Both subcutaneous semaglutide and calorie restriction improves pancreatic cell hyperplasia and gut microbiota in high-fat diet-induced obese mice

Abstract

Background: Obesity has emerged as a global health crisis, with its prevalence having increased alarmingly over recent decades. There is significant damage to pancreatic islets due to obesity, as well as metabolic syndrome. Improving the function of β-cells in obese patients is meaningful for treatment. Thus, GLP-1 receptor agonists like semaglutide may be beneficial for islet structural remodeling and their endocrine function in diet-induced obese mice and associated with food intake. However, whether the specific impact of semaglutide on obesity is the same as calorie restriction(CR) has not been investigated.

Methods: In this study, Five-week-old male C57BL/6 mice were divided into two dietary groups and fed for 12 weeks a control diet or a high-fat diet (HFD). Then, for an additional four weeks, the main groups were resampled to include treatment (Semaglutide, SME, 40 µg/kg), or CR, totaling four groups: Control, Model, Model + SME, Model + CR. Immunofluorescence, Western blot, and RT-qPCR were used in the study.

Results: Semaglutide or CR was capable of ameliorating hyperglycemia and insulin sensitivity, and reduces the lesion on the islet, increases islet cell proliferation, and recovers islet size and alpha- and beta-cell masses. Moreover, the changes include improvement of METTL3/14, pancreatic duodenal homeobox 1 (PDX-1), and insulin signaling. Meanwhile, Semaglutide or CR significantly decreases the abundance of Firmicutes, Proteobacteria, and Verrucomicrobia, but increases the Bacteroides content.

Conclusions: Semaglutide plays a positive role in alleviating β-cell dysfunction by regulating gut microbiota, and METTL3/14, PDX-1, insulin signal pathway-related genes may be associated with CR.

Keywords: Calorie restriction; Gut microbiota; High-fat diet; METTL3/14; Pancreatic β-cell function; Semaglutide.

Fig. 1

Effect of semaglutide or calorie restriction administration on blood glucose homeostasis in obese mice (n = 10). (A) Mice’s body weight. (B) Time course of blood glucose levels during the IPGTT. (C) AUCs of IPGTT. (D) Serum insulin levels at the end of 4-week administration. (E) HOMA-IR at the end of 4-week administration. (F, G) Pancreatic tissue weight and the ratio of pancreatic tissue to body weight. (H) Weekly feed data for each cage of mice in each group for 4 weeks before and after drug administration (2 cages per group, 5 mice per cage). Data are presented as mean ± SD. *p < 0.05 versus control group, #p < 0.05 versus model group, respectively. Control, normal mice group; Model, high-fat diet-induced obese mice group; Model + SME, obese mice group administrated with semaglutide; Model + CR, obese mice group administrated with calorie restriction

Fig. 2

Restoration of pancreatic islet function by 4-week semaglutide or calorie restriction administration in obese mice (n = 5).(A, E) Pancreatic tissue was stained with hematoxylin and eosin (H&E) and quantification of islet area was calculated based on H&E staining(Scale bar, 20 μm). (B, F,G) Histological analysis of double-immunostained with anti-insulin antibody (green) and anti-glucagon antibody (red), while the nuclei were counterstained with DAPI(blue) in the pancreatic tissues and the ratios of β-cell or α-cell area to total islet area were calculated based on insulin or glucagon positive area in the whole pancreas sections(Scale bar, 100 μm). (C, H) Representative images of pancreatic sections that were stained with TUNEL reagent (red), anti-insulin antibody (green), DAPI(blue), the percentage quantification of β-cell apoptosis was calculated based on TUNEL positive area and insulin-positive area(Scale bar, 100 μm). (D, I) Representative photograph of pancreatic sections that were stained with C-peptide(green), BrdU(red), DAPI(blue), the percentage quantification of β-cell proliferating was calculated based on BrdU positive area and C-peptide positive area (Scale bar, 100 μm). Data are presented as mean ± SD. *p < 0.05 versus control group, #p < 0.05 versus model group, respectively. Control, normal mice group; Model, high-fat diet-induced obese mice group; Model + SME, obese mice group administrated with semaglutide; Model + CR, obese mice group administrated with calorie restriction

Fig. 3

Effects of 4-week semaglutide or calorie restriction administration on METTL3/14 signaling and β-cell proliferation marker genes in the pancreatic of obese mice. (A) Western blot was used to analyze the expression of METTL3, METTL14, and PDX1. (B) Relative mRNA expression levels of METTL3/14 and β-cell proliferation marker genes. (C) Relative m6A levels in the pancreatic islets (n = 3). Data are presented as mean ± SD. *p < 0.05 versus control group, #p < 0.05 versus model group, respectively. Control, normal mice group; Model, high-fat diet-induced obese mice group; Model + SME, obese mice group administrated with semaglutide; Model + CR, obese mice group administrated with calorie restriction

Fig. 4

Effects of semaglutide or calorie restriction on the gut microbiota in obese mice(n = 5). (A) Change of gut microbiota at the phylum level. (B) Change of gut microbiota at the genus level. (C) Chao1 index, Faith_pd, Goods_coverage, Obeserved_species (α-diversity) of gut microbiota. (D) Nonmetric multidimensional scaling(NMDS) analysis (when stress is < 0.2, NMDS accurately reflects the difference between the groups) was performed for weighted UniFrac distance matrices. (E, F) β-diversity, PCoA plots of beta diversity based on weighted UniFrac analysis in different groups. (G) ASV/OTU analysis. Control, normal mice group; Model, high-fat diet-induced obese mice group; Model_S, obese mice group administrated with semaglutide; Model_L, obese mice group administrated with calorie restriction

Fig. 5

The effect of semaglutide on PA-induced β-TC-6 cell model.(A)β-TC-6 cells were treated with 0.1, 0.2, 0.5, 1, or 2mM palmitic acid for 48 h and cell survival was measured by CCK-8 kits(n = 6). (B) β-TC-6 cells were treated with 0.5, 1, 2, or 4 µg/ml semaglutide in the presence of 1mM PA for 48 h. Cell survival was analyzed using CCK-8 kits(n = 6). (C, D) The mRNA and protein levels of METTL3, METTL14, and PDX1 were measured by RT-qPCR and western blot(n = 3). (E) Effect of semaglutide on GSIS in PA-induced β-TC-6 cells(n = 6). *p < 0.05 versus control group, #p < 0.05 versus PA group, respectively