Striatal Rgs4 regulates feeding and susceptibility to diet-induced obesity

Abstract

Consumption of high fat, high sugar (western) diets is a major contributor to the current high levels of obesity. Here, we used a multidisciplinary approach to gain insight into the molecular mechanisms underlying susceptibility to diet-induced obesity (DIO). Using positron emission tomography (PET), we identified the dorsal striatum as the brain area most altered in DIO-susceptible rats and molecular studies within this region highlighted regulator of G-protein signaling 4 (Rgs4) within laser-capture micro-dissected striatonigral (SN) and striatopallidal (SP) medium spiny neurons (MSNs) as playing a key role. Rgs4 is a GTPase accelerating enzyme implicated in plasticity mechanisms of SP MSNs, which are known to regulate feeding and disturbances of which are associated with obesity. Compared to DIO-resistant rats, DIO-susceptible rats exhibited increased striatal Rgs4 with mRNA expression levels enriched in SP MSNs. siRNA-mediated knockdown of striatal Rgs4 in DIO-susceptible rats decreased food intake to levels comparable to DIO-resistant animals. Finally, we demonstrated that the human Rgs4 gene locus is associated with increased body weight and obesity susceptibility phenotypes, and that overweight humans exhibit increased striatal Rgs4 protein. Our findings highlight a novel role for involvement of Rgs4 in SP MSNs in feeding and DIO-susceptibility.

Figure 1. Behavioral and metabolic abnormalities in DIO-susceptible OM rats coincide with decreased metabolic activity in the dorsal striatum

(a) High fat (HF)-fed OM rats (n=8) exhibited significantly increased caloric intake (kcal) relative to both chow (CH)-fed OM rats (n=8) and HF-fed S5B rats (n=8) (two-way repeated measures (RM) ANOVA, interaction effect, F_{(21, 196)}=11.2). (b) HF-fed OM rats (n=10) exhibited significantly increased body weight relative to both CH-fed OM rats (n=10) and HF-fed S5B rats (n=10) (two-way RM ANOVA, interaction effect, F_{(21, 252)}=30.1). (c) HF-fed OM rats (n=4) exhibited significantly increased adiposity compared to CH-fed OM rats (n=4) and to HF-fed S5B rats (n=4) (two-way ANOVA, interaction effect, F_{(1, 12)}=19.04). (d) CH-fed OM rats (n=10) (separate cohort from above) weighed significantly more than S5B (n=10) (separate cohort from above) rats (unpaired t-test; t=10.2, df=18). (e) CH-fed OM (n=8) rats consumed significantly more food than S5B (n=8) rats (unpaired t-test; t=3.59, df=14). (f) CH-fed OM (n=4) and S5B (n=4) rats did not differ in adiposity (n=4) (unpaired t-test). (g) CH-fed OM rats (n=7) exhibited greater glucose intolerance compared to S5B (n=8) rats (two-way RM ANOVA, interaction effect F_(3,39)=2.56, p=0.06, 60 min: t=3.1, df=52). (h) SDH rats (n=6) weighed significantly more than SDL rats (n=6) (unpaired t-test, t=6.92, df=10). (i) SDH rats exhibited significantly greater food intake compared to SDL rats (unpaired t-test, t=2.29, df=10). (j) SDH rats exhibited increased adiposity compared to SDL rats (unpaired t-test; t=3.87, df=10). (k) SDH and SDL rats did not differ in glucose tolerance (unpaired t-test). (l) CH-fed OM (n=6) and S5B (n=7) rats did not differ in CH intake during limited access sessions (two-way RM ANOVA). (m) CH-fed OM rats (n=6) exhibited rapid escalation of Froot Loops® (FL) intake and consumed significantly more FL than CH-fed S5B (n=7) rats (two-way RM ANOVA, interaction effect, F_(3,33)=9.48, p<0.001, session 3: t=3.49, df=44, session 4: t=5.81, df=44). (n, o) SDL and SDH rats did not differ in CH or FL intake (two-way RM ANOVA). (p) Statistical parametric map from FDG uptake images overlaid onto MRI showing that CH-fed OM rats (n=5) exhibited significantly lower FDG uptake than CH-fed S5B rats (n=5) (unpaired t-test, t=2.71; df=8; p≤0.05, uncorrected) in several regions (hippocampus, CA1, dorsal subiculum, DS, dentate gyrus, DG, cerebellum, CB, tegmental nuclei, Tg, reticular nuclei, Rt, amygdala, AM, brainstem nuclei, BN, nucleus accumbens, NAc, vertical diagonal band, lateral preoptic, VDB/LPO) with the most notable effect in dorsal striatum (caudate putamen, CPu); L=left, R=right.*p≤0.05, **p≤0.01, ***p≤0.001

Figure 2. Striatal Rgs4 regulates feeding and is associated with DIO-susceptibility

(a) Transcriptional profiling in dorsal striatum demonstrated that OM rats (n=7) exhibited significantly greater mRNA expression (unpaired t-tests) of Rgs4 (t=3.22; df=12), Drd1a (t=2.32; df=12), Drd2L (t=2.46; df=12), Grm5 (t=2.81; df=12), and Cnr1 (t=2.48; df=12) than S5B rats (n=7). (b) OM rats (n=8) had significantly greater Rgs4 protein in dorsal striatum compared to S5B rats (n=8) (unpaired t-test, t=7.74; df=14) (representative blots shown). (c) Compared to S5B (n=8), OM rats (n=8) exhibited significantly lower enrichment of the repressive 3meH3K9 histone mark (unpaired t-test, t=2.28; df=14) but not (d) the permissive 3meH3K4 at a highly conserved genomic region (~1.5 kb upstream of transcriptional start site (TSS)) of Rgs4. (e) OM (n=6) and S5B (n=6) rats did not differ in basal [³⁵S]GTPγS binding but the Rgs4 inhibitor CCG-63802 (0.5 μM) led to a significant increase in [³⁵S]GTPγS binding in dorsal striatum in S5B rats (n=6) (paired t-test, t=6.29; df=5) and to a smaller, marginally significant increase in [³⁵S]GTPγS binding in OM rats (n=6) (paired t-test, t=2.38; df=5; p=0.06). (f, g) Representative extracellular recordings from the dorsal striatum of S5B and OM rats. The glutamatergic-driven population spike (PS) increased with increasing stimulation intensity. Summary input-output (I/O) curves from all slices revealed no significant group differences between S5B (n = 12 slices, 3 rats) and OM rats (n = 12 slices, 3 rats; two-way repeated measures (RM) ANOVA, F_(1,22) = 0.87, p = 0.36). (h) Summary time course of long-term depression (LTD) elicited by high frequency stimulation (HFS; 100 Hz, 1s duration trains delivered 4 times at 10 s intervals). Inset shows representative traces from a control (S5B) brain slice, prior to (pre) and 30 minutes following HFS (post). No significant differences in LTD were observed between S5B and OM rats (two-way RM ANOVA, F_(1,22) = 0.29, p = 0.59). (i) Coronal and sagittal planes of SPM images shown in Figure 1p indicating that the lower FDG uptake observed in dorsal striatum of OM rats extends to the globus pallidus (white arrow). Red line denotes location of sagittal plane. L=left, R=right. (j) Strategy for dissecting striatonigral (SN) (green) and striatopallidal (SP) (red) projection pathways using retrograding latex beads. (k) Representative microscopy image (20×) of latex bead accumulation in SN and SP MSNs. (l, m) Rgs4 mRNA is selectively enriched in SP compared to SN MSNs (unpaired t-test, t=3.15; df=4) of OM (n=3) but not S5B (n=3) rats. (n) Targeted cannula placement for siRNA delivery to dorsal striatum. (o) Representative in situ hybridization autoradiograms from rats injected with scrambled (n=4) or Rgs4-targeted siRNA (n=4). (p) siRNA-mediated inhibition of striatal Rgs4 in SD rats significantly decreased striatal Rgs4 mRNA (unpaired t-test, t=4.55; df=6) and (q) 24-hr food intake (unpaired t-test, t=2.51; df=6). (r) OM and S5B rats differed in 24-hr food intake (unpaired t-test, Pre; t=3.01; df=40) but siRNA-mediated Rgs4 inhibition decreased food intake (two-way RM ANOVA, interaction effect, F_(12,48)=1.45, p=0.17; Session 4 (t=2.60; df=56), Session 6 (t=3.38; df=56), and Session 7 (t=2.52; df=56)) in OM rats (n=4) but not in scramble controls (OM; n=4; S5B; n=3). (s) Cumulative food intake from days 4-7 from adjacent figure (one way ANOVA, F_{(2, 8)}=6.94; p=0.018). *p≤0.05, **p≤0.01, ***p≤0.001

Figure 3. Striatal Rgs4 is associated with increased body weight in humans

(a) Quantitative trait locus (QTL) data showing that human RGS4 is associated with obesity-related traits including body weight and adiposity. (b) Body mass index (BMI) used to stratify normal weight (NW; n=8) and overweight (OW; n=6) human subjects (unpaired t-test, t=10.81, df=12). (c) Coronal human brain section showing representative region of dorsal striatum sampled for downstream analysis (red asterisk, putamen). (d) Overweight human subjects (n=6) exhibit significantly greater striatal Rgs4 (~35 kDa) protein levels than normal weight controls (n=8) (unpaired t-test; t=2.23; df=12). *p≤0.05, ***p≤0.001