Mice lacking GPR3 receptors display late-onset obese phenotype due to impaired thermogenic function in brown adipose tissue

Abstract

We report an unexpected link between aging, thermogenesis and weight gain via the orphan G protein-coupled receptor GPR3. Mice lacking GPR3 and maintained on normal chow had similar body weights during their first 5 months of life, but gained considerably more weight thereafter and displayed reduced total energy expenditure and lower core body temperature. By the age of 5 months GPR3 KO mice already had lower thermogenic gene expression and uncoupling protein 1 protein level and showed impaired glucose uptake into interscapular brown adipose tissue (iBAT) relative to WT littermates. These molecular deviations in iBAT of GPR3 KO mice preceded measurable differences in body weight and core body temperature at ambient conditions, but were coupled to a failure to maintain thermal homeostasis during acute cold challenge. At the same time, the same cold challenge caused a 17-fold increase in Gpr3 expression in iBAT of WT mice. Thus, GPR3 appears to have a key role in the thermogenic response of iBAT and may represent a new therapeutic target in age-related obesity.

Figure 1. Phenotype differences among GPR3 mice.

(A) Tissue expression profile of GPR3 mRNA in 12- week-old wild type male mice. Each circle represents a single value relative to heart (filled circles). (B) Body weights of 1-year-old male wild type (WT), heterozygous (Het) and knockout (KO) mice along with genotyping results and representative images of WT (33.29 g), Het (36.52 g) and KO (41.48 g) mice. Values are mean ± s.e.m of n = 16–19 animals. *P < 0.05, **P < 0.01, ***P < 0.001 compared to age-matched WT littermates. (C) Adiposity index, relative mass of subcutaneous fat and epididymal fat, (D) subcutaneous adipocyte diameters, (E) epididymal adipocyte diameters and (F) liver triglyceride level in GPR3 WT, Het and KO mice. Values are mean ± s.e.m of n = 7–14 samples. *P < 0.05, **P < 0.01 compared to age-matched WT littermates. Panels D-F are accompanied by the representative H&E stained tissue sections. Pictures were taken at 10x magnification (scale bar indicates 100 μm). (G) Glucose tolerance (GTT) was tested after intraperitoneal (i.p.) injection of 1.5 g/kg glucose (at 0 min; (A) to the overnight-fasted 12-month-old male GPR3 WT and KO mice. On the following day, mice were fasted for 6 hour and received 0.75 mU/g insulin i.p. at time 0 (insulin sensitivity test; ITT). Changes in blood glucose level were monitored for 2 hours. Values are mean ± s.e.m of n = 8–14 samples. *P < 0.05, **P < 0.01 compared to age-matched WT littermates.

Figure 2. Metabolic profiles of older GPR3 mice as analyzed by means of indirect calorimetry.

The O₂ consumption and CO₂ production were monitored by indirect calorimetry in older (12-month-old) male GPR3 wild type (WT) and knockout (KO) mice and presented as average of a 24-hour recording period (left panels) or as hourly observations (right panels; shaded area indicates lights off). Shown are total energy expenditure (TEE; (A)), respiratory quotient (RQ; (B)), food intake (C) and ambulatory activity (D). Values are mean ± s.e.m of n = 5–8 samples *P < 0.05, ***P < 0.001 compared to age-matched WT littermates.

Figure 3. GPR3 KO mice display late onset obese phenotype with unchanged metabolic profile until adulthood.

(A) Body weight monitoring for 18 months in male GPR3 wild type (WT) and knock out (KO) mice. Values are mean ± s.e.m of n = 7–23 animals. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the age-matched WT littermates. (B–E) Results of the monitoring of O₂ consumption and CO₂ production by indirect calorimetry in adult (5-month-old) male GPR3 wild type (WT) and knockout (KO) mice. Data are presented as average of a 24-hour recording period. Shown are: total energy expenditure (TEE; (B)), respiratory quotient (RQ; (C)), food intake (D) and ambulatory activity (E). Values are mean ± s.e.m of n = 6–8 samples.

Figure 4. Glycemic control in adult GPR3 mice.

(A–E) Hyperinsulinemic euglycemic clamps were performed in conscious, unrestrained 5-month-old male GPR3 WT and KO mice. Time course of blood glucose levels and glucose infusion rates (GIR; (A)) during the course of clamps; Glucose Infusion Rate (B), whole body glucose clearance (Rd; (C)), levels of hepatic glucose production (hGP; (D) were measured during the steady-state period of the clamp. (E) Glucose uptake into individual tissues: interscapular brown adipose tissue (iBAT), epididymal (epidyd. ad. t.), subcutaneous (subcut ad. t.) and perirenal adipose tissues (ad. t.), heart, liver, gastrocnemius muscle (gastrocnemius m.) and hypothalamus. Tissue uptake was determined as described in materials and methods. Values are mean ± s.e.m of n = 6–11 samples (except hypothalamus for which n = 5–7). ***P < 0.001 compared to age-matched WT littermates.

Figure 5. Thermogenic features of adult GPR3 mice.

(A) Core body temperature was recorded in 2.5-, 5-, 12- and 18-month-old male GPR3 wild type (WT) and knock out (KO) mice. Values are mean ± s.e.m of n = 6–13 samples. *P < 0.05, **P < 0.01 compared to age-matched WT littermates. (B) Relative abundance of GPR3 transcript in iBAT obtained from adult male GPR3 WT at different ages. Values are mean ± s.e.m of n = 4–6 animals. ^#P < 0.05, compared to 5-month-old mice. (C) Relative abundance of indicated transcripts in interscapular brown adipose tissue (iBAT) of adult (5-month-old) male GPR3 WT and KO mice at room temperature. Values are mean ± s.e.m of n = 3–9 samples. *P < 0.05, **P < 0.01, compared to the age-matched WT littermates. (D) The UCP1, MTCO2 and α-tubulin (tubulin) proteins detected in iBAT from two groups of male GPR3 WT and KO mice (1–3 and 4–6). Protein sizes corresponding to UCP1, COX and α-tubulin were 33 kDa, 25 kDa and 52 kDa, respectively. Representative fluorescent immunostaining images against UCP1 in iBAT of GPR3 WT and KO mice. Pictures were taken at 10× magnification (scale bar indicates 100 μm). Western blot band and staining intensities were calculated as described in Material and Methods section and presented as bar graphs next to images. Values are mean ± s.e.m of n = 6–8 samples. *P < 0.05, **P < 0.01 compared to age-matched WT littermates. (E) Changes in core body temperature in 5-month-old male GPR3 WT and KO mice undergoing cold challenge. Body temperature was measured at ambient conditions for baseline as well as 1, 2, 3 and 4 hours after the exposure to cold (4 °C, cold challenge) and 0.5, 1 and 4 hours during the recovery period at room temperature (rt). Values are mean ± s.e.m of n = 5–6 samples. *P < 0.05 compared to age-matched WT littermates. (F) Relative abundance of GPR3 transcript in iBAT obtained from adult male GPR3 WT and GPR3 KO mice obtained at room temperature (rt) and at the 4^th-hour of cold challenge (cold). Values are mean ± s.e.m of n = 3–8 samples. ***P < 0.001, compared to age-matched GPR3 WT littermates at room temperature (rt). GPR3 transcripts were not detectable (nd) in GPR3 KO mice. All target genes in panels (B,C,F) were corrected via reference Rpl19 mRNA.

Figure 6. Summary of phenotype changes in GPR3 deficient mice.