric surgery but a leading cause of postoperative mortality. Studying the factors predictive of low-incidence complications requires the analysis of large cohorts. The Bariatric Outcomes Longitudinal Database, the worlds largest prospective database for bariatric surgery, has provided a suitable medium for analyzing low-frequency events. METHODS The data in the Bariatric Outcomes Longitudinal Database from 73,921 research-consented patients who had undergone bariatric surgery by a participant in the American Society for Metabolic and Bariatric Surgery Bariatric Surgery Center of Excellence program before September 22, 2009, were analyzed for VTE events within 90 days after surgery. RESULTS The overall risk of VTE within 90 days after surgery was .42%, and 73% of these events occurred after discharge, most within 30 days after surgery. The risk of VTE was greater in the patients undergoing gastric bypass than in those undergoing adjustable gastric banding (.55% versus .16%). VTE was more frequent when the procedure was performed using an open than a laparoscopic approach (1.54% versus .34%). Patients with a VTE event were older (+4.9 yr), had had a greater preoperative body mass index (+3.9 kg/m(2)), and were more likely to have a history of VTE (16.5% versus 3.7%). The risk of VTE was greater in men (hazard ratio 2.32, 95% confidence interval 1.81-2.98) and in patients with an inferior vena cava filter (hazard ratio 7.66, 95% confidence interval 4.55-12.91). CONCLUSION The overall risk of VTE was low in the population treated by participants in the Bariatric Surgery Center of Excellence program, where clinical pathways to prevent VTE have been mandated. Analysis of this large study population allowed the identification of patient characteristics correlating with increased risk of postoperative VTE and the variable effectiveness of VTE prophylaxis methods.

Rap1-b is phosphorylated by protein kinase a in intact human platelets

Agonists that increase cAMP levels in platelets promote the phosphorylation of a 24 kDa GTP-binding protein that is immunoreactive with a monoclonal antibody (M90) to the H-ras p21 protein. Evidence is presented which indicates that this protein is rap-1b, not rap1-a as previously suggested (Ohmori, T., Kikuchi, A., Yamamoto, K., Kawata, M., Kondo, J. and Takai, Y. (1988) Biochem. Biophys. Res. Commun. 157, 670-676). The amino acid sequence of labeled peptides obtained by proteolytic cleavage of the purified phosphorylated protein was identical with that of rap-1b. Furthermore, a comparison of the kinetics of phosphorylation of synthetic peptides corresponding to the C-terminal region of rap-1a and rap-1b proteins indicated that rap-1b is the preferred substrate for phosphorylation by cAMP-dependent protein kinase.

Serum adiponectin as a biomarker for in vivo PPARgamma activation and PPARgamma agonist-induced efficacy on insulin sensitization/lipid lowering in rats

BackgroundPPARγ agonists ameliorate insulin resistance and dyslipidemia in type 2 diabetic patients. Adiponectin possesses insulin sensitizing properties, and predicts insulin sensitivity of both glucose and lipid metabolism. In diet-induced insulin resistant rats and ZDF rats, the current studies determined the correlation between PPARγ agonist-upregulated fatty acid binding protein(FABP3) mRNA in adipose tissue and PPARγ agonist-elevated serum adiponectin, and the correlation between PPARγ agonist-elevated serum adiponectin and PPARγ agonist-mediated efficacy in insulin sensitization and lipid lowering.ResultsParallel groups of SD rats were fed a high fat/sucrose (HF) diet for 4 weeks. These rats were orally treated for the later 2 weeks with vehicle, either PPARγ agonist GI262570 (0.2–100 mg/kg, Q.D.), or GW347845 (3 mg/kg, B.I.D). Rats on HF diet showed significant increases in postprandial serum triglycerides, free fatty acids (FFA), insulin, and area under curve (AUC) of serum insulin during an oral glucose tolerance test, but showed no change in serum glucose, adiponectin, and glucose AUC. Treatment with GI262570 dose-dependently upregulated adipose FABP3 mRNA, and increased serum adiponectin. There was a positive correlation between adipose FABP3 mRNA and serum adiponectin (r = 0.7350, p < 0.01). GI262570 dose-dependently decreased the diet-induced elevations in triglycerides, FFA, insulin, and insulin AUC. Treatment with GW347845 had similar effects on serum adiponectin and the diet-induced elevations. There were negative correlations for adiponectin versus triglycerides, FFA, insulin, and insulin AUC (For GI262570, r = -0.7486, -0.4581, -0.4379, and -0.3258 respectively, all p < 0.05. For GW347845, r = -0.6370, -0.6877, -0.5512, and -0.3812 respectively, all p < 0.05). In ZDF rats treated with PPARγ agonists pioglitazone (3–30 mg/kg, B.I.D.) or GW347845 (3 mg/kg, B.I.D.), there were also negative correlations for serum adiponectin versus glucose, triglycerides, FFA (for pioglitazone, r = -0.7005, -0.8603, and -0.9288 respectively; for GW347845, r = -0.9721, -0.8483, and -0.9453 respectively, all p < 0.01).ConclusionsThis study demonstrated that (a) PPARγ agonists improved insulin sensitivity and ameliorated dyslipidemia in HF fed rats and ZDF rats, which were correlated with serum adiponectin; (b) Serum adiponectin was positively correlated with adipose FABP3 mRNA in GI262570-treated rats. These data suggest that serum adiponectin can serve as a biomarker for both in vivo PPARγ activation and PPARγ agonist-induced efficacy on insulin resistance and dyslipidemia in rats.

Discovery of GSK837149A, an inhibitor of human fatty acid synthase targeting the β‐ketoacyl reductase reaction

GSK837149A has been identified as a selective inhibitor of human fatty acid synthase (FAS). The compound was first isolated as a minor impurity in a sample found to be active against the enzyme in a high‐throughput screening campaign. The structure of this compound was confirmed by NMR and MS studies, and evaluation of the newly synthesized molecule confirmed its activity against FAS. The compound and other analogs synthesized, all being symmetrical structures containing a bisulfonamide urea, act by inhibiting the β‐ketoacyl reductase activity of the enzyme. GSK837149A inhibits FAS in a reversible mode, with a Ki value of ∼ 30 nm, and it possibly binds to the enzyme–ketoacyl‐ACP complex. Although initial results suggest that cell penetration for these compounds is impaired, they still can be regarded as useful tools with which to probe and explore the β‐ketoacyl reductase active site in FAS, helping in the design of new inhibitors.