Lack of FFAR1/GPR40 does not protect mice from high-fat diet-induced metabolic disease

Abstract

Objective: FFAR1/GPR40 is a G-protein-coupled receptor expressed predominantly in pancreatic islets mediating free fatty acid-induced insulin secretion. However, the physiological role of FFAR1 remains controversial. It was previously reported that FFAR1 knockout (Ffar1(-/-)) mice were resistant to high-fat diet-induced hyperinuslinemia, hyperglycemia, hypertriglyceridemia, and hepatic steatosis. A more recent report suggested that although FFAR1 was necessary for fatty acid-induced insulin secretion in vivo, deletion of FFAR1 did not protect pancreatic islets against fatty acid-induced islet dysfunction. This study is designed to investigate FFAR1 function in vivo using a third line of independently generated Ffar1(-/-) mice in the C57BL/6 background.

Research design and methods: We used CL-316,243, a beta3 adrenergic receptor agonist, to acutely elevate blood free fatty acids and to study its effect on insulin secretion in vivo. Ffar1(+/+) (wild-type) and Ffar1(-/-) (knockout) mice were placed on two distinct high-fat diets to study their response to diet-induced obesity.

Results: Insulin secretion was reduced by approximately 50% in Ffar1(-/-) mice, confirming that FFAR1 contributes significantly to fatty acid stimulation of insulin secretion in vivo. However, Ffar1(+/+) and Ffar1(-/-) mice had similar weight, adiposity, and hyperinsulinemia on high-fat diets, and Ffar1(-/-) mice showed no improvement in glucose or insulin tolerance tests. In addition, high-fat diet induced comparable levels of lipid accumulation in livers of Ffar1(+/+) and Ffar1(-/-) mice.

Conclusions: FFAR1 is required for normal insulin secretion in response to fatty acids; however, Ffar1(-/-) mice are not protected from high-fat diet-induced insulin resistance or hepatic steatosis.

FIG. 1.

Generation of Ffar1^−/− mice. A: Schematic view of the regions depleted in Ffar1 gene. A 152-bp fragment corresponding to position 143–294 of the open reading frame of the Ffar1 gene (NM_194057) was replaced by a neo cassette. The two black bars below the bottom panel represent the positions of the 5′ and 3′ probes used for Southern blotting. B: Southern blotting of ES cell clones. Shown are DNA samples from two nontargeted ES clones and a targeted clone (ES 1315) digested by EcoRV and probed with a 5′ probe and by HindIII and probed with a 3′ probe, respectively. C: Representative PCR genotyping of DNA samples from tail clips of Ffar1^+/+ (WT) and Ffar1^−/− (KO) mice. The WT allele is 234 bp and the KO allele is 399 bp in size. D: Expression of Ffar1 mRNA in pancreatic islets and spleen of Ffar1^+/+ (WT) and Ffar1^−/− (KO) mice accessed by real-time quantitative PCR.

FIG. 2.

Plasma parameters of Ffar1^+/+ and Ffar1^−/− mice in response to β3 agonist CL-316,243 (CL) injections. Mice received intraperitoneal injection of either saline or CL-316,243. The four treatment groups are: Ffar1^+/+ mice receiving saline, Ffar1^+/+ mice receiving CL-316,243, Ffar1^−/− mice receiving saline, and Ffar1^−/− mice receiving CL-316,243. n = 5–8 mice per group. A: Free fatty acids. B: Insulin. C: Amylin. D: Leptin. E: GLP-1 (7-36)amide. F: Glucagon. The data are analyzed by one-way ANOVA followed by t tests with Bonferroni correction. *P < 0.05; **P < 0.01.

FIG. 3.

Metabolic characterizations of Ffar1^+/+ and Ffar1^−/− mice after 8 weeks on a low-fat diet and high-fat diet. Ffar1^+/+ and Ffar1^−/− mice at N6 generation of backcrossing into B6 background were fed for 8 weeks on a Surwit diabetogenic diet (58% of calories from lard) or a semipurified low-fat diet (10% of calories from fat) ad libitum. n = 5 mice per group. Body weight and body fat were monitored biweekly. Food intake was measured weekly. Glucose tolerance tests (GTT) were performed before mice were killed for blood samples and tissue collection. A: Body weight of low-fat diet–fed mice. B: Body weight of high-fat diet–fed mice. C: Accumulated food intake of low-fat diet–fed mice. D: Accumulated food intake of high-fat diet–fed mice. E: Body fat of low-fat diet–fed mice. F: Body fat of low-fat diet–fed mice. G: Glucose tolerance test of low-fat diet–fed mice. H: Glucose tolerance test of low-fat diet–fed mice. Data are presented as means ± SE. *P < 0.05, t test comparing Ffar1^+/+ and Ffar1^−/− mice at each time point.

FIG. 4.

Metabolic characterizations of Ffar1^+/+ and Ffar1^−/− mice after 10 weeks on 60% high-fat diet (HFD). Ffar1^+/+ and Ffar1^−/− mice after 10 generations of backcrossing into B6 background were fed for 10 weeks on semipurified high-fat diet (60% calories from fat). n = 11, Ffar1^+/+ male; n = 9, Ffar1^−/− male; n = 10, Ffar1^+/+ female; n = 10, Ffar1^−/− female. Body weight was monitored weekly, and body fat was assessed every 4 weeks. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed at the end of the study. A: Body weight of male mice. B: Body weight of female mice. C: Body fat of male mice. D: Body fat of female mice. E: GTT of male mice. F: ITT of male mice. Data are presented as means ± SE.