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Association between regulator of G protein signaling 9-2 and body weight

PLoS ONE (2011) 6(11)
DOI: 10.1371/journal.pone.0027984
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Abstract

Regulator of G protein signaling 9-2 (RGS9-2) is a protein that is highly enriched in the striatum, a brain region that mediates motivation, movement and reward responses. We identified a naturally occurring 5 nucleotide deletion polymorphism in the human RGS9 gene and found that the mean body mass index (BMI) of individuals with the deletion was significantly higher than those without. A splicing reporter minigene assay demonstrated that the deletion had the potential to significantly decrease the levels of correctly spliced RGS9 gene product. We measured the weights of rats after virally transduced overexpression of RGS9-2 or the structurally related RGS proteins, RGS7, or RGS11, in the nucleus accumbens (NAc) and observed a reduction in body weight after overexpression of RGS9-2 but not RGS7 or 11. Conversely, we found that the RGS9 knockout mice were heavier than their wild-type littermates and had significantly higher percentages of abdominal fat. The constituent adipocytes were found to have a mean cross-sectional area that was more than double that of corresponding cells from wild-type mice. However, food intake and locomotion were not significantly different between the two strains. These studies with humans, rats and mice implicate RGS9-2 as a factor in regulating body weight. © 2011 Waugh et al.

Figures

  • Table 1. Table outlining the body mass index (BMI) associated with each ethnic group and with DTTTCT deletion positive (D+) and DTTTCT (D-) deletion negative groups.
  • Table 2. Table outlining the mean body mass index (BMI) associated with males and females and with DTTTCT deletion positive (D+) and DTTTCT (D-) deletion negative male and female subjects.
  • Figure 1. Effect of RGS9 gene elements containing the DTTTCT deletion on the splicing of a minigene reporter construct. (A) Schematic representation of the minigene reporter system used to assess the effect of the intron 13 TTTCT deletion (represented as D) on RGS9 gene splicing. A section of the RGS9 gene spanning the 39 end of exon 13 (gray shaded box) and intron 13 (gray dashed line between Exon 2 and 3) was inserted at the indicated restriction enzyme sites. Note that the 39 end of the RGS9 gene exon 13 (gray shaded box) forms the 39 end of Exon 2 of the new minigene construct. In subsequent panels ‘‘DTTTCT+ve’’ refers to the minigene construct made by inserting the RGS9 gene fragment with the TTTCT deletion while ‘‘DTTTCT-ve’’ refers to a minigene construct that is identical to DTTTCT except that it does not have the TTTCT deletion. (B) The DTTTCT-ve and DTTTCT+ve RGS9 gene fragment-containing minigenes were transfected separately into HEK293 cells. The minigene transcripts were visualized by reverse transcription, PCR amplification followed by agarose gel electrophoresis. The gel image (left) is a representative image of DNA bands produced from the separate minigene transcripts that are depicted schematically on the right (labeled ‘‘a’’ and ‘‘b’’, respectively). The binding sites of the PCR primers used to amplify ‘‘a’’ and ‘‘b’’ are indicated by the forward and reverse arrows in A. We sequenced bands ‘‘a’’ and ‘‘b’’ to determine that they are derived, respectively, from the minigene products shown schematically to the right of the gel image. (C) Densitometric quantification of the relative intensities of bands ‘‘a’’ and ‘‘b’’, (*p,0.01, t test). doi:10.1371/journal.pone.0027984.g001
  • Figure 2. Body weight and locomotor activity comparisons of RGS9 wild-type (rgs9+/+) and RGS9 knockout mice (rgs9-/-). (A) 12 wk old male rgs9-/- (on right) with wild-type, rgs9+/+ littermate at left. (B) Comparison of weight of female rgs9-/- and rgs9+/+ littermates from weaning through day 50 post-weaning (number of mice at day 50, rgs9+/+ 9, rgs9-/- 6). (C) Comparison as in B with male mice (number of mice, rgs9+/+ 11, rgs9-/- 8). Points represent mean6SEM. Solid line, p,0.005, repeated measures ANOVA. (D) Comparison of ambulatory locomotor activity of 8 week old male and female rgs9-/- (black bars) and rgs9+/+ (open bars) mice, after placement of mice in activity measurement chambers over a 2 hr period. Bars represent mean6SEM number of beam breaks per mouse after 30 min and after 2 hr (number of male mice, rgs9+/+ 8, rgs9-/- 8, number of female mice, rgs9+/+ 8, rgs9-/- 8, *p,0.02, unpaired t test male mice, rgs9+/+ compared to rgs9-/-). doi:10.1371/journal.pone.0027984.g002
  • Figure 3. Comparison of visceral fat and mesenteric adipocytes from RGS9 wild-type (rgs9+/+) and RGS9 knockout mice (rgs9-/-). (A) Combined weight of epididymal and mesenteric fat depots expressed as a percentage of body weight. (number of mice, rgs9+/+ 5, rgs9-/- 6,*p,0.02, t test). (B) Western blot (WB) of visceral fat (Fat) and striatal (Str.) tissue from an 8 week old female wild-type mouse probed with an antibody directed against RGS9. Numbers to the left of the blot indicate position of the 75 kDa and 50 kDa protein molecular weight markers. The blot is representative of experiments performed with 2 female and 2 male mice. (C) representative images of epididymal adipocytes from rgs9+/+ (left panel) and rgs9-/- (right panel) mice. Scale bar represents 50 mm. (D) Apparent area of adipocytes observed in hematoxylin and eosin stained epididymal fat sections. n, rgs9+/+ 463 cells, rgs9-/- 497 cells pooled from sections from two separate animals each, (*p,0.01, t test). (E) Cumulative frequency distribution of apparent epididymal adipocyte cross-sectional area. Adipocyte crosssectional area is plotted on the x-axis, and y-axis indicates the percent of total cells with cross-sectional area less than the value indicated on the x-axis. doi:10.1371/journal.pone.0027984.g003
  • Figure 4. Body-weight changes in rats following Herpes simplex virus (HSV)-mediated over-expression of RGS proteins in the nucleus accumbens shell. Graphs depict the difference in body weight following intracranial injection of HSV constructs encoding RGS7 (open circles), RGS9-2 (filled squares) and RGS11 (closed triangles) into the nucleus accumbens (day 0), relative to the weight of control group HSV-LacZ-injected rats. The weights at day 0 for each of the paired comparisons were as follows: LacZ, 281.764.4 (n = 18); RGS7, 283.362.7 (n = 20); LacZ, 289.763.6 (n = 51); RGS9-2, 290.264.26 (n = 48); LacZ, 287.563.2 (n = 19); RGS11 292.263.4 (n = 22). Not all the animals were followed until day 15, hence ‘‘n’’s at day 15 were as follows: LacZ, 22; RGS9-2, 14; RGS11, 11. *p,0.05, **p,0.01, repeated measures ANOVA followed by Fisher’s least squares test. doi:10.1371/journal.pone.0027984.g004

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Waugh, J. L., Celver, J., Sharma, M., Dufresne, R. L., Terzi, D., Risch, S. C., … Kovoor, A. (2011). Association between regulator of G protein signaling 9-2 and body weight. PLoS ONE, 6(11). https://doi.org/10.1371/journal.pone.0027984

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