Activator of G protein signaling 3 null mice: I. Unexpected alterations in metabolic and cardiovascular function

Abstract

Activator of G protein signaling (AGS)-3 plays functional roles in cell division, synaptic plasticity, addictive behavior, and neuronal development. As part of a broad effort to define the extent of functional diversity of AGS3-regulated-events in vivo, we generated AGS3 null mice. Surprisingly, AGS3 null adult mice exhibited unexpected alterations in cardiovascular and metabolic functions without any obvious changes in motor skills, basic behavioral traits, and brain morphology. AGS3 null mice exhibited a lean phenotype, reduced fat mass, and increased nocturnal energy expenditure. AGS3 null mice also exhibited altered blood pressure control mechanisms. These studies expand the functional repertoire for AGS3 and other G protein regulatory proteins providing unexpected mechanisms by which G protein systems may be targeted to influence obesity and cardiovascular function.

Figure 1

Strategy for generation of Gpsm1 null mice. A, Targeting vector for the conditional null Gpsm1 allele and PCR genotyping strategy to confirm the loss of Gpsm1 exon 3 from mouse tail genomic DNA using primers (a and b) flanking the floxed exon. An expected 565-bp wild-type band is amplified from WT DNA (+/+), whereas a single 310-bp band representing the loss of exon 3 is produced from Gpsm1 null mice (−/−). Heterozygous null (+/−) mice contain both fragments. The asterisk (*) represents the stop codon introduced by a shift in reading frame due to the loss of exon 3. C, ClaI; A, AflIII; B, BamHI; H, HincII. Additional information regarding Gpsm1 gene targeting can be found in supplemental information. B, Left panel, Brain lysates (75 μg per lane) from WT and Gpsm1^−/− were prepared and subjected to SDS-PAGE and immunoblotting with AGS3, LGN, and Giα3-specific antisera as described in Materials and Methods. Right panel, Lysates (75 μg per lane) prepared from COS7 cells transfected with 10 μg pcDNA3 empty vector (V) or pcDNA3::AGS3-Short (SH) (25) as well as heart tissue lysates (150 μg per lane) from WT and Gpsm1^−/− were subjected to SDS-PAGE and immunoblotting with AGS3 antisera (PEP22), which detects both full-length AGS3 (AGS3-FL) and AGS3-short (25). A prominent immunoreactive species of Mr approximately 45,000 is also evident in the heart and in untransfected COS7 cells (Fig. 1B, right panel). The Mr approximately 45,000 species is observed in both WT and AGS3 null mice and is variably and inconsistently detected with the different panel of AGS3 antibodies available (PEP22, PEP32, PEP98, AGS3 monoclonal) and as such appears to be unrelated to full-length AGS3.

Figure 2

Adipose tissue expression and metabolic profile of Gpsm1^−/− mice. A, AGS3 immunoblot of adipose tissue from WT, Gpsm1^+/−, and Gpsm1^−/− mice. Lysates were isolated, prepared, and subjected to SDS-PAGE and immunoblotting with AGS3-specific antisera as described in Experimental Procedures. BAT, Brown adipose tissue; IWAT, inguinal white adipose tissue; EWAT, epididymal white adipose tissue; RPWAT, retroperitoneal white adipose tissue. B, Body weight for wild-type and Gpsm1^−/− mice was measured weekly. C, Food consumption was measured three times per week and is presented as grams per day per gram body weight (left side of graph;**, p = 0.003) and as absolute food consumption as grams per day per mouse (right side of graph; *, p = 0.04). D, Repeated measures of body composition were determined in each animal using a small animal NMR as described in Materials and Mehtods, and group means for each ratio were calculated from the respective ratios of individual mice in each group at each time point. The differences in both body weight (B) and body composition (D) between WT and Gpsm1^−/− mice were statistically significant as determined by ANOVA (p = 0.0001). E, EE was measured using indirect calorimetry during a 4-d period at 21 wk of age. Oxygen consumption and carbon dioxide production were measured at 48-min intervals. Group means for both WT and Gpsm1^−/− were calculated for each interval, and means for each interval were binned in groups of three intervals. EE is expressed as kilojoules per hour per unit of FFM (kilograms). Gray bars indicate night.

Figure 3

Cardiovascular profile of Gpsm1^−/− mice. A, Top panel, Heart rate (HR) and MAP were averaged from all animals (WT, n = 4; Gpsm1^−/−, n = 5) over a 24-h period. WT HR = 561 ± 9 beats per minute (bpm); Gpsm1^−/− HR = 562 ± 6 bpm; WT MAP = 117 ± 1 mm Hg; Gpsm1^−/− MAP = 107 ± 2 mmHg. *, p = 0.0001. Also shown is the difference in HR and MAP between the peak nocturnal increase and the day time nadir. WT ΔHR = 170 ± 23 bpm; Gpsm1^−/− ΔHR = 107 ± 16 bpm; p = 0.055. WT ΔMAP = 37 ± 4 mm Hg; Gpsm1^−/− ΔMAP = 13 ± 2 mm Hg. *, p < 0.05. Bottom panel, Telemetry recordings of arterial pressure and heart rate from conscious, unrestrained WT (n = 4) and Gpsm1^−/− (n = 5) mice over a 50-h period. MAP and HR recordings were taken for 10 sec every 10 min, and tracings plotted over time are presented. Black and white bars underneath the tracings represent night and day, respectively. B, Spontaneous baroreflex gain (SBG) was measured as described in Materials and Methods. WT (n = 4) SBG mean = 1.28 ± 0.07 msec/mm Hg; Gpsm1^−/− (n = 5) SBG mean = 1.88 ± 0.21 msec/mm Hg. *, p < 0.05. C, MAP and HR telemetry recordings from WT and Gpsm1^−/− mice at 14 wk of age after administration of 87.5 μg/kg SNP. Recordings were taken continuously and the time of drug delivery is marked by the vertical line in the tracing. Data are representative of three WT and five Gpsm1^−/− mice.