Abstract
A PPARγ fluorescence polarization (FP) assay was used to measure the release of fatty acid products from triglyceride emulsions during digestion with pancreatic and yeast lipases in a real-time, homogenous assay. Using the same FP assay we show the anti-obesity drug Orlistat is a PPARγ ligand with an IC50 of 2.84 ± 0.16 μM. Analytical Mass Spectrometry confirms that Orlistat does not bind covalently to PPARγ. The PPARγ FP assay is shown to be a simple method for measuring real-time lipase activity using a number of triglyceride substrates including olive oil and grape seed oil emulsions. Incubation of Orlistat with the human intestinal epithelial cell line Caco-2, at concentrations of 1 - 100 μM, leads to induction of genes regulated by PPARγ. At 100 μM Orlistat, transcription of β-defensin 1 (hDB1) & Adipose Differentiation Related Protein (ADRP) increase by up to 2.6 fold and 6.8 fold, respectively. Although at 1 μM and 100 μM Orlistat did not significantly increase defensin protein synthesis, at 10 μM Orlistat induced a 1.5 fold increase in hDB1 protein secretion in the human colonic adenocarcinoma cell line HT-29. Thus Orlistat is similar to the anti-diabetic drug Rosiglitazone in its ability to induce defensin gene expression. The antimicrobial peptide β-defensin 1 protects against pathogenic micro-organisms in the gut and PPARγ suppresses inflammatory gene expression. These may be beneficial side effects of Orlistat consumption on gut epithelial cells.
Highlights
The PPARγ agonist and anti-diabetic drug Rosiglitazone is known to induce expression of the human β-defensin 1 gene in human intestinal epithelial Caco-2 cells [1]
Real time lipase assay using PPARγ fluorescence polarization method Candida rugosa lipase and porcine pancreatic lipase were incubated at concentrations ranging from 30 μg/mL to 3.3 μg/mL with various triglyceride emulsions in the presence of the FP reagents and polarization readings were taken at 1–2 minute intervals for up to 30 minutes
The PPARγ binding products released from the triglyceride emulsions were detected by the FP assay
Summary
The PPARγ agonist and anti-diabetic drug Rosiglitazone is known to induce expression of the human β-defensin 1 (hBD1) gene in human intestinal epithelial Caco-2 cells [1]. Defensins are cationic peptides expressed in phagocytic and epithelial cells that lyse micro-organisms by forming pores in their membranes. Deficiency of defensin expression is associated with colonisation of the gut with Candida albicans [2] and with Crohn’s disease [3]. The lipase inhibiting drug Orlistat is marketed by Roche under the trade name Xenical. It has a worldwide distribution and is used as an oral treatment for obesity [6].
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