Several transgenic and knockout congenic mouse models
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 , , . Such differences may be explained by undesirable side effects generated by insertion of exogenous DNA, VX-950 synthesis 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) , . Congenic mice differ not only in the ablated gene but also in a flanking segment on either side of the ablated locus . 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 , . 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 , . 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.
Materials, animals and methods
Discussion This study presents mice with a missense point mutation in R258 of FFAR1 that has functional consequences. Firstly, in islets of Ffar1R258W/R258W mice, both the physiological agonist palmitate and the synthetic agonist TUG-469 were unable to augment GIIS. Secondly, palmitate-mediated increase of Ppara mRNA levels was abrogated. The findings that FFAR1 mediates FFA effects on insulin secretion and Ppara mRNA are consistent with previous observations , , . The loss of function of FFAR1 in Ffar1R258W/R258W mouse islets was not accompanied by a change of Ffar1 mRNA levels, indicative of a normal expression of the non-functional receptor. Whether protein trafficking to the plasma membrane remains unaltered needs further experimental evidence. HEK-EM 293 cells that overexpress R258A-mutated FFAR1 exhibit an unaltered receptor abundance at the plasma membrane and an abrogation of FFAR1-agonist GW9508-induced Ca2+-flux when compared to cells expressing wild-type receptors . In contrast, in isolated islets of Ffar1(−/−) mice, Ffar1 mRNA was undetectable. That the deletion affected the expression of adjacent genes is suggested by concomitant reduction of Ffar3 mRNA levels. The functional consequence of the reduction of Ffar3 and the increase of Gpr119 mRNA levels is unknown. In respect to undesirable side effects, the mutant mouse represents a more reliable model. The functional impact of Ffar1R258W/R258W became visible only in homozygous mice, while heterozygous mice did not develop any phenotype revealing a recessive character of the mutation (data not shown). Furthermore, GIIS was unaffected. Comparably, in humans, mutations (single nucleotide polymorphisms) in FFAR1 link to minor, but significant metabolic changes . The Ffar1R258W/R258W mouse model provided new insight into FFAR1-dependent and -independent effects of palmitate. Thus, in contrast to the FFAR1-mediated effect on Ppara mRNA, the palmitate-induced reduction of Slc2a2 mRNA levels was independent of functional FFAR1. An effect of palmitate on Glut-2 expression has been previously reported but the underlying signaling pathways remained unexplored . However, HFD did not alter Slc2a2 mRNA levels, suggesting that the in vitro observation may not translate to the in vivo situation and, consequently, does not link to HFD-induced glucose intolerance. Chronic stimulation of G-protein coupled receptors, including FFAR1, is known to induce a downregulation of receptors and receptor function . The exposure of wild-type islets to palmitate for 24 h was not sufficient for a significant reduction of Ffar1 mRNA levels. Nevertheless, after 8 weeks HFD feeding Ffar1 mRNA levels were reduced 5-fold indicating that chronic stimulation may attenuate receptor function.