Discovery of melanin-concentrating hormone receptor 1 in brown adipose tissue

Abstract

Although extensive research on brown adipose tissue (BAT) has stimulated optimism in the battle against obesity and diabetes, BAT physiology and organ crosstalk are not fully understood. Besides BAT, melanin-concentrating hormone (MCH) and its receptor (MCHR1) play an important role in energy homeostasis. Because of the link between hypothalamic MCH neurons and sympathetic BAT activation via β-adrenoceptors, we investigated the expression and physiological role of the MCHR1 in BAT. MCHR1 was detected in rodent and human BAT with RT-qPCR and western blot analyses. In vivo imaging in rats used the glucose analog [¹⁸ F]FDG and the MCHR1-tracer [¹¹ C]SNAP-7941. We found that the β3-adrenoceptor (ADRB3) agonist CL316,243 increased [¹¹ C]SNAP-7941 uptake in BAT. Additionally, a pharmacological concentration of SNAP-7941-a low-affinity ADRB3 ligand-stimulated [¹⁸ F]FDG uptake, reflecting BAT activation. In cultured human adipocytes, CL316,243 induced MCHR1 expression, further supporting a direct interaction between MCHR1 and ADRB3. These findings characterized MCHR1 expression in rodent and human BAT for the first time, including in vitro and in vivo data demonstrating a link between MCHR1 and the β3-adrenergic system. The presence of MCHR1 in BAT emphasizes the role of BAT in energy homeostasis and may help uncover treatment approaches for obesity.

Keywords: MCHR1; PET; brown adipose tissue; imaging; obesity.

Figure 1

The μPET scan shows [¹¹C]SNAP‐7941 uptake in rat BAT. Exemplary BAT uptake of [¹¹C]SNAP‐7941 in rats shown with a transversal (left), sagittal (center), and coronal (right) μPET/CT summation image (0–45 minutes). White arrows indicate corresponding BAT uptake.

Figure 2

This figure represents the timeline of small animal imaging. After a μCT scan of 7 min, the respective tracer was applied and the μPET scan started. In the case of imaging with [¹¹C]SNAP‐7941, SNAP‐7941, CL316,243, or vehicle was added 15 min after tracer application. The μPET scan was terminated after 45 minutes. As for the [¹⁸F]FDG scans, SNAP‐7941 or vehicle was added 60 min after tracer application and the μPET scan was stopped after 120 minutes.

Figure 3

Mchr1 mRNA is expressed in rodent BAT. Relative Mchr1 expression in BALB/c brain and BAT of two different mouse strains (A, two animals each) and in the brain and BAT of HIM:OFA rats (B, three and four animals, respectively). Data are represented as the mean ± SEM from independent experiments. The Ct values were normalized to β‐actin, relative mRNA expression was calculated with respect to the negative control tissue spleen using the formula log(2^–ΔΔCt). Mchr1 expression on the genome level is significantly lower in rats aged ≥50 weeks (^* P < 0.05).

Figure 4

MCHR1 mRNA is expressed in human BAT, but does not correlate with UCP1 mRNA expression. Mean relative MCHR1 and UCP1 expression (±SEM) in human fat depot biopsies of the carotid sheath, thymus, and longus colli muscle (A). The Ct values were normalized to 36B4, relative mRNA expression was calculated with respect to a subcutaneous WAT as the positive control with the formula log(2^–ΔΔCt) (see Fig. S4 for the ΔCt values, online only). The respective 2^–ΔΔCt values for MCHR1 were plotted against UCP1 levels of the carotid sheath (B), thymus (C), and longus colli muscle (D) fat depots. Trend analyses (E–G) show a significant positive trend with regard to MCHR1 versus UCP1 for the carotid sheath (R ² = 0.8196) and longus colli muscle samples (R ² = 0.8529), but not for the thymus. Dotted red lines correspond to the confidence interval (95%). Only deep cervical fat samples with a significantly higher UCP1 expression compared with white fat depots were used for analysis (n = 6 for the carotid sheath, n = 19 for the thymus, and n = 15 for the longus colli muscle).

Figure 5

UCP1 and MCHR1 mRNA expression increases during differentiation of hAPCs and is induced by CL316,243. The mean relative MCHR1 (A) and UCP1 (B) mRNA expression (±SEM) on day 0 (before differentiation), day 3 (during differentiation), and day 6 (fully differentiated). MCHR1 significantly (^* P < 0.05) increases with CL316,243 stimulation.

Figure 6

MCHR1 expression is comparable in two different mouse strains and dependent on age in HIM:OFA rats. MCHR1 expression in the brain and spleen of BALB/c mice and in BAT of two different mouse strains (A and B), as well as in the brain, spleen, and BAT of HIM:OFA rats of different age (C–E). (A, C, and D) The original western blots and matching relative pixel densities of the respective protein bands, normalized to Ponceau S (see Fig. S1, online only). In (C) and (D), all rats aged ≤40 weeks are shown in light blue and the ones aged ≥50 weeks in darker blue. Relative pixel densities of the protein bands of rat BAT probes in (C) were calculated for the 35–40‐kDa bands, not for the weaker ones visible at ∼55 kDa.

Figure 7

[¹⁸F]FDG uptake in BAT is significantly induced through SNAP‐7941. A representative BAT uptake of [¹⁸F]FDG shown in axial rat μPET summation images before and after treatment with 15 mg/kg SNAP‐7941 (A) and the dedicated vehicle (B), respectively. Corresponding TACs (center) and paired test statistics (right) are presented for both treatments with 15 mg/kg SNAP‐7941 (A) and the dedicated vehicle (B). Data are plotted as the mean ± SEM from independent experiments (n = 4). Differences among groups were tested using a two‐tailed parametric paired t‐test (^** P ≤ 0.01). If not visible, error bars are within the margin of the symbols. Red arrows indicate the time point of SNAP‐7941 or vehicle addition.

Figure 8

[¹¹C]SNAP‐7941 uptake in BAT is significantly induced through SNAP‐7941 and CL316,243. Representative BAT uptake of [¹¹C]SNAP‐7941 shown in axial rat μPET summation images before and after treatment with 2 mg/kg CL316,243 (A), 15 mg/kg SNAP‐7941 (B), and the dedicated vehicle (C). Corresponding TACs (center) and paired test statistics (right) are presented for the BAT uptake under treatment with 2 mg/kg CL316,243 (A), 15 mg/kg SNAP‐7941 (B), and the dedicated vehicle (C), respectively. Data are plotted as the mean ± SEM from independent experiments (n = 4). Differences among groups were tested using a two‐tailed parametric paired t‐test (^* P ≤ 0.05). If not visible, error bars are within the margin of the symbols. Red arrows indicate the time point of SNAP‐7941, CL316,243, or vehicle addition.