A mouse model for a partially inactive obesity-associated human MC3R variant

Bonggi Lee, Jashin Koo, Joo Yun Jun, Oksana Gavrilova, Yongjun Lee, Arnold Y Seo, Dezmond C Taylor-Douglas, Diane C Adler-Wailes, Faye Chen, Ryan Gardner, Dimitri Koutzoumis, Roya Sherafat Kazemzadeh, Robin B Roberson, Jack A Yanovski, Bonggi Lee, Jashin Koo, Joo Yun Jun, Oksana Gavrilova, Yongjun Lee, Arnold Y Seo, Dezmond C Taylor-Douglas, Diane C Adler-Wailes, Faye Chen, Ryan Gardner, Dimitri Koutzoumis, Roya Sherafat Kazemzadeh, Robin B Roberson, Jack A Yanovski

Abstract

We previously reported children homozygous for two MC3R sequence variants (C17A+G241A) have greater fat mass than controls. Here we show, using homozygous knock-in mouse models in which we replace murine Mc3r with wild-type human (MC3R(hWT/hWT)) and double-mutant (C17A+G241A) human (MC3R(hDM/hDM)) MC3R, that MC3R(hDM/hDM) have greater weight and fat mass, increased energy intake and feeding efficiency, but reduced length and fat-free mass compared with MC3R(hWT/hWT). MC3R(hDM/hDM) mice do not have increased adipose tissue inflammatory cell infiltration or greater expression of inflammatory markers despite their greater fat mass. Serum adiponectin levels are increased in MC3R(hDM/hDM) mice and MC3R(hDM/hDM) human subjects. MC3R(hDM/hDM) bone- and adipose tissue-derived mesenchymal stem cells (MSCs) differentiate into adipocytes that accumulate more triglyceride than MC3R(hWT/hWT) MSCs. MC3R(hDM/hDM) impacts nutrient partitioning to generate increased adipose tissue that appears metabolically healthy. These data confirm the importance of MC3R signalling in human metabolism and suggest a previously-unrecognized role for the MC3R in adipose tissue development.

Conflict of interest statement

J.A.Y. is a Commissioned Officer in the United States Public Health Service (PHS), Department of Health and Human Services. The remaining authors declare no competing financial interests.

Figures

Figure 1. Validation of mouse MC3R replacement…
Figure 1. Validation of mouse MC3R replacement by human MC3R.
Quantitative real-time PCR for relative hypothalamic mRNA expression normalized by β-actin expression by the 2–ΔΔCt method (n=3/group) in 4-month-old female C57BL/6 mice (MC3R+/+) or knock-in mice that were homozygous (MC3RhWT/hWT) or heterozygous (MC3RhWT/+) for the common alleles for the human MC3R or homozygous (MC3RhDM/hDM) or heterozygous (MC3RhDM/+) for the human MC3R sequence variants C17A+G241A. Expression was determined using human-specific MC3R primers (a,b) and mouse-specific MC3R primers (c,d). MC3R protein expression (e) was measured by western blotting (for MC3RhWT/hWTn=5; for MC3RhDM/hDMn=4) in homozygous mice and in the hypothalamic N8 murine cell line, which does not express MC3R mRNA. MC3R protein expression adjusted for GAPDH was quantified using Image J (f). Data are represented as mean±s.e.m. A different letter represents significant differences at P<0.05 compared with the other groups. Similar results were found for male mice (data not shown). Groups were compared by one-way analysis of variance followed by Bonferroni post-tests (a–d) and Student's t-test (two-tailed) (f).
Figure 2. Greater fat mass and reduced…
Figure 2. Greater fat mass and reduced fat-free mass in MC3RhDM/hDM mice.
(a) Female (5-month old) MC3RhDM/hDM mice had increased adiposity compared with MC3RhWT/hWT. (b,c) Female MC3RhWT/hWT (n=7, open bars) or MC3RhDM/hDM (n=10, closed bars) mice were fed chow diet or (d,e) high-fat diet (MC3RhWT/hWTn=6; MC3RhDM/hDMn=9) for 2 months. Body fat mass and fat-free mass were measured by MRI at age 2–3 months and 4–5 months for chow-fed and high-fat-fed mice, respectively. Data are represented as mean±s.e.m. *P<0.05 and **P<0.01 MC3RhDM/hDM versus MC3RhWT/hWT mice. Groups were compared by Student's t-tests (two-tailed) (b–e).
Figure 3. Micro-CT shows decreased bone in…
Figure 3. Micro-CT shows decreased bone in femurs of MC3RhDM/hDM mice.
(a) Trabecular number (per mm) of chow-fed female (12-week old) MC3RhDM/hDM and MC3RhWT/hWT mice. (b) Trabecular bone-volume fraction: Trabecular Bone Volume/Total Volume (BV/TV), (c) Trabecular thickness, (d) average cortical thickness, and (e) Cortical area per Total area fraction were reduced in MC3RhDM/hDM; (f) Medullary (marrow) area was increased in MC3RhDM/hDM; (g) representative reconstructed 3D images of femur trabecular bones; (h) representative cross-sectional cortical bones (h). Data are represented as mean±s.e.m.; MC3RhWT/hWTn=7; MC3RhDM/hDMn=8. *P<0.05 MC3RhDM/hDM versus MC3RhWT/hWT mice. Groups were compared by Student's t-tests (two-tailed) (a–f).
Figure 4. Increased feeding efficiency in MC3R…
Figure 4. Increased feeding efficiency in MC3RhDM/hDM mice.
Feeding efficiency (the ratio of body weight to energy intake (g Kcal−1) × 20) was determined in female MC3RhWT/hWT (open bars) and MC3RhDM/hDM (closed bars) mice during (a) 5 weeks of chow diet feeding or (b) 7 weeks of high-fat-diet feeding. (c) For the high-fat pair-feeding study, the daily energy intake (11.83 Kcal per day) of 7- to 8-week-old female high-fat-fed MC3RhWT/hWT mice (n=7) was supplied to the age matched female high-fat-fed MC3RhDM/hDM mice (n=5), and their body weight change was monitored weekly. (d,e) Body composition of MC3RhWT/hWT and MC3RhDM/hDM mice was determined immediately after the end of pair-feeding period at age 12–13 weeks. Data are represented as mean±s.e.m. *P<0.05 and **P<0.01 MC3RhDM/hDM versus MC3RhWT/hWT mice. Groups were compared by Student's t-tests (two-tailed) (a,b,d and e) and two-way analysis of variance followed by Bonferroni post-tests (c).
Figure 5. Maintained leptin sensitivity in MC3R…
Figure 5. Maintained leptin sensitivity in MC3RhDM/hDM mice.
Blood was collected from tail veins of female MC3RhWT/hWT (open symbols) and MC3RhDM/hDM (closed symbols) mice. (a) Serum leptin concentrations were measured in fed (n=10 per group) and fasted (n=7 per group) conditions. (b) Serum leptin concentrations were significantly correlated with body fat mass in both MC3RhWT/hWT (dotted line; P<0.0041) and MC3RhDM/hDM (solid line, P<0.0043) mice, but these slopes were not significantly different (P=0.29) and leptin values were not different after results were adjusted for fat mass. (c,d) 2-month-old chow-fed mice (n=5 per group) were injected twice daily with mouse leptin (1 μg g−1 body weight, arrows) for 3 days. (c) Food intake and (d) body weight were measured daily. Inset bar graphs (e,f) show body weight and fat mass of the mice given leptin. Data are represented as mean±s.e.m. A different letter represents significant differences at P<0.05 compared with the other groups. Groups were compared by one-way (a) and two-way (c,d) analysis of variance followed by Bonferroni post-tests, Student's t-tests (two-tailed) (e–f), and analysis of covariance (b).
Figure 6. Maintained insulin sensitivity and circulating…
Figure 6. Maintained insulin sensitivity and circulating lipid and hormone profile in MC3RhDM/hDM mice.
Serum samples were used to measure (a) triglycerides, (b) total cholesterol, (c) glucose, (d) insulin, (e) free fatty acids and (f) corticosterone in 3-month-old female MC3RhWT/hWT (open symbols) and MC3RhDM/hDM (closed symbols) mice. For MC3RhWT/hWTn=8 except n=6 for triglycerides and corticosterone; for MC3RhDM/hDMn=9. For glucose (g) and insulin (h) challenge tests (n=7 per group), glucose and insulin were injected at time 0. Fat mass (i) and fat-free mass (j) were determined on the day of the glucose tolerance test. Data are represented as mean±s.e.m. A different letter represents significant differences at P<0.05 compared to the other groups. Groups were compared with one-way (a–f) and two-way (g–h) analysis of variance followed by Bonferroni post-tests and Student's t-tests (two-tailed) (i,j).
Figure 7. Increased serum adiponectin in MC3R…
Figure 7. Increased serum adiponectin in MC3RhDM/hDM mice and humans.
(a) Serum adiponectin levels were measured in 3-month-old female MC3RhWT/hWT (open bars) and MC3RhDM/hDM (closed bars) mice under fed (n=8 per group) and fasted (n=11 per group). (b) Plasma samples from a matched cohort of MC3RhWT/hWT and MC3RhDM/hDM children (Supplementary Table 4) were analysed for adiponectin (n=13 per group). Data are represented as mean±s.e.m. A different letter represents significant differences at P<0.05 compared with the other groups. Groups were compared by one-way analysis of variance followed by Bonferroni post-tests (a) and analysis of covariance (b).
Figure 8. Maintained adipose tissue function in…
Figure 8. Maintained adipose tissue function in MC3RhDM/hDM mice.
Gonadal fat was isolated from fasted female mice fed a high-fat diet for (a) quantitative real-time PCR (MC3RhWT/hWTn=5; MC3RhDM/hDMn=8/group), (b–d) western blotting analysis (MC3RhWT/hWTn=5; MC3RhDM/hDMn=4), and (e–l) FACS analysis (MC3RhWT/hWTn=4; MC3RhDM/hDMn=5) in MC3RhWT/hWT (open bars) and MC3RhDM/hDM (closed bars) mice. (a) The expression levels of genes related to macrophage infiltration, inflammation and adipose tissue metabolism were normalized for β-actin expression. Western protein expression results for (b) peroxisome proliferator-activated receptor gamma (PPARγ), (c) adiponectin and (d) phosphospecific 5′-adenosine monophosphate-activated protein kinase (p-AMPK), divided by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and normalized relative to average for MC3RhWT/hWT. The stromal vascular fraction was isolated from gonadal fat (∼2 g) from MC3RhWT/hWT and MC3RhDM/hDMmice. For detecting macrophages by flow cytometry, a gating strategy was used to enrich samples from MC3RhWT/hWT and MC3RhDM/hDMmice for adipose tissue macrophages by selecting cells in the gate 1 area (e,g). The gate 1 area was stained with a F4/80 antibody (f and h) and the dot plots depict forward scatter (FSC) and side scatter (SSC) (left). For detecting neutrophils, whole cells (non-gated) were stained with CD11b and Gr-1 (i,j). Bar graphs show the average values±s.e.m. for percentage of cells in the stromal vascular fraction that were (k) macrophages and (l) neutrophils. *P<0.05 MC3RhDM/hDMversus MC3RhWT/hWT. (m,n) Haematoxylin and eosin stained sections showed no apparent differences in adipocyte morphology between groups. Groups were compared by Student's t-tests (two-tailed) (a–d and k–i). Scale bar, 100 μm. Data are represented as mean±s.e.m. for a-d, k, and l. FACS, fluorescence-activated cell sorting
Figure 9. Decreased osteoblast differentiation in MSCs…
Figure 9. Decreased osteoblast differentiation in MSCs of MC3RhDM/hDM mice.
MSCs were isolated from compact bone of tibia and femur from chow-fed female MC3RhWT/hWT and MC3RhDM/hDM mice at 7 weeks of age. Cells were isolated from one mouse for each group, and six independent experiments were performed (n=6/group). Isolated MSCs were cultured in 25-cm cell culture dishes for 4 passages (see methods for detailed information). MSCs were used (a) for western blotting to examine MC3R protein expression or (b–f) MSCs were differentiated into osteoblasts for 14 days to examine differentiation capacity. (b–c) Cultured MSCs differentiated into osteoblasts after Alizarin red S staining. (d–e) Microscopic images (10X) of osteoblasts after Alizarin red S staining. Scale bar, 100 μm. (f) Stained Alizarin red S was extracted from osteoblast and quantified at 450 nm. (g–j) Confocal microscopic images of osteoblasts stained with Alizarin red S (red) (g,h) or osteocalcin (red) (i,j). Nuclei were stained with DAPI (blue). Representative maximum intensity projection images are shown. Scale bar, 25 μm. (k) qPCR analysis of genes related to osteoblast differentiation (7 days after differentiation). Similar results were found for male mice (data not shown). Data are represented as mean±s.e.m. for f and k. Groups were compared by Student's t-tests (two-tailed) (f,k).
Figure 10. Increased adipocyte differentiation in MSCs…
Figure 10. Increased adipocyte differentiation in MSCs of MC3RhDM/hDM mice.
MSCs were isolated as described in Fig. 8. (a–f) MSCs were differentiated into adipocytes to examine differentiation capacity. Cells were isolated from one mouse for each group, and six independent experiments were performed (n=6 per group). (a) Microscopic images (× 10) of MSCs differentiated into adipocytes for 18 days. (b) Microscopic images of differentiated adipocytes after Oil red O staining. Scale bar, 100 μm (c) Oil red O was extracted and quantified at 520 nm. (d–f) Confocal microscopic images of MSCs differentiated into adipocytes. Cells were stained with Nile Red and Hoechst 33342 at indicated days. Representative maximum intensity projection images of lipid droplets (LDs, red) and nuclei (blue) are shown. Scale bar, 25 μm (g) The percentage of cells displaying LDs>10 μm in diameter was quantified (n=∼350). (h) qPCR analysis of genes related to adipocyte differentiation (7 days after differentiation). Similar results were found for male mice (data not shown). Data are represented as mean±s.e.m. for c and h. Groups were compared by Student's t-tests (two-tailed) (c,h).

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