Mc4r-KO

Fig.1 Body weight change of male Mc4r-KO mice under CD and HFD conditions. Male 10-week-old WT C57BL/6 mice and homozygous Mc4r-KO mice were put on a CD or HFD feeding for body wight (A) and body weight change (B) recording. (n=4-10, Mean±SEM).

Abbr. WT, wild type; HO, homozygous; CD, chow diet; HFD, high-fat diet.

Fig.2 Food intake of male Mc4r-KO mice under CD and HFD conditions. Male 10-week-old WT C57BL/6 mice and homozygous Mc4r-KO mice were put on a CD or HFD feeding for food intake detection. (n=4-10, Mean±SEM). 

Abbr. WT, wild type; HO, homozygous; CD, chow diet; HFD, high-fat diet.

Fig.3 Detection of fasting blood glucose after 8 weeks (16 h fast) or 9 weeks (6 h fast) of CD or HFD feeding. (n=10 per group, Mean±SEM, Welch’s t test, Unpaired t test or Mann-Whitney test, *p＜0.05, **p＜0.01, ***p＜0.001, ****p＜0.0001, ns, no significance). 

Abbr. WT, wild type; HO, homozygous; CD, chow diet; HFD, high-fat diet.

Fig.4 Detection of fasting blood glucose after 20 weeks (16 h fast) or 22 weeks (6 h fast) of CD or HFD feeding. (n=6 per group, Manisem, Welch’s t test, Unpaired t test or Mann-Whitney test, *p＜0.05, **p＜0.01, ***p＜0.001, ns, no significance). 

Abbr. WT, wild type; HO, homozygous; CD, chow diet; HFD, high-fat diet.

Fig.5 Detection of glucose tolerance in WT and Mc4r-KO mice after 8 weeks of CD or HFD feeding. Male, 10-week-old WT mice and homozygous Mc4r-KO mice were maintained on either CD or HFD for 8 weeks. An intraperitoneal glucose tolerance test (IPGTT) was subsequently performed. Following a 16-h fast, baseline blood glucose was measured (0 min), followed by an intraperitoneal glucose injection (2 g/kg). (A) Blood glucose levels were monitored at 15, 30, 60, 90, and 120 min post-injection, and (B) the area under the curve (AUC) was calculated. (n=10 per group, Manisem, Welch’s t-test, Unpaired t-test or Mann-Whitney test, ****p＜0.0001)

Abbr. WT, wild type; HO, homozygous; CD, chow diet; HFD, high-fat diet; ns, no significance.

Fig.6 Detection of glucose tolerance in WT and Mc4r-KO mice after 20 weeks of CD or HFD feeding. Male, 10-week-old WT mice and homozygous Mc4r-KO mice were maintained on either CD or HFD for 20 weeks. An intraperitoneal glucose tolerance test (IPGTT) was subsequently performed. Following a 16-h fast, baseline blood glucose was measured (0 min), followed by an intraperitoneal glucose injection (2 g/kg). (A) Blood glucose levels were monitored at 15, 30, 60, 90, and 120 min post-injection, and (B) the area under the curve (AUC) was calculated. (n=6 per group, Manisem, Welch’s t-test, Unpaired t-test or Mann-Whitney test, *p＜0.05, **p＜0.01, ***p＜0.001)

Fig.7 Detection of insulin tolerance of Mc4r-KO mice after 9 weeks of CD or HFD feeding. Male, 10-week-old WT mice and homozygous Mc4r-KO mice were maintained on either CD or HFD for 9 weeks. Insulin tolerance test (ITT) was then performed. All mice were fasted for 6 h, detected blood glucose (0 min), then received an intraperitoneal insulin injection (0.5 IU/kg body weight). (A) Blood glucose levels were measured at 15, 30, 60, 90, 120 and 150 min post-injection and (B) area under curve (AUC) was calculated. (n=10 per group, Mean±SEM, Unpaired t-test, ***p＜0.001, ****p＜0.0001)

Fig.8 Detection of insulin tolerance of Mc4r-KO mice after 22 weeks of CD or HFD feeding. Male, 10-week-old WT mice and homozygous Mc4r-KO mice were maintained on either CD or HFD for 22 weeks. Insulin tolerance test (ITT) was then performed. All mice were fasted for 6 h, detected blood glucose (0 min), then received an intraperitoneal insulin injection (1.25 IU/kg body weight). (A) Blood glucose levels were measured at 15, 30, 60, 90, 120 and 150 min post-injection and (B) area under curve (AUC) was calculated. (n=10 per group, Mean±SEM, Welch’s t-test, Unpaired t-test or Mann-Whitney test, ***p＜0.001, ****p＜0.0001)

Fig.9 Detection of glucose-stimulated insulin secretion of Mc4r-KO mice after 10 weeks of CD or HFD feeding. Male, 10-week-old WT mice and homozygous Mc4r-KO mice were maintained on either CD or HFD for 10 weeks. Following a 16-h fast, mice received an intraperitoneal glucose injection (2 g/kg). Plasma was collected at baseline (0 min) and 15 min post-injection. (A) Insulin levels were determined by ELISA (CRYSTAL CHEM INC , 90080) and (B) the fold induction was calculated. (n=4 per group, Mean±SEM, Welch’s t test, Unpaired t test or Mann-Whitney test, *p＜0.05, **p＜0.01)

Fig.10 Detection of glucose-stimulated insulin secretion of Mc4r-KO mice after 19 weeks of CD or HFD feeding. Male, 10-week-old WT mice and homozygous Mc4r-KO mice were maintained on either CD or HFD for 19 weeks. Following a 16-h fast, mice received an intraperitoneal glucose injection (2 g/kg). Plasma was collected at baseline (0 min) and 15 min post-injection. (A) Insulin levels were determined by ELISA and (B) the fold induction was calculated. (n=6 per group, Mean±SEM, Welch’s t test, Unpaired t test or Mann-Whitney test, *p＜0.05, **p＜0.01)

Fig.11 Blood lipid profiles of Mc4r-KO mice after CD or HFD feeding. Male, 10-week-old WT mice and homozygous Mc4r-KO mice were maintained on either CD or HFD for 11 or 23 weeks. Serum samples were collected following a 6-hour fast to measure levels of (A) triglycerides (TG), (B) total cholesterol (T-CHO), (C) nonestesterified fatty acid (NEFA), (D) high-density lipoprotein cholesterol (HDL-C) and (E) low-density lipoprotein cholesterol (LDL-C) (n=4 per group, Mean ± SEM, t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig.12 White adipose tissue indices of Mc4r-KO mice after CD or HFD feeding. Male, 10-week-old WT mice and homozygous Mc4r-KO mice were maintained on either CD or HFD for 11 or 23 weeks, then were sacrificed. Tissue weights of (A-B) epididymal (eWAT), (C-D) subcutaneous (sWAT), and (E-F) mesenteric (mWAT) white adipose tissue were recorded. (n=4 per group, Mean ± SEM, t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig.13 Analysis of hepatic lipid accumulation in Mc4r-KO mice after CD or HFD feeding. Male, 10-week-old WT mice and homozygous Mc4r-KO mice were maintained on either CD or HFD for 11 or 23 weeks, then were sacrificed. (A-B) The isolated livers were weighted and (C) used to detect the level of TG. (n=4 per group, Mean ± SEM, t-test, *p < 0.05, **p < 0.01)

Fig.14 Representative Oil red O staining images of Liver in CD- or HFD-fed Mc4r-KO mice. After 23 weeks of dietary intervention,  mice were sacrificed, and the isolated livers were subjected to embed in tissue freezing medium for Oil red O staining. Each image represents one mouse (n=3 per group, male).  Scale bar, 100 μm.

Fig.15 Analysis of hepatic function in Mc4r-KO mice under CD or HFD feeding. Male, 10-week-old WT and homozygous Mc4r-KO mice were maintained on either CD or HFD for 11 or 23 weeks.  Serum samples were collected following a 6-hour fast to measure levels of (A) alanine aminotransferase (ALT) and (B) aspartate aminotransferase (AST). (n = 4 per group, Mean ± SEM, t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig.16 Representative image of HE staining in CD- or HFD-fed Mc4r-KO mice. After 11 weeks of dietary treatment, mice were sacrificed, and the isolated livers were subjected to embed in paraffin for HE staining. Each image represents one mouse (n=3 per group, male). 

Fig.17 Representative image of Masson-trichrome staining in CD- or HFD-fed Mc4r-KO mice. After 11 weeks of dietary treatment, mice were sacrificed, and the isolated livers were subjected to embed in paraffin for Masson-trichrome staining. Each image represents one mouse (n=3 per group, male).  

Fig.18 Representative image of HE staining in CD- or HFD-fed Mc4r-KO mice. After 23 weeks of dietary treatment, mice were sacrificed, and the isolated livers were subjected to embed in paraffin for HE staining. Each image represents one mouse (n=3 per group, male). 

Fig.19 Representative image of Masson-trichrome staining in CD- or HFD-fed Mc4r-KO mice. After 23 weeks of dietary treatment, mice were sacrificed, and the isolated livers were subjected to embed in paraffin for Masson-trichrome staining. Each image represents one mouse (n=3 per group, male).   

Fig.20 The liver image of CD- or HFD-fed Mc4r-KO mice. Following 442 days of dietary intervention, only mouse #49, a Mc4r-KO mouse fed HFD, exhibited a grossly visible abnormal lesions in the liver; no lesions were observed at any other anatomical site. 

After 442 days of dietary treatment, mice were sacrificed, and the isolated livers were photographed and subjected to embed in paraffin for HE staining (n=1-2 per group, male).   

Abbr. WT, wild type; CD, chow diet；HFD, high-fat diet.  

Fig.22 Representative image of liver HE staining in CD- or HFD-fed Mc4r-KO mice. After 442 days of dietary treatment, mice were sacrificed, and the isolated livers were subjected to embed in paraffin for HE staining (n=1-2 per group, male).   

Abbr. WT, wild type; CD, chow diet；HFD, high-fat diet.  

WT + CD: Extensive, moderate steatosis with vacuolar degeneration of hepatocytes was observed.

WT + HFD: Mild inflammatory cell infiltration was noted within the hepatic lobules, accompanied by Kupffer cell hyperplasia in the interstitial stroma.

Mc4r-KO + CD: Hepatocytes showed extensive mild vacuolar degeneration and moderate ballooning degeneration, with mild inflammatory cell infiltration in the hepatic interstitium.

Mc4r-KO + HFD: Hepatocytes exhibited mild steatosis and extensive vacuolar degeneration, with focal areas of hepatocellular necrosis and inflammatory cell infiltration in the lobules. The overall hepatic volume was increased, with hepatocellular hypertrophy. Focally, atypical cells were increased in number and displayed an elevated nucleus-to-cytoplasm (N/C) ratio with hyperchromatic nuclei; moderate cellular dysplasia was observed.

Conclusion of HE staining for liver pathology:

Following 442 days of dietary intervention, only mouse #49, a Mc4r-KO mouse fed HFD, exhibited focal hepatocellular necrosis, increased overall liver volume with hepatocellular hypertrophy, and focally increased atypical cells displaying an elevated nucleus-to-cytoplasm (N/C) ratio and hyperchromatic nuclei, with moderate dysplasia observed. These findings suggest that long-term HFD feeding induces spontaneous hepatic tumorigenesis in Mc4r-KO mice.