Gqα/G11α deficiency in dorsomedial hypothalamus leads to obesity resulting from decreased energy expenditure and impaired sympathetic nerve activity

Abstract

The G-protein subunits G_qα and G₁₁α (G_q/11α) couple receptors to phospholipase C, leading to increased intracellular calcium. In this study we investigated the consequences of G_q/₁₁α deficiency in the dorsomedial hypothalamus (DMH), a critical site for the control of energy homeostasis. Mice with DMH-specific deletion of G_q/11α (DMHGq/11KO) were generated by stereotaxic injection of adeno-associated virus (AAV)-Cre-green fluorescent protein (GFP) into the DMH of G_qα^flox/flox:G₁₁α^-/- mice. Compared with control mice that received DMH injection of AAV-GFP, DMHGq/11KO mice developed obesity associated with reduced energy expenditure without significant changes in food intake or physical activity. DMHGq/11KO mice showed no defects in the ability of the melanocortin agonist melanotan II to acutely stimulate energy expenditure or to inhibit food intake. At room temperature (22°C), DMHGq/11KO mice showed reduced sympathetic nervous system activity in brown adipose tissue (BAT) and heart, accompanied with decreased basal BAT uncoupling protein 1 (Ucp1) gene expression and lower heart rates. These mice were cold intolerant when acutely exposed to cold (6°C for 5 h) and had decreased cold-stimulated BAT Ucp1 gene expression. DMHGq/11KO mice also failed to adapt to gradually declining ambient temperatures and to develop adipocyte browning in inguinal white adipose tissue although their BAT Ucp1 was proportionally stimulated. Consistent with impaired cold-induced thermogenesis, the onset of obesity in DMHGq/11KO mice was significantly delayed when housed under thermoneutral conditions (30°C). Thus our results show that G_qα and G₁₁α in the DMH are required for the control of energy homeostasis by stimulating energy expenditure and thermoregulation.NEW & NOTEWORTHY This paper demonstrates that signaling within the dorsomedial hypothalamus via the G proteins G_qα and G₁₁α, which couple cell surface receptors to the stimulation of phospholipase C, is critical for regulation of energy expenditure, thermoregulation by brown adipose tissue and the induction of white adipose tissue browning.

Keywords: G proteins; hypothalamus; obesity; thermogenesis.

Graphical abstract

Figure 1.

Mice with dorsomedial hypothalamus-specific deletion of G_q/11α (DMHGq/11KO) develop obesity. A: representative image confirming bilateral injection of adeno-associated virus (AAV)-green fluorescent protein (GFP) by green fluorescence into the DMH. VMH, ventromedial hypothalamus; 3V, third ventricle. B: G_qα mRNA levels in GFP⁺ and GFP⁻ cells isolated by FACS from the hypothalamus of control and DMHGq/11KO mice (n = 4/group). C: body weight curves of DMHGq/11KO and control mice after viral injections (n = 10–12/group). D: body composition of DMHGq/11KO and control mice at 3–4 mo postviral injection (n = 6–7/group). E: representative histologic images (hematoxylin and eosin staining) of brown adipose tissue (BAT), epidydimal white adipose tissue (eWAT), and inguinal white adipose tissue (iWAT) from DMHGq/11KO and control mice. Scale bar = 100 µm. F and G: adipocyte area (F; µm²) and number/section (G) quantified for BAT, eWAT, and iWAT from DMHGq/11KO mice and controls (n = 3/group; each number is the average of 2 sections/mouse). H: body lengths of DMHGq/11KO and control mice at 4–6 mo postviral injection (n = 10–11/group). I and J: body weight (I) and composition (J) of DMHGqKO and control mice (n = 5/group). Data are shown as means ± SE. *P < 0.05 or **P < 0.01 vs. controls.

Figure 2.

Mice with dorsomedial hypothalamus-specific deletion of G_q/11α (DMHGq/11KO) have reduced energy expenditure (EE). A: daily food intake (left) and mean body weight (right) in DMHGq/11KO and control mice measured from 2 to 4 wk postviral injection (n = 10–11/group). B: total and ambulatory (Amb) activity levels in DMHGq/11KO and control mice at 3–4 mo postviral injection measured at 22°C and 30°C (n = 10/group). C: linear regression analysis of body weight vs. total EE (TEE) at 22°C (left) or 30°C (right) for DMHGq/11KO and control mice at 3–4 mo postviral injection (n = 10–11/group). D: body weight curves of DMHGq/11KO and control mice maintained at 30°C for 7 wk starting 1 wk postviral injection (n = 8/group). E: food intake after melanotan II (MTII) intraperitoneal administration in DMHGq/11KO and control mice expressed as %intake vs. after saline injection (n = 7/group). F: percent increase in EE (O₂ consumption) after MTII intraperitoneal injection in DMHGq/11KO and control mice (n = 6–7/group). Data are shown as means ± SE. *P < 0.05 or **P < 0.01 vs. controls.

Figure 3.

Mice with dorsomedial hypothalamus-specific deletion of G_q/11α (DMHGq/11KO) have impaired tolerance to acute and chronic cold conditions. A: rectal temperature in DMHGq/11KO and control mice (3–6 mo postviral injection) at room temperature (0 h) or at the indicated time points after being placed at 6°C ambient temperature (n = 8–10/group). B: serum norepinephrine (NE) levels in DMHGq/11KO and control mice at 22 °C or following exposure to 6°C for 5 h (n = 5/group). C: brown adipose tissue (BAT) uncoupling protein 1 (Ucp1) mRNA levels at room temperature (22°C) or after acute exposure to 6°C for 5 h (6°C-5 h) or after chronic cold adaptation to 6°C (6°C-adapt) in DMHGq/11KO and control mice (n = 5–6/group). D: daily rectal temperature during cold adaptation in DMHGq/11KO and control mice at 4.5–6 mo postviral injection (n = 6–7/group). The ambient temperature on each day is indicated at the top. Mice were removed from chronic cold adaptation when rectal temperature fell below 25°C (indicated by the dotted line). E: representative histologic images (hematoxylin and eosin staining) of BAT, epidydimal white adipose tissue (eWAT), and inguinal white adipose tissue (iWAT) from DMHGq/11KO and control mice after chronic cold adaptation. Scale bar = 100 µm. F: representative images of UCP1 immunostaining in iWAT sections from DMHGq/11KO and control mice after chronic cold adaptation (6°C). G and H: adipocyte area (G; µm²) and number/section (H) quantified for BAT (n =2–3/group), eWAT (n = 3–4/group), and iWAT (n = 3–4/group) from DMHGq/11KO mice and controls (each number is the average of 2 sections/mouse). Data are shown as means ± SE. *P < 0.05 or **P < 0.01 vs. control; ##P < 0.01 vs. 22 °C.

Figure 4.

Reduced sympathetic nervous system activity and cardiovascular function in mice with dorsomedial hypothalamus-specific deletion of G_q/11α (DMHGq/11KO). A–C: cells expressing tyrosine hydroxylase (TH⁺) were visualized and quantified by immunohistochemistry in brown adipose tissue (BAT; A; n = 11/group), inguinal white adipose tissue (iWAT; B; n = 11–13/group), and heart (C; n = 12–13/group) of DMHGq/11KO and control mice at 6 mo postviral injection. Mice were maintained at 22°C. In A–C, representative TH, DAPI, and merged images are shown on the left and quantification (normalized to controls) is shown on the right. D and E: heart rate (D) and systolic and diastolic blood pressure (E) were measured in DMHGq/11KO and control mice at 7–11 wk postviral injection (n = 8–9/group). Data are shown as means ± SE. *P < 0.05 or **P < 0.01 vs. control.

Figure 5.

Glucose metabolism in mice with dorsomedial hypothalamus-specific deletion of G_q/11α (DMHGq/11KO). A: fasting blood glucose levels at 2–3 mo postviral injection (n = 11–14/group). B: glucose tolerance tests were performed in DMHGq/11KO and control mice at 2–3 mo postviral injection (n = 11–14/group). Areas under the curve (AUC) are shown to the right. C: serum insulin levels at time 0 and 40 min during the glucose tolerance tests shown in B. D: insulin tolerance tests (left) and AUC values (right) in DMHGq/11KO and control mice at 2–2.5 mo postviral injection (n = 11–13/group). E and F: body weights (E) and results of glucose tolerance tests (F) performed in young DMHGq/11KO and control mice at 2 wk postviral injection after 3 h of fasting (n = 10–11/group). Data are shown as means ± SE. *P < 0.05 vs. controls.