Active compounds and were further

Active compounds , , and were further tested and EC and pEC values were determined as shown in . Compound showed EC of 0.97μM (pEC 6.01) with 84.5% maximal response, which suggests that introduction of alkyl chain on aromatic nucleus of , resulted in improved GPR40 agonistic activity than that of (EC 6μM, pEC 5.22, 102.2% maximal response). Compound showed potent activity with EC value of 0.07±0.004μM (pEC 7.12, 100.2% maximal response). The efficacy of the compound is shown as a percentage of the maximal agonistic response elicited by the test compound with respect to the maximal response evoked by the internal standard (linoleic acid) at a dose of 10μM. In order to determine the selectivity, compound was also tested for hPPAR-γ agonistic activity and it showed very weak agonistic activity for hPPAR-γ. DMPK study on one of representative of the scaffold (compound ) was performed and pharmacokinetic parameters ( 0.25h, 0.88μg/mL, AUC 3–13hμg/mL, AUC 3.41hμg/mL and half-life 6.59h) showed that this scaffold has good PK properties. Activation of the GRP40 receptor is known to play a role in pancreatic and neurological function and the receptor is specifically localized in the brain and pancreas. In the pancreas the receptor expression is restricted to insulin producing β-cells. Since the key objective of GPR40 agonists is to prime islet β-cells to respond to glucose by inducing insulin secretion, we further evaluated these active compounds for their ability to induce insulin secretion in pancreatic islet cells. Further, the GPR40 mRNA is known to be expressed significantly in most pancreatic β-cell lines with highest expression levels in MIN6, followed by β-TC and HIT-T15. We therefore selected the HIT-T15 cell line for insulin secretion studies. HIT-T15 from hamsters were treated with 10μM of compound , and Each of these induced insulin secretion comparable to the effect of linoleic Solithromycin (). Further, the inactive compounds did not show any antagonist effect for linoleic acid induced calcium flux (data not included).
Introduction Free fatty acid receptor-1 (FFAR1, formerly GPR40) promotes long chain fatty acid-mediated augmentation of glucose-induced insulin secretion (GIIS) [1], [2], [3]. In humans and rodents, high expression of FFAR1 is restricted to pancreatic and gastric endocrine cells, while expression in other tissues, including brain, is much lower [1], [2], [4], [5]. These features make FFAR1 an attractive drug target for the treatment of insufficient insulin secretion, which is the ultimate cause for the onset of hyperglycemia and type-2 diabetes mellitus [6], [7]. Until today, multiple agonists have been generated and tested for their efficacy to treat hyperglycemia in humans [6]. Although FFAR1 agonists counteract glucose intolerance in mice and humans, the beneficial effect of these new therapeutic drugs is still a matter of debate [8], [9]. Thus, the promising drug TAK875 was discontinued after clinical phase III due to its liver toxicity. Confirming this side effect, FFAR1-deficient mice are protected against diet-induced liver steatosis [10]. This observation prompted the investigation of FFAR1-antagonists as therapeutic tools against fatty liver disease. In addition, different FFAR1 agonists exert their effects through different cellular pathways. Thus, fatty acids stimulate insulin secretion mainly via Gq proteins, while TAK875 stimulation is mediated by β-arrestin-2 [11]. An additional, but indirect, stimulatory effect of FFAR1-agonists on insulin secretion is caused by the activation of FFAR1 expressed in intestinal endocrine cells which leads to GLP-1 secretion [12]. Several transgenic and knockout/congenic mouse models have been generated in order to assess the role of FFAR1 for proper insulin secretion and maintenance of glucose homeostasis. The results obtained with three different receptor knockout mouse models were not consistent. The protection against high fat feeding-induced fatty liver and glucose intolerance, as observed by Steneberg and colleagues, could not be reproduced using other Ffar1 KO mouse models [10], [13], [14]. Such differences may be explained by undesirable side effects generated by insertion of exogenous DNA, deletion of non-coding regions with specific functions, e.g. microRNA, and the additional role of the Ffar1 promoter for the expression of FFAR2 (GPR43) and FFAR3 (GPR41) [15], [16]. Congenic mice differ not only in the ablated gene but also in a flanking segment on either side of the ablated locus [17]. Furthermore, a complete deletion of a protein may generate a compensatory up-regulation of other proteins. To circumvent such problems, we searched for a coisogenic mouse model with a minimal genetic alteration producing a maximal effect. Using site-directed mutagenesis, several point mutations in Ffar1 with functional consequences have been identified, including R258 [18], [19]. We screened the Munich ENU-mutagenesis-derived F1 sperm and corresponding DNA archive for point mutations in Ffar1. The archive comprises more than 16,800 samples from individual F1-mutagenized mice on the C3HeB/FeJ genetic background [20], [21]. Two mouse models carrying point mutations in the coding region of Ffar1 are presented in this study of which the R258W mutation prevents the stimulation of insulin secretion by palmitate and the FFAR1 agonist TUG-469.