Objective The obesity epidemic and its metabolic complications continue to be a major global public health threat with limited effective treatments, especially drugs that can be taken orally. Peptides are a promising class of molecules that have gained increased interest for their applications in medicine and biotechnology. In this study, we focused on looking for peptides that can be administrated orally to treat obesity and exploring its mechanisms.
Design Here, a 9-amino-acid peptide named D3 was designed and administered orally to germ-free (GF) mice and wild-type (WT) mice, rats and macaques. The effects of D3 on body weight and other basal metabolic parameters were evaluated. The effects of D3 on gut microbiota were evaluated using 16S rRNA amplicon sequencing. To identify and confirm the mechanisms of D3, transcriptome analysis of ileum and molecular approaches on three animal models were performed.
Results A significant body weight reduction was observed both in WT (12%) and GF (9%) mice treated with D3. D3 ameliorated leptin resistance and upregulated the expression of uroguanylin (UGN), which suppresses appetite via the UGN-GUCY2C endocrine axis. Similar effects were also found in diet-induced obese rat and macaque models. Furthermore, the abundance of intestinal Akkermansia muciniphila increased about 100 times through the IFNγ-Irgm1 axis after D3 treatment, which may further inhibit fat absorption by downregulating Cd36.
Conclusion Our results indicated that D3 is a novel drug candidate for counteracting diet-induced obesity as a non-toxic and bioactive peptide. Targeting the UGN-GUCY2C endocrine axis may represent a therapeutic strategy for the treatment of obesity.
According to the WHO, 650 million adults had obesity in 2016, and 39 million children under the age of 5 were overweight or obese in 2020. Obesity is becoming a worldwide epidemic and poses major therapeutic challenges due to its associated high risk for developing non-communicable diseases, including type 2 diabetes, cardiovascular disease, stroke, cancer and depression. Obesity is also linked with decreased life expectancy and increased morbidity and mortality and economic burden on healthcare systems. Various strategies have been developed to combat obesity and overweight conditions, especially through reducing energy intake or enhancing metabolic expenditure rate. However, recent studies found that excessive dietary interventions may lead to physical and mental health disorders, which further increase the demand for effective, safe and acceptable therapeutic options.
Extensive studies have shown that obese humans and mice display an altered gut microbiota with reduced diversity and increased capacity to absorb energy. The intestinal microbiota is essential in regulating host energy homeostasis, fat accumulation and mucosal barrier integrity. Disordered microbiota are closely associated with metabolic disorders such as obesity, insulin resistance and glucose intolerance. Intervention strategies that modulate the gut microbiota have been proposed to prevent and treat obesity. For example, tempol can decrease the weight gain of mice by preferentially reducing the genus Lactobacillus. Cranberry extract and metformin can protect mice from diet-induced obesity by increasing Akkermansia muciniphila in the gut microbiota. Several recent studies have shown that daily administration of A. muciniphila can counteract the development of high-fat-diet (HFD)-induced obesity. Additionally, oral gavage with either Bacteroides thetaiotaomicron or Lactobacillus rhamnosus GG can also alleviate diet-induced body weight gain and adiposity in mice.
Consisting of 20 natural amino acids and a multitude of non-natural amino acids, peptides have attractive pharmacological profiles, excellent safety and tolerability, which represent an excellent starting point for the design of novel therapeutics. Currently, a number of peptides have been developed to prevent obesity, such as glucagon-like peptide-1 (GLP-1), atrial natriuretic peptide and brain natriuretic peptide. Many of these peptides, however, are usually longer than 20 amino acids and are therefore less likely to escape degradation by proteases in the gastrointestinal tract. On the other hand, recent studies on apelin and semaglutide indicate that oral drugs represent the trend in weight-loss drug development for the preference of general patients for oral medicines compared with injectable therapies. In addition, small molecules may have some intrinsic disadvantages, including the accumulation of peptides in organs and potential toxic metabolic products causing side effects. Most of the peptides are membrane impermeable, which makes the therapeutic application of them restricted to extracellular and transmembrane targets, and the inability to permeate the intestinal mucosa necessitates parenteral administration via subcutaneous or intravenous injection, with the corresponding detriment to patient convenience and compliance. Based on the principle of biocompatibility, improving the hydrophobicity of peptides can improve their ability to interact with cell surface receptors and even promote their penetration of the membrane and binding to internal targets. Further studies are still needed to find or modify more endogenous peptides that are much smaller and lack cumulative toxicity for the treatment of obesity.
In this study, we designed and optimised a 9-amino-acid peptide with high hydrophobicity from human α-defensin 5 (HD-5), which was found to reverse dyslipidaemia and improve glucoregulatory capacity in diet-induced obese (DIO) mice through feeding directly mixed with feed. By using both pharmacological and genetic approaches, we identified and confirmed the role and mechanism of the modified peptide (D3) in inhibiting obesity development. Metabolic analysis revealed that D3 can ameliorate insulin and leptin resistance. By performing 16S rRNA amplicon sequencing and faecal microbial administration experiments, we observed increased abundance of gut commensal A. muciniphila and a causal relationship between it and the reduced anabolism of adipose tissue. Our study reports a new and promising candidate drug with high safety that can be taken orally for the prevention and treatment of obesity.
Increasing electric charge and hydrophobicity can improve the membrane permeability of peptides. Therefore, to enhance hydrophobicity, four peptides named D1–4 were modified from one of the fragments of HD5 degraded by proteases in human duodenal fluid, HD5(1–9), the first nine amino acids at the N-terminus of defensin HD5. All these peptides have a positive charge, among which D3 shows the strongest potential to penetrate cell membranes (online supplemental table 1 and figure 1A). We then evaluated the cytotoxicity of D1–4 in mammalian cells. D1–3 showed a slight inhibition of cell activity (2.71%–5.42%) at a very high concentration (2 9 µM), indicating their low cytotoxicity (online supplemental figure 1a). Additionally, D1–4 was assayed for potential toxic effects on mouse red blood cells in vitro. As shown in online supplemental figure 1b, the haemolytic rate of D1–4 at 2 8 µM was 1.06%–3.61%, respectively, indicating their low haemolytic effect. Therefore, the safe concentration range of these small peptides was set to 0–2 7 µM.
Figure 1 Oral administration of D3 counteracts obesity. (A) Design of HD5(1–9)-derived peptides D1–4 and their amphipathic surface. Blue, red and white represent positive, negative and neutral charges, respectively. Molecular models were generated with PyMOL 3.0. (B) Experimental outline. (C–H) Mice were divided into three groups (normal chow (NC), high-fat-diet (HFD) and D3) and fed a standard normal chow diet or 60% fat diet for 10 weeks; the D3 group was gavaged orally with D3, while saline was used as a parallel control (HFD). (C) Representative pictures of specific pathogen-free (SPF) mice at the eighth week. Mice in the D3 group exhibited reduced weight and better grooming. (D–F) SPF is shown on top and germ-free (GF) is shown on bottom. (D) Grams of weight gain measured over time, starting at 4 weeks of age; SPF mice (n=10–12 per group); GF mice (n=6–8 per group). Representative of three independent experiments. (E, F) insulin tolerance test (ITT) (E) and oral glucose tolerance test (OGTT) (F) of SPF mice and GF mice. (G) Total weight of white fat content in tissues from different organs. Representative of three independent experiments. (H) H&E staining of liver and epididymal fat from mice in the HFD or D3 group, taken at ×20 magnification. Scale bars: 100 µm. n=10 for each group. (I, J) Experimental outline (above); grams of weight gain measured over time, starting at 4 weeks of age, and the feed was changed at the 14th week. For D–G, I, and J, p values were determined by a two-tailed Wilcoxon test, and data are presented as the means±SEM; *p
