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).