Gpr75-deficient mice are protected from high-fat diet-induced obesity

Abstract

Objective: G-protein coupled receptor 75 (GPR75) has been identified as the high-affinity receptor of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive and proinflammatory lipid, and mice overproducing 20-HETE have been shown to develop insulin resistance when fed a high-fat diet (HFD), which was prevented by a 20-HETE receptor blocker. Simultaneously, a large-scale exome sequencing of 640,000 subjects identified an association between loss-of-function GPR75 variants and protection against obesity.

Methods: Wild-type (WT) and Gpr75-deficient mice were placed on HFD for 14 weeks, and their obesity phenotype was examined.

Results: Male and female Gpr75 null (knockout [KO]) and heterozygous mice gained less weight than WT mice when placed on HFD. KO mice maintained the same level of energy expenditure during HFD feeding, whereas WT mice showed a significant reduction in energy expenditure. Diet-driven adiposity and adipocyte hypertrophy were greatly lessened in Gpr75-deficient mice. HFD-fed KO mice did not develop insulin resistance. Adipose tissue from Gpr75-deficient mice had increased expression of thermogenic genes and decreased levels of inflammatory markers. Moreover, insulin signaling, which was impaired in HFD-fed WT mice, was unchanged in KO mice.

Conclusions: These findings suggest that GPR75 is an important player in the control of metabolism and glucose homeostasis and a likely novel therapeutic target to combat obesity-driven metabolic disorders.

Figure 1:. Body weight, energy intake, and energy expenditure over 14-weeks of HFD.

Body weight trajectories for (A) male (n=8–11), and (B) female (n=7–8) WT, HET and KO mice. Results are mean±SE; n=8–11/group; *p=0.05 from WT by unpaired T-Test. Change in body weight from baseline after 14 weeks of HFD feeding for males (C) and females (D). Total energy intake before and after HFD feeding for (E) male and (F) female WT, HET, and KO mice. Energy expenditure (Kcal/day) at baseline and week 14 of HFD feeding for (G) males and (H) females. Results are mean±SE, ns, not significant; **p<0.01, *** P<0.001 and ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test.

Figure 2:. Visceral and subcutaneous fat pad composition over 14-weeks of HFD.

Representative MicroCT images of male (A) and female (B) WT, HET and KO mice at weeks 0, 7 and 14 of HFD feeding (blue, visceral fat; purple, subcutaneous fat; scale bar = 20 mm). Visceral (VAT) and subcutaneous (SAT) fat volumes at baseline, week 7, and week 14 for males (C-D) and females (E-F) WT, HET and KO mice. Results are mean±SE, ns, not significant; **p<0.01, *** P<0.001 and ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test.

Figure 3:. Visceral, subcutaneous, and brown fat morphology after 14-weeks of HFD.

(A) Representative images of H&E staining in visceral (VAT), subcutaneous (SAT) and brown (BAT) adipose tissues of HFD (14 weeks)-fed male and female WT and KO mice (20X; scale bar = 110 μm). Visceral adipocyte size at baseline and week 14 of HFD for males (B) and females (C). Subcutaneous adipocyte size at baseline and week 14 of chow diet or HFD for males (D) and females (E). Results are mean±SE (n=4); ns, not significant; **p<0.01, *** P<0.001 and ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test.

Figure 4:. Fasting blood glucose, glucose tolerance, and insulin tolerance over 14-weeks of HFD.

Twelve-hour fasting blood glucose of (A) males and (B) female WT, HET, and KO mice at weeks 0, 7 and 14 of HFD feeding. Glucose tolerance test (GTT) area under curve (AUC) of (C) males and (D) female WT, HET, and KO mice at weeks 0, 7 and 14 of HFD feeding. Insulin tolerance test (ITT) area under curve (AUC) of (E) males and (F) female WT, HET, and KO mice at weeks 0, 7 and 14 of HFD feeding. Results are mean±SE (n=8); ns, not significant; **p<0.01, *** P<0.001 and ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test.

Figure 5:. Levels of markers of insulin resistance in response to diet.

Plasma insulin (A), leptin (B), adiponectin (C), resistin (D) and PAI-1 (E) after 14 weeks of chow diet (CD) or HFD feeding in WT, HET and KO mice. (F) Homeostatic model assessment of insulin resistance (HOMA-IR) after 14 weeks of HFD-feeding. (G) Leptin-adiponectin ratio after 14 weeks of HFD feeding. Results are mean±SE (n=10–20); ns, not significant; **p<0.01, *** P<0.001 and ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test.

Figure 6:. Expression of inflammatory and thermogenic markers in adipose tissues.

Levels of mRNA of (A) interleukin-6 (Il-6), (B) tumor necrosis factor-alpha (Ucp1), (C) chemokine ligand 5 (Ccl5), (D) peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α), (E) uncoupling protein-1 (Ucp1) and (F) PR domain containing 16 (Prmd16) in VAT of WT and KO mice after 14 weeks of chow diet (CD) or HFD feeding. Expression of Pgc1α (G) Ucp1 (H) and Ucp1 (I) in subcutaneous (SAT) of WT and KO animals after 14 weeks of chow diet (CD) or HFD-feeding. Expression of Pgc1α (J) Ucp1 (K) and Ucp1 (L) in BAT of WT and KO animals after 14 weeks of chow diet (CD) or HFD-feeding. Results are mean±SE (n=8–10); ns, not significant; *p<0.05, **p<0.01, *** P<0.001 and ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test.

Figure 7:. UCP1 protein levels and mitochondria respiration in BAT from HFD-fed WT and KO mice.

(A) Western blot and densitometry analysis of UCP1 in BAT from WT and KO mice after 14 weeks of HFD feeding. Results are mean±SE, n=6/group; *p<0.05 compared to WT by unpaired T-Test. (B) ADP-stimulated oxygen respiration in isolated mitochondria from BAT of WT and KO mice after 14 weeks of HFD feeding. Results are mean±SE, n=4/group, ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test.

Figure 8:. Skeletal muscle thermogenic marker expression and insulin signaling.

(A) Gpr75 gene expression in WT, HET and KO mice after 14 weeks of chow diet (CD) or HFD-feeding. (B) Expression levels of uncoupling protein-3 (Ucp3) and (C) Mitofusin-1 (Mfn1) in WT and KO after 14 weeks of CD- or HFD-feeding. (D) representative western blot and densitometry analysis of phosphorylated insulin receptor at tyrosine 972 (pIR) normalized to total insulin receptor (IR) levels in skeletal muscle from HFD-fed WT, HET, and KO mice. (E) western blot and (F) densitometry analysis of phospho-AKT (serin 473) normalized to total AKT in skeletal muscle from HFD-fed WT and KO mice. Results are mean±SE (n=3–5); ns, not significant; *p<0.05, **p<0.01, *** P<0.001 by unpaired t-test (B, C and E) or two-way ANOVA with Tukey’s multiple comparison test (A and D).