Gq/11α and Gsα mediate distinct physiological responses to central melanocortins (original) (raw)
Generation of PVNGq/11KO mice. To study the role of Gq/11α signaling in the PVN on metabolic regulation, we generated mice that lacked both Gqα and G11α in the PVN (Sim1-Cre Gnaqfl/fl Gna11–/– mice, herein referred to as PVNGq/11KO mice), which was confirmed by in situ hybridization (Figure 1, A and B). Expression of Sim1-Cre is relatively specific for PVN but is also expressed in the supraoptic nucleus, the amygdala, and the anterior periventricular nucleus of the hypothalamus (4). As previously reported (14), Gnaqfl/fl Gna11–/– mice were indistinguishable from WT littermates and had a normal metabolic phenotype (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI76348DS1) and therefore were used as controls. The number of mutants alive at weaning was consistent with expected Mendelian ratios.
Loss of Gqα and G11α expression in the PVNs of PVNGq/11KO mice. (A) In situ hybridization with antisense Gqα-, G11α-, and Gsα-specific probes and a sense Gqα-specific probe in the PVNs of control (Gnaqfl/fl Gna11–/–), PVNGq/11KO, and WT mice (original magnification, ×20) (see Supplemental Table 2 for probe sequences). Hybridization with sense G11α- and Gsα-specific probes also showed no significant signal (data not shown). (B) Quantitation of in situ hybridization studies, with results expressed as percentage of WT (n = 6 per group). Our results show that in controls expression of G11α but not Gqα is lost, while in PVNGq/11KO mice expression of both Gqα and G11α is lost. Gsα expression is unaffected in all mouse lines. *P < 0.05 by 1-way ANOVA. (C) IP1 levels (normalized to protein) in PVNs from control and PVNGq/11KO mice after a 1-hour exposure to saline or MTII (50 nM) ex vivo (n = 6 per group). *P < 0.05 vs. control, #P < 0.05 vs. saline by Student’s t test. Data are expressed as mean ± SEM.
As PLC is the major effector of Gqα and G11α, we examined the effect of knocking out these G proteins on PLC signaling in the PVN. PVNs from control and PVNGq/11KO mice were isolated and incubated with either PBS or MTII, and accumulation of inositol monophosphate (IP1), a metabolite of inositol triphosphate (IP3), was examined in the presence of LiCl, which inhibits the metabolism of IP1 (19). Baseline IP1 levels (after PBS administration) in the PVNs from PVNGq/11KO mice were only 21% of those present in controls (Figure 1C), consistent with Gqα and G11α being lost in the PVN. Moreover, while MTII stimulated IP1 levels 7.3-fold in the PVNs from control mice, MTII incubation had no effect on IP1 levels in the PVNs from PVNGq/11KO mice (Figure 1C), indicating that a melanocortin MC3/4R agonist can stimulate Gq/11α/PLC signaling in the PVN.
PVNGq/11KO mice develop hyperphagia and obesity. PVNGq/11KO mice developed severe obesity (Figure 2, A and B), with markedly increased fat mass (Figure 2, C and D). Similar to Mc4r–/– (3) and SIM1 mutant mice (20), female mice had a greater disparity in body weight between mutant and control mice. Consistent with increased adiposity, PVNGq/11KO mice also developed severe hyperleptinemia (Table 1). Absolute lean mass was also increased, although less strikingly than fat mass, and was decreased when expressed as a percentage of total body weight (Figure 2D). Some of the increase in lean mass was due to PVNGq/11KO mice having increased body length (Figure 2E), a characteristic associated with both MC4R (2, 3) and SIM1 (5, 7) mutations but not observed in mBrGsKO mice (9).
PVNGq/11KO mice develop hyperphagia and severe obesity. (A) Body weight curves of male (n = 6–7 per group) and female (n = 8–13 per group) PVNGq/11KO mice and respective controls. (B) Representative image of a 24-week-old female PVNGq/11KO mouse and a control mouse. (C–E) Body composition showing (C) absolute lean and fat mass, (D) fat and lean mass expressed as a percentage of body weight, and (E) body length of 24- to 28-week-old female PVNGq/11KO and control mice (n = 8–9 per group). (F and H) Absolute food intake and (G and I) food intake normalized to body weight in 6- to 8-week-old female PVNGq/11KO mice and controls measured at (F and G) 24°C and at (H and I) thermoneutrality (30°C) (n = 6–8 per group). (J) Resting (Rest) and TEE and (K) total and ambulatory (Amb) motor activity at 24°C and 30°C in 6- to 8-week-old female PVNGq/11KO and control mice (n = 8 per group). (L) Percentage inhibition of food intake after MTII administration, as compared to that after injection with PBS vehicle in 8- to 10-week-old female mice (n = 6–7 per group). (M) Percentage increase in TEE (O2 utilization ) at 30°C after MTII administration, as compared to that after injection with PBS in 8- to 10-week-old female mice (n = 7 per group). Data are expressed as mean ± SEM. *P < 0.05 vs. controls by Student’s t test.
Serum chemistries in female PVNGq/11KO and control mice in the fed state
At 6 to 8 weeks of age, before the onset of obesity, PVNGq/11KO mice ate significantly more than controls at both room temperature (24°C; Figure 2, F and G) and at thermoneutrality (30°C, Figure 2, H and I). In contrast, there were no differences in resting energy expenditure and total energy expenditure (TEE) or activity levels between groups at either 24°C or 30°C (Figure 2, J and K). Similarly, PVNGq/11KO mice also showed hyperphagia at 12 to 16 weeks of age (Supplemental Figure 2, B and C), at a time when obesity was established (Supplemental Figure 2A), while energy expenditure and activity levels remained unaffected (Supplemental Figure 2, D and E). These effects on food intake and energy expenditure were similar to those observed in SIM1 mutant mice (5) but opposite to those observed in obese mBrGsKO mice (9).
Impaired anorectic response to melanocortin agonist in PVNGq/11KO mice. We next examined the acute effects of MTII on food intake and energy expenditure. Similar to SIM1 mutant mice (5) and opposite to that observed in mBrGsKO mice (9), PVNGq/11KO mice showed impaired ability to reduce food intake in response to MTII (Figure 2L), while the ability of MTII to stimulate TEE was maintained (Figure 2M).
MC3/4R activation of Gsα in the PVN has been shown to increase sympathetic nerve activity to the cardiovascular system and increase blood pressure (BP) and heart rate (HR) (21), and we recently confirmed this by showing that mPVNGsKO mice (also generated using Sim1-Cre) have reduced BP and HR (10). In contrast, BP and HR were unaffected in PVNGq/11KO mice (Figure 3, A and B), showing that even within the PVN distinct physiological effects of MC3/4R may be mediated through different G proteins.
Differential effects of MTII injections in the PVNs of PVNGq/11KO and mPVNGsKO mice. (A) Systolic BP and (B) HR in male PVNGq/11KO and control mice at 8 to 10 weeks (n = 8 per group). (C) Food intake and (E) systolic BP after injection of saline or MTII into the PVNs of PVNGq/11KO and control mice (n = 5–8 per group). (D) Food intake and (F) systolic BP after injection of saline or MTII into the PVNs of mPVNGsKO and control mice (n = 5–6 per group). Data are expressed as mean ± SEM. *P < 0.05 vs. controls at baseline, #P < 0.05 vs saline by Student’s t test.
To directly examine the effects of PVN-specific Gsα and Gq/11α deficiency on melanocortin-induced actions, cannulas were placed unilaterally in mPVNGsKO and PVNGq/11KO mice to allow direct delivery of MTII to the PVN, and its acute effects on food intake and systolic BP were examined. The dose of MTII was chosen based upon pilot experiments in control mice to determine the optimal dose for inhibition of food intake. In PVNGq/11KO mice, the ability of MTII injected into the PVN to inhibit food intake was lost in PVNGq/11KO mice (Figure 3C), while the ability of the same MTII injection to increase systolic BP was unaffected (Figure 3E). As we have shown previously (10), baseline systolic BP was lower than normal in mPVNGsKO mice (Figure 3F). In mPVNGsKO mice, the ability of MTII injected into the PVN to inhibit food intake was unaffected (Figure 3D), while the ability of MTII to stimulate systolic BP was lost (Figure 3F). These experiments directly confirm that melanocortin actions on food intake and BP in the PVN are mediated via distinct G protein pathways and that inhibition of food intake by melanocortins in the PVN is mediated by Gq/11α. The residual inhibition of food intake observed in response to i.p. MTII administration (Figure 2L) may represent the effects of the melanocortin agonist on peripheral sites (e.g., the gastrointestinal tract) (22) or other CNS sites (23, 24) or may be secondary to other metabolic changes resulting from systemic MTII administration.
Altered glucose and cholesterol metabolism in PVNGq/11KO mice. Older PVNGq/11KO mice developed hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance after becoming obese (24–28 weeks of age; Figure 4, C and D, and Table 1). However, glucose metabolism and insulin action were unaffected prior to the onset of obesity (6–8 weeks of age; Figure 4, A and B, and Table 1), in contrast to Mc4r–/– (1) and mBrGsKO mice (9), which have both been shown to develop glucose intolerance and insulin resistance at a young age, prior to the onset of obesity. This suggests that the primary effects of central melanocortins on glucose metabolism are mediated by Gsα rather than Gq/11α.
Glucose metabolism and cardiovascular function in PVNGq/11KO mice. (A and C) Glucose tolerance tests performed in (A) 6- to 8-week-old (n = 11–12 per group) and (C) 24- to 28-week-old female PVNGq/11KO and control mice (n = 9–10 per group). (B and D) Insulin tolerance tests performed in (B) 6- to 8-week-old (n = 7–10 per group) and (D) 24- to 28-week-old female PVNGq/11KO and control mice (n = 8–15 per group). AUCs for glucose and insulin tolerance tests were significantly different at 24 to 28 weeks (P < 0.05 by Student’s t test) but not at 6 to 8 weeks. Data are expressed as mean ± SEM.
Impaired melanocortin signaling has been shown to raise serum cholesterol levels (25). Serum cholesterol levels were significantly elevated in both young and old PVNGq/11KO mice (Table 1), while cholesterol levels were unaffected in 6- to 8-week-old mBrGsKO mice (mBrGsKO, 99 ± 10 mg/dl, vs. control, 91 ± 11 mg/dl, n = 5 per group). Serum-free fatty acid, triglyceride, and adiponectin levels were unaffected in PVNGq/11KO mice (Table 1). These results suggest that central melanocortin effects on cholesterol are mediated primarily by Gq/11α rather than Gsα.
Altered gene expression in the PVNs of PVNGq/11KO mice. We next examined the expression of various genes in the PVN by qRT-PCR. In PVNGq/11KO mice, Mc4r gene expression was unaffected, while Sim1 expression was significantly (26%) reduced (Figure 5A). Reduced Sim1 expression may be a direct effect of loss of MC4R/Gq/11α signaling and may contribute to hyperphagic obesity and increased linear growth, as Sim1 expression in the PVN has been shown to be induced by melanocortin signaling (5) and Sim1 haploinsufficiency is sufficient to lead to hyperphagic obesity and increased linear growth (5, 7). In contrast, mPVNGsKO mice showed no loss of Sim1 expression (Figure 5B), providing further evidence that regulation of Sim1 by MC4R in the PVN is mediated by Gq/11α rather than Gsα. Although it has been reported that SIM1 deficiency leads to hyperphagic obesity via reduction of oxytocin (Oxt) expression (26), Oxt expression in the PVNs of PVNGq/11KO mice was unaffected (Figure 5A), suggesting that other mechanisms downstream or independent of SIM1 may also be important in regulating food intake. Except for the gene encoding corticotropin-releasing hormone (Crh), which is discussed below, we observed no other changes in gene expression in the PVNs of PVNGq/11KO mice.
Reduced Sim1 and Crh expression in PVNGq/11KO mice. (A and B) PVN mRNA levels of various genes in (A) 8- to 10-week-old female PVNGq/11KO mice and (B) 12- to 16-week-old male mPVNGsKO mice and their littermate controls (n = 3–4 per group). Avp, arginine vasopressin; Trh, thyrotropin-releasing hormone; Bdnf, brain-derived neurotrophic factor; Sst, somatostatin. (C–E) Relative mRNA expression in the PVN of (C) Sim1, (D) Crh, and (E) Oxt 2 hours after administration of saline or MTII (5 mg/kg i.p.) in 8- to 10-week-old female control and PVNGq/11KO mice (n = 6 per group). Data are all normalized to controls and expressed as mean ± SEM. *P < 0.05 vs. controls, #P < 0.05 vs. saline by Student’s t test corrected for repeated measures.
To directly answer whether MTII can stimulate Sim1 and Crh gene expression and whether this action is mediated by Gq/11α, expression of these genes in the PVN were examined 2 hours after i.p. administration of PBS or MTII (5 mg/kg). In this experiment, baseline (after PBS administration) Sim1 and Crh mRNA levels were reduced by 40% and 48%, respectively, in PVNGq/11KO mice (Figure 5, C and D), confirming our original findings. MTII administration increased Sim1 and Crh mRNA levels by 38% and 75%, respectively, in controls, while MTII had no effect in PVNGq/11KO mice (Figure 5, C and D). These results show that MC4R activation leads to induction of Sim1 and Crh expression in a Gq/11α-dependent manner. In contrast, Oxt gene expression was unaffected by MTII and was similar in control and PVNGq/11KO mice (Figure 5E).
Impaired hypothalamic-pituitary-adrenal axis in PVNGq/11KO mice. Expression of the Crh gene, another gene that is upregulated by MC4R activation (27), was significantly (30%) reduced in PVNGq/11KO mice but not in mPVNGsKO mice (Figure 5, A and B), indicating that CRH induction by melanocortins (and likely other factors) in the PVN is mediated by Gq/11α. In line with reduced Crh expression, PVNGq/11KO mice had severe adrenal insufficiency, with markedly reduced serum adrenocorticotropin hormone (ACTH) and corticosterone levels (Figure 6), while we reported previously that corticosterone levels were unaffected in mBrGsKO mice (9). In addition, the stimulation of ACTH and corticosterone release at 1 hour after injection of insulin observed in control mice was significantly blunted in PVNGq/11KO mice (Figure 6), consistent with dysregulation of the hypothalamo-pituitary-adrenal axis at the level of the CNS.
Impaired hypothalamic-pituitary-adrenal axis in PVNGq/11KO mice. (A) Serum ACTH and (B) corticosterone levels in female 16- to 20-week-old PVNGq/11KO mice at baseline (0900, fed) and 1 hour after insulin administration (0.75 mIU/g, i.p.). Data are expressed as mean ± SEM (n = 5–8 per group). *P < 0.05 vs. controls, #P < 0.05 vs. basal by Student’s t test.
AAV-Cre–mediated Gq/11α signaling deficiency in the PVN leads to hyperphagic obesity. As Sim1 expression, which was used to drive Cre expression in PVNGq/11KO mice, is not limited to the PVN, we generated mice with Gq/11α deficiency specifically in the PVN by bilateral stereotaxic injection of adeno-associated virus–expressing Cre (AAV-Cre-GFP) into the PVNs of Gnaqfl/fl Gna11–/– mice to generate AAV-PVNGq/11KO mice. The same mice injected with AAV-GFP were used as controls. The injection sites were verified by fluorescence imaging (Figure 7A), and AAV-Cre-GFP injection led to an approximately 84% reduction in the PVN Gnaq mRNA (Figure 7B). By 10 weeks after injection, AAV-PVNGq/11KO mice developed severe obesity with a 2-fold increase in body weight relative to that of controls (Figure 7C), associated with significant hyperphagia (Figure 7D, 33% increase in food intake), which was measured at 4 weeks after injection (22% increase in body weight, data not shown). Similar to that in PVNGq/11KO mice, AAV-PVNGq/11KO mice also showed marked reductions in the PVN Sim1 and Crh mRNA levels, with no change in Mc4r mRNA levels (Figure 7B). As a control, we also knocked out Gnaq and Gna11 in the basomedial amygdala (BMA), another region in which Sim1 is expressed, by bilateral stereotaxic injection of AAV-Cre-GFP into this region in Gnaqfl/fl Gna11–/– mice, which led to an 80% reduction in Gqα expression (Supplemental Figure 3, A and B). This manipulation produced no effect on either body weight or food intake (Supplemental Figure 3, C and D).
AAV-PVNGq/11KO mice are hyperphagic and obese. (A) Representative image (original magnification, ×10; scale bar: 200 μM) showing fluorescence localized to PVN at 10 weeks after bilateral injection of AAV-Cre-GFP. (B) PVN mRNA levels of Gqα (Gnaq), Mc4r, Crh, and Sim1 measured 10 weeks after viral injection in AAV-PVNGq/11KO and control mice (n = 4 per group). (C) Body weight of AAV-PVNGq/11KO and control mice measured before and 10 weeks after viral injection (n = 6 per group). (D) Daily food intake of AAV-PVNGq/11KO and control mice measured 4 weeks after viral injection (n = 6 per group). Data are expressed as mean ± SEM. *P < 0.05 vs. controls by Student’s t test.