Objective

The fatty acid receptor 1 (FFAR1/GPR40) mediates fatty acid-dependent augmentation of glucose-induced insulin secretion (GIIS) in pancreatic β-cells. Genetically engineered Ffar1-knockout/congenic mice univocally displayed impaired fatty acid-mediated insulin secretion, but in vivo experiments delivered controversial results regarding the function of FFAR1 in glucose homeostasis and liver steatosis. This study presents a new coisogenic mouse model carrying a point mutation in Ffar1 with functional consequence. These mice reflect the situations in humans in which point mutations can lead to protein malfunction and disease development.

Methods

The Munich N-ethyl-N-nitrosourea (ENU) mutagenesis-derived F1 archive containing over 16,800 sperms and corresponding DNA samples was screened for mutations in the coding region of Ffar1. Two missense mutations (R258W and T146S) in the extracellular domain of the protein were chosen and homozygote mice were generated. The functional consequence of these mutations was examined in vitro in isolated islets and in vivo in chow diet and high fat diet fed mice.

Results

Palmitate, 50 μM, and the FFAR1 agonist TUG-469, 3 μM, stimulated insulin secretion in islets of Ffar1^T146S/T146S mutant mice and of wild-type littermates, while in islets of Ffar1^R258W/R258W mutant mice, these stimulatory effects were abolished. Insulin content and mRNA levels of Ffar1, Glp1r, Ins2, Slc2a2, Ppara, and Ppard were not significantly different between wild-type and Ffar1^R258W/R258W mouse islets. Palmitate exposure, 600 μM, significantly increased Ppara mRNA levels in wild-type but not in Ffar1^R258W/R258W mouse islets. On the contrary, Slc2a2 mRNA levels were significantly reduced in both wild-type and Ffar1^R258W/R258W mouse islets after palmitate treatment. HFD feeding induced glucose intolerance in wild-type mice. Ffar1^R258W/R258W mutant mice remained glucose tolerant although their body weight gain, liver steatosis, insulin resistance, and plasma insulin levels were not different from those of wild-type littermates. Worth mentioning, fasting plasma insulin levels were lower in Ffar1^R258W/R258W mice.

Conclusion

A point mutation in Ffar1 abrogates the stimulatory effect of palmitate on GIIS, an effect that does not necessarily translate to HFD-induced glucose intolerance.

中文翻译：

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Ffar1的点突变消除了脂肪酸依赖性胰岛素的分泌，但可防止HFD诱导的葡萄糖耐受不良。

目的

脂肪酸受体1（FFAR1 / GPR40）介导胰腺β细胞中脂肪酸依赖性葡萄糖诱导的胰岛素分泌（GIIS）的增加。基因改造的Ffar1基因敲除/转基因小鼠单方面显示出脂肪酸介导的胰岛素分泌受损，但体内实验对FFAR1在葡萄糖稳态和肝脂肪变性中的作用提出了有争议的结果。这项研究提出了一个新的同基因小鼠模型，携带Ffar1中的点突变并具有功能性后果。这些小鼠反映了人类的情况，在这种情况下，点突变可导致蛋白质功能异常和疾病发展。

方法

筛选了慕尼黑N-乙基-N-亚硝基脲（ENU）诱变的F1档案，其中包含超过16,800个精子和相应的DNA样品，以寻找Ffar1编码区中的突变。选择蛋白质的胞外域中的两个错义突变（R258W和T146S），并产生纯合子小鼠。这些突变的功能性结果，检查在体外在分离的胰岛和体内在食物饮食和高脂肪饮食喂养的小鼠。

结果

50μM棕榈酸酯和3μMFFAR1激动剂TUG-469刺激了Ffar1 ^{T146S / T146S}突变小鼠和野生型同窝小鼠的胰岛中的胰岛素分泌，而在Ffar1 ^{R258W / R258W}突变小鼠的胰岛中，这些刺激作用是废除。胰岛素含量和mRNA水平Ffar1，GLP1R，INS2，SLC2A2，PPARA和PPARD没有野生型之间显著不同Ffar1 ^{R258W / R258W}小鼠胰岛。棕榈酸酯暴露量为600μM，可显着增加野生型中Ppara mRNA的水平，而Ffar1中则没有^{R258W / R258W}小鼠胰岛。相反，棕榈酸处理后，野生型和Ffar1 ^{R258W / R258W}小鼠胰岛中的Slc2a2 mRNA水平均显着降低。HFD喂养在野生型小鼠中诱发了葡萄糖不耐症。尽管Ffar1 ^{R258W / R258W}突变小鼠的体重增加，肝脂肪变性，胰岛素抵抗和血浆胰岛素水平与野生型同窝小鼠没有差异，但它们仍具有葡萄糖耐量。值得一提的是，Ffar1 ^{R258W / R258W}小鼠的空腹血浆胰岛素水平较低。

结论

Ffar1中的点突变消除了棕榈酸酯对GIIS的刺激作用，这种作用不一定转化为HFD诱导的葡萄糖耐受不良。