br Materials animals and methods br Results

Materials, animals and methods
Results
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 [1], [3], [10]. 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 szl mg 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 [19]. 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 [24]. 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 [25]. 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 [26]. 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. The improved glucose tolerance of Ffar1R258W/R258W mice on HFD was unexpected. In combination with similar fasting blood glucose levels, the significant lower fasting plasma insulin of mutant mice compared to wild-type mice is indicative of improved insulin sensitivity. However, peripheral insulin resistance assessed with ipITT was not different between wild-type and mutant mice. The degree of liver steatosis was also independent of the expression of a functional FFAR1. Indeed, any change of liver steatosis and insulin resistance can only be attributed to an indirect effect of FFAR1, since the receptor is not expressed in rodent liver, muscle and adipose tissue (Refs. [1], [27]; data not shown). The lower basal plasma insulin levels of Ffar1R258W/R258W compared to wild-type mice could be attributed to FFAR1 deficiency, because fatty acids are increased after overnight fasting and blood glucose levels were elevated, i.e. at 6 mM. In view of similar HFD-induced insulin resistance and liver steatosis, the significantly higher glucose excursions in wild-type mice during ipGTT cannot be explained by β-cell dysfunction only. It is more likely that additional, insulin-independent factors regulating blood glucose levels, e.g. via the regulation of hepatic glucose production, account for differences in glucose tolerance between wild-type and FFAR1 mutant mice.