Fig 1.
Obesity phenotypes of Lep KO and HFD-treated mice.
(A) Morphology of the animals. The animals were photographed using a digital camera after anesthesia. (B) Body and fat weights. Five to six mices were selected from each group, and their weights were assayed in duplicate using an electric balance. (C) Histological structure of fat and liver tissue. After hematoxylin and eosin (H&E) staining of the fat and liver tissue, their histopathological features were observed at 400× magnification using an optical microscope. The area of each adipocyte in fat and the number of lipid droplets in the liver were measured using the Image J program. Five to six mices per group were used in histological analysis, and each parameter was measured in duplicate on two different slides. (D) Expression of the lipogenic proteins. The levels of ATGL, Perilipin, p-Perilipin, HSL, and p-HSL expression were measured by Western blot analysis using the specific primary antibodies and HRP-labeled anti-rabbit IgG antibody. Three to five mices per group were used to prepare the total tissue homogenate, and Western blot analyses were assayed in duplicate in each sample. (E) Expression of Lep mRNA and glucose concentration. The relative levels of Lep mRNA were determined in the liver tissue using RT-qPCR, while the glucose concentration was measured in the serum of whole blood. The data are reported as the mean ± SD. * indicates p < 0.05 compared to the Con group. Abbreviations: Con, Control group; Lep KO, Leptin knockout mice; HFD, High fat diet; ATGL, Adipose triglyceride lipase; HSL, Hormone-sensitive lipase.
Fig 2.
Excretion parameters and feeding behavior.
(A) The levels of the four excretion parameters, including number of stools, the weight of stools, water contents of stool, and urine volume, were measured from mice bred in a metabolic cage. Five to six mices per group were used for the stool and urine sample collection, and each parameter was assayed in duplicate. (B) The food intake and water consumption were also calculated using the amount of feed (water) supplied and the amount of feed (water) remaining. The data are reported as the mean ± SD. * indicates p < 0.05 compared to the Con group. Abbreviations: Con, Control group; Lep KO, Leptin knockout mice; HFD, High fat diet.
Fig 3.
GI tract length and GI transit ratio.
(A) Length of the GI tract. Three to five mice per group were used to prepare the GI tract, and their length was measured in duplicate. (B) Charcoal transit ratio. The charcoal meal transit ratio was then calculated using the total length of the intestine and the distance of the charcoal meal. Three to five mices per group were used in the GI transit ratio test, and the charcoal meal transit distance and intestine length were measured in duplicate. The data are reported as the mean ± SD. * indicates p < 0.05 compared to the Con group. Abbreviations: Con, Control group; Lep KO, Leptin knockout mice; HFD, High fat diet; GI, gastrointestinal.
Fig 4.
Histological structures of the mid colon.
(A) Histological structures of the hematoxylin and eosin (H&E)-stained mid colon. H&E-stained sections of the mid colon from the Lep KO mice and HFD-treated mice were observed at 100× and 400× magnification using an optical microscope. (B) The thickness of the mucosal layer and muscle. The histopathological parameters were determined using the Leica Application Suite (Leica Microsystems). Three to five mices per group were used for histological analysis, and each parameter was measured in duplicate on two different slides. The data are reported as the mean ± SD. * indicates p < 0.05 compared to the Con group. Abbreviations: Con, Control group; Lep KO, Leptin knockout mice; HFD, High fat diet.
Fig 5.
Level of mucin secretion and water channel expression.
(A) Level of mucin secretion. Mucin in the mid colon section was stained with alcian blue at pH 2.5. Their images were observed at 400× magnification. Five to six mice per group were used in the slide section, and mucin staining was assessed in duplicate in two different slides. (B) Levels of MUC2, AQP3, and AQP8 expression. The mRNA levels of the three genes were calculated based on the transcript level of β-actin as an endogenous control. Three to five mices per group were used to prepare the total RNA, and RT-qPCR analyses were assayed in duplicate for each sample. The data are reported as the mean ± SD. * indicates p < 0.05 compared to the Con group. Abbreviations: Con, Control group; Lep KO, Leptin knockout mice; HFD, High fat diet; RT-qPCR, Quantitative real time-PCR; MUC2, Mucin 2; AQP, Aquaporin.
Fig 6.
Expression of c-kit, nNOS, NSE, and PGP9.5.
The expression levels of four proteins were measured by Western blot analysis using the specific primary antibodies and HRP-labeled anti-rabbit IgG antibody. Three to five mices per group were used to prepare the total tissue homogenate, and Western blot analyses were assayed in duplicate in each sample. The data are reported as the mean ± SD. * indicates p < 0.05 compared to the Con group. Abbreviations: Con, Control group; Lep KO, Leptin knockout mice; HFD, High fat diet; c-kit, Receptor tyrosine kinase; nNOS, Neuronal nitric oxide synthase; NSE, Neuron-specific enolase; PGP9.5, Protein gene product 9.5.
Fig 7.
Expression of mAChR M1, M2, M3, M4, and M5 mRNA in the mid colon.
The levels of the five mAChRs mRNA in the total mRNA of the mid colon were measured by RT-qPCR using the specific primers. The mRNA levels of these genes were calculated based on the intensity of β-actin as an endogenous control. Three to five mices per group were used to prepare the total RNA. The RT-qPCR analyses were assayed in duplicate for each sample. The data are reported as the mean ± SD. * indicates p < 0.05 compared to the Con group. Abbreviations: Con, Control group; Lep KO, Leptin knockout mice; HFD, High fat diet; mAChR, muscarinic acetylcholine receptors.
Fig 8.
Expression of mAChRs proteins and the key mediators within their downstream signaling pathway.
Expression levels of mAChR M2/M3 and the key mediators, including Gα, PKC, p-PKC, PI3K, p-PI3K, MLC, and p-MLC were measured by Western blot analysis using the specific primary antibodies and HRP-labeled anti-rabbit IgG antibody. The intensity of each lane for a specific protein was calculated based on the intensity of β-actin. Three to five mices per group were used to prepare the total tissue homogenate, and Western blot analyses were assayed in duplicate in each sample. The data are reported as the mean ± SD. * indicates p < 0.05 compared to the Con group. Abbreviations: Con, Control group; Lep KO, Leptin knockout mice; HFD, High fat diet; mAChR, muscarinic acetylcholine receptors; PKC, Protein kinase C; PI3K, Phosphoinositide 3-kinases; MLC, Myosin light chain.
Fig 9.
Expression of 5HT-2AR, 2BR, 3AR, and 3BR mRNA in the mid colon.
The levels of four serotonin receptors mRNA in the total mRNA of the mid colon were measured by RT-qPCR using the specific primers. The mRNA levels of three genes were calculated based on the intensity of β-actin as an endogenous control. Three to five mices per group were used to prepare the total RNA. The RT-qPCR analyses were assayed in duplicate for each sample. The data are reported as the mean ± SD. * indicates p < 0.05 compared to the Con group. Abbreviations: Con, Control group; Lep KO, Leptin knockout mice; HFD, High fat diet.
Table 1.
Similarities and differences in obesity and constipation phenotypes between the Lep KO and HFD-treated mice.
The minus sign indicates a decrease compared to the Con mice, and the plus sign indicates an increase in the same condition.