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  • GLP is an intestinal peptide hormone that is

    2022-08-05

    GLP-1 is an intestinal peptide hormone that is released by the gut following ingestion of food, particularly calcifediol mg and fats. Following its release, GLP-1 stimulates pancreatic β-cells to secrete insulin (Hirasawa et al., 2005, Covington et al., 2006). Originally, GLP-1 acted in a similar manner to glucagon in fish and was secreted by the intestine and pancreas (Irwin, 2001). However, GLP has evolved in mammals to become an important peptide involved in glucose homeostasis. GLP-1 levels in Type II diabetic (T2D) patients were found to be lower compared to normal patients (Toft-Nielsen et al., 2001). It is believed that having lower levels of GLP-1 could cause a decrease in insulin secretion in T2D patients. In addition, researchers have demonstrated that GLP-1 can promote β-cell preservation as well as increasing insulin secretion (Tanaka et al., 2008). Exendin-4, a known GLP-1 agonist, was shown to cause expansion of β-cells in the rat (Xu et al., 1999). These studies suggest that GPR120 may be an important regulator of GLP-1 release. The sequences for the human, mouse, and rat GPR120 receptors have been reported in the literature (Hirasawa et al., 2005, Tanaka et al., 2008). Two sequences have been reported for the human GPR120 receptor (NM_181745 and BC101175), with the latter being shorter by 16 amino acids. Only one sequence, corresponding to BC101175, has been found for the mouse and rat. In addition, the rhesus monkey sequence has been inferred from BLAST searches and thought to be comparable to the human BC101175 sequence. Cynomolgus monkeys are often used in preclinical drug discovery studies to determine the effects of compounds in non-human primates. Therefore understanding which sequence(s) of the GPR120 receptor is in this species could be important for future drug discovery efforts around this target. In this paper, the sequence of cynomolgus monkey (Macaca fascicularis) GPR120 receptor (FJ810855) will be introduced and its functional characteristics will be explored using fatty acids of varying lengths and GW9508, a synthetic GPR120/GPR40 receptor agonist. In addition, a pharmacological comparison of intracellular calcium signaling in response to free fatty acids between the cynomolgus monkey GPR120 receptor and the human BC101175 variant is reported and compared with that previously reported for the NM_181745 human splice variant described by Hirasawa et al. (2005). To our knowledge, this is the first report providing not only a pharmacological characterization of the BC101175 human splice variant, but also the first to compare the functional activity between primate and human GPR120 receptors.
    Materials and methods
    Results
    Discussion In drug development it is often desirable to compare results obtained from human expression systems with those of other species. In the case of the free fatty acid GPCR GPR120, it was known that the genetic sequences available for the rat and mouse GPR120 receptor had low homology (86.4 and 85.1%, respectively) compared with the known human GPR120 receptor sequences, which could affect the affinity of small molecules when compared between these species. In this study, the GPR120 receptor from cynomolgus monkey was cloned and characterized to see if it better matched the sequence and pharmacological profile of the human GPR120 receptor. According to this study, the percent similarity between the human GPR120 (BC101175) receptor sequence and the cynomolgus monkey sequence (FJ810855) is 97.5%. Only eight amino acid differences separate the human and cynomolgus monkey GPR120 sequences. Compared to the mouse and rat GPR120 sequences, the cynomolgus monkey GPR120 sequence is only 85.3% and 83.7% similar, suggesting that the cyno monkey is a more appropriate species for comparison of GPR120- mediated effects to the human GPR120 receptor. Two splice variants of the human GPR120 receptor have been reported: the original sequence published by Hirasawa's group (2005) (NM_181745) and; a variant without exon 3 published by the same group (BC101175) (Tanaka et al., 2008). This study sought to determine if the cynomolgus monkey genome contains a similar splice variant. Based on the fact that a frame shift mutation occurs in the region where the extra exon (in NM_181745) would occur, and the fact that this region is a suboptimal splicing donor site (~50 fold less likely to make an effective splice donor site) (Burge and Karlin, 1997), it was determined that there is no evidence of this GPR120 splice variant in the rhesus monkey genome. Numerous clones sequenced from the cynomolgus monkey colon cDNA library did not show a higher molecular weight band on a DNA gel that would correspond to the longer splice variant of human GPR120. It appears that the longer splice variant of GPR120 is specific to humans.