Dysfunction of the Maid gene induced hyperobesity in Mc4r gene-deficient mice. Mice with a Maid gene single knockout did not show obesity at the same level as wild-type (Supplemental Fig. 1), however, mice with a combined Maid and Mc4r gene deletion showed obesity even without a high-fat diet (Fig. 1A). The mean body weight at 28 weeks of age was 36.8 ± 7.9 g in Mc4r-KO, 55.6 ± 3.2 g in Maid-KO; Mc4r-KO mice and 40.7 ± 4.4 g in Mc4r-KO mice fed a high-fat diet (Mc4r-HFD) (p < 0.01, Fig. 1B). In addition, dietary intake was significantly higher in Mc4r-HFD mice compared to Mc4r-KO and Maid-KO; Mc4r-KO mice (4.2 ± 0.2g/day in Mc4r-KO, and 3.9 ± 0.5g/day in Maid-KO; Mc4r -KO, and 4.8 ± 0.7 g/day in Mc4r-HFD, respectively, p < 0.05, Fig. 1C).
According to these findings, Maid-KO; Mc4r-KO mice were found to be hyperobese even when not overfed or given a high-fat diet.
Dysfunction of Maid did not worsen metabolic status or induce liver fibrosis in Mc4r gene-deficient mice. Next, to assess MASLD and related metabolic abnormalities caused by hyperobesity, 28-week-old mice were subjected to liver weight/body weight ratio, liver histological evaluation, and blood biochemical analysis, comparing Mc4r-KO, Maid-KO; Mc4r-KO, and Mc4r-HFD.
Though no significant differences were found in these three groups, MC4R-KO; Maid-KO and Mc4r-HFD mice showed higher liver weight/body weight ratio than Mc4r-KO (4.2 ± 0.5% in Mc4r-KO, 5.6 ± 1.0% in Maid-KO; Mc4r-KO, and 5.7 ± 1.2% in MC4R-HFD, respectively, p = 0.09, Fig. 2A). Additionally, Maid-KO; Mc4r-KO and Mc4r-HFD mice had significantly higher liver fat deposition than Mc4r-KO mice (14.5% ± 1.9% in Mc4r-KO, 20.7% ± 4.5% in Maid-KO; Mc4r-KO, and 23.4% ± 2.5% in Mc4r-HFD, respectively, p < 0.01, Fig. 2B, C). Furthermore, Mc4r-HFD mice had significantly higher liver fibrosis area than Mc4r-KO and Maid-KO; Mc4r-KO mice (0.04 ± 0.03% in Mc4r-KO, 0.04% ± 0.03% in Maid-KO; Mc4r-KO, and 0.15% ± 0.08% in Mc4r-HFD, respectively, p < 0.01, Fig. 2B, D).
Blood biochemical analysis showed that Mc4r-HFD showed hyperlipidemia compared to Mc4r-KO and Maid-KO; Mc4r-KO (p < 0.01, Fig. 2E), but no significant differences in blood sugar and insulin levels and homeostasis model assessment for insulin resistance (HOMA-IR) were found. Furthermore, in 12-week-old mice, Mc4r-HFD demonstrated not only hyperlipidemia but also hyperglycemia and hyperinsulinemia when compared to Mc4r-KO and Maid-KO; Mc4r-KO (supplemental Fig. 2). Although Mc4r-HFD showed a decrease in blood insulin levels with hyperglycemia since youth, indicating the complication of insulin insufficiency.
These results revealed that Maid-KO; Mc4r-KO mice exhibited hyperobesity without liver fibrosis or metabolic abnormalities in contrast to Mc4r-HFD mice.
Maid gene dysfunction reduced the infiltration of macrophages in white adipose tissue. To assess fat accumulation in each adipocyte, the diameter of adipocytes in the visceral white adipose tissue (WAT) of 12-week-old mice was measured. Mc4r-HFD mice had a significantly larger size of adipocytes than Mc4r-KO and Maid-KO; Mc4r-KO mice (2140 ± 900 µm2 in Mc4r -KO, 3880 ± 1290 µm2 in Maid-KO; Mc4r-KO, and 8160 ± 2530 µm2 in Mc4r-HFD, respectively, p < 0.01, Fig. 3A, B). Furthermore, Maid-KO; Mc4r-KO mice also had a larger size of adipocytes than Mc4r-KO mice (p = 0.08 of the Tukey’s multiple comparison test). Maid gene dysfunction increased the size of adipocytes as well as their capacity for fat accumulation in each adipocyte, which is similar to high-fat loading.
To investigate tissue inflammation in visceral WAT, cluster of differentiation (CD) 206 and CD11c staining with immunofluorescence was performed. There were no significant differences in the number of CD206-positive M2 macrophages in the three groups (62.2 ± 12.2 cells/field of view (FOV) in Mc4r-KO, 40.8 ± 20.9 cells/FOV in Maid-KO; Mc4r-KO, and 51.6 ± 25.8 cells/FOV in Mc4r-HFD, respectively, p = 0.23, Fig. 3A, C), while CD11c-positive M1 macrophages in Maid-KO; Mc4r-KO were significantly fewer than in Mc4r-KO and Mc4r-HFD mice (3.4 ± 1.7 cells/FOV in Mc4r-KO, 0.4 ± 0.9 cells/FOV in Maid-KO; Mc4r-KO, and 3.6 ± 0.9 cells/FOV in Mc4r-HFD, respectively, p < 0.01, Fig. 3A, C).
In addition, the expression levels of both inflammatory cytokines related M1 macrophages (interleukin (IL) -6 and tumor necrosis factor (TNF)-α) and anti-inflammatory cytokines related M2 macrophages (IL-10 and transforming growth factor (TGF)-β) in Maid-KO; Mc4r-KO were lower than those in Mc4r-KO and Mc4r-HFD (p < 0.01 in IL-10, p < 0.05 in TGF-β, Fig. 3D) by quantitative PCR.
These findings demonstrated that reducing M1 macrophages in Maid-KO; Mc4r-KO mice suppressed adipose tissue inflammation, which contributed to the preservation of large adipocytes while also preventing inflammation from spreading to the liver and promoting liver fibrosis.
Maid gene dysfunction increased fat deposition by adipocytes via sympathetic nerve signaling. Quantitative PCR was used to investigate the cause of increased fat accumulation in adipose tissue, specifically adipocytokines and sympathetic nerve signaling in WAT. Adiponectin and resistin expression in WAT were lower in Maid-KO; Mc4r-KO and Mc4r-HFD mice than in Mc4r-KO mice, while leptin expression was significantly higher in Mc4r-HFD mice than in Mc4r-KO and Maid-KO; Mc4r-KO mice (p < 0.05 in adiponectin, p < 0.01 in leptin, Fig. 3D). This indicated hyperleptinemia and leptin resistance in Mc4r-HFD. Furthermore, the expression of β-3 adrenergic receptor was lower in Maid-KO; Mc4r-KO and Mc4r-HFD mice compared to Mc4r-KO mice, indicating WAT hypertrophy (Fig. 3D). To assess sympathetic nerve signaling, uncoupling protein-1 (UCP1) expression and fat deposition in brown adipose tissue (BAT) were measured. Maid-KO; Mc4r-KO and Mc4r-HFD mice showed significantly higher fat deposition in BAT than in Mc4r-KO mice (34.1% ± 2.6% in Mc4r-KO, 58.1% ± 10.4% in Maid-KO; Mc4r-KO, and 58.6% ± 1.2% in Mc4r-HFD, respectively, p < 0.01, Fig. 4A, B). In addition, UCP1 expression in Mc4r-KO mice was significantly higher compared to Maid-KO; Mc4r-KO in female mice (56.3% ± 2.9% in Mc4r-KO, 43.6 ± 0.8% in Maid-KO; Mc4r-KO, and 39.2% ± 1.2% in Mc4r-HFD, respectively, p < 0.01, Fig. 4A, C).
These results indicated BAT in Maid-KO; Mc4r-KO were more higher fat deposition and lower expression of UCP1 than in MC4R-KO mice via sympathetic nerve signaling and β-3 adrenergic receptor.