Article Text

Original research
A novel peptide protects against diet-induced obesity by suppressing appetite and modulating the gut microbiota
  1. Zhanzhan Li1,2,
  2. Bing Zhang1,2,
  3. Ning Wang1,2,
  4. Zhenqiang Zuo1,
  5. Hong Wei3,
  6. Fangqing Zhao1,2,4,5
  1. 1Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China
  2. 2University of Chinese Academy of Sciences, Beijing, China
  3. 3Laboratory Animal Department, College of Basic Medicine Army Medical University, Chongqing, China
  4. 4Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, China
  5. 5Key Laboratory of Systems Biology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China
  1. Correspondence to Professor Fangqing Zhao, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing 100045, China; zhfq{at}biols.ac.cn; Professor Hong Wei; weihong63528{at}163.com

Abstract

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.

  • OBESITY
  • INTESTINAL MICROBIOLOGY
  • DRUG DEVELOPMENT

Data availability statement

Data are available upon reasonable request.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

Statistics from Altmetric.com

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

WHAT IS ALREADY KNOWN ON THIS TOPIC

  • Uroguanylin (UGN) is an anorexic hormone that can target the GUCY2C receptor of the hypothalamus and activate anorexigenic pathways.

  • Regulation of the gut microbiota can improve host metabolism and reduce obesity.

  • Polypeptides such as glucagon-like peptide-1 (GLP-1) analogue used to prevent obesity represents a new direction for drug design, but with poor adherence of patients to injectable therapies.

WHAT THIS STUDY ADDS

  • Oral administration of D3, a 9-amino-acid peptide but not a GLP-1 analogue, can counteract diet-induced obesity in mice, rat and rhesus macaca.

  • D3 can effectively inhibit appetite through the UGN-GUCY2C endocrine axis.

  • D3 restores the gut microbiota disorder caused by obesity and specifically increases the abundance of Akkermansia muciniphila in the gut via the IFNγ-Irgm1 axis.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

  • D3 is a promising candidate of weight reducing drug that can be taken orally to manage excess weight and obesity.

  • The UGN-GUCY2G axis represents a potential therapeutic target for the development of anti-obesity drugs.

Introduction

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 2020.1 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.2 Obesity is also linked with decreased life expectancy and increased morbidity and mortality and economic burden on healthcare systems.3 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,4 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.5 The intestinal microbiota is essential in regulating host energy homeostasis, fat accumulation and mucosal barrier integrity.6 Disordered microbiota are closely associated with metabolic disorders such as obesity, insulin resistance and glucose intolerance.7 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.8 Cranberry extract9 and metformin10 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.11 Additionally, oral gavage with either Bacteroides thetaiotaomicron12 or Lactobacillus rhamnosus GG13 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.14 Currently, a number of peptides have been developed to prevent obesity, such as glucagon-like peptide-1 (GLP-1),15 atrial natriuretic peptide and brain natriuretic peptide.16 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 apelin17 and semaglutide18 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.19 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.20 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.21 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.

Results

Oral administration of D3 counteracts 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 HD5.22 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 (29 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 28 µM was 1.06%–3.61%, respectively, indicating their low haemolytic effect. Therefore, the safe concentration range of these small peptides was set to 0–27 µM.

Supplemental material

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<0.05; **p<0.01; ***p<0.001.

We next compared the effects of daily administration of D1–4 on HFD-fed mice. Strikingly, HFD-fed mice orally administered D3 rather than other peptides caused a 12.06%±2.35% decrease in body weight gain compared with the HFD-fed control mice over 8 weeks of HFD exposure (figure 1B–D). Recent findings indicate that obesity and its accompanying metabolic state are increasingly implicated in the composition of the gut microbiota.1 To examine whether the gut microbiota may contribute to this effect, the same experiment was repeated in gnotobiotic (antibiotic cocktail, ABX) and germ-free (GF) mice. Similarly, 9.65%±4.26% and 9.14%±2.93% weight loss was observed in ABX (online supplemental figure 1C) and GF mice (figure 1D), respectively, suggesting that the reduction in adiposity by D3 oral gavage may be partially independent of the gut microbiome.

When challenged with additional insulin and glucose, D3-treated mice showed similar kinetics for glucose clearance as mice in the NC group, indicating that D3 could reduce the insulin resistance caused by obesity (figure 1E,F). Significant decreases in the tissue weight of inguinal, epididymal and perirenal fat were detected after D3 treatment (p<0.05, Wilcoxon test), which may be attributed to the reduced volume of fat cells (figure 1H). Additionally, D3 treatment also protected the liver from steatosis (figure 1H).

Based on these findings, we further examined the impact of 4 weeks of oral administration of D3 in a new cohort of DIO mice (n=5) after 10 weeks of HFD feeding. A significant reduction in body weight was observed in DIO mice (p<0.01, Wilcoxon test) (figure 1I), whereas the trend tended to be modest in the lean mice (n=5) fed a chow diet for 10 weeks in parallel (p<0.05, Wilcoxon t-test) (figure 1J). Notably, similar effects were observed after exchanging their diets at the 14th week (figure 1I,J). Taken together, these findings indicated that D3 treatment can prevent the onset and worsening of DIO, particularly on a HFD (online supplemental figure 1e).

D3 maintains energy homeostasis by inhibiting appetite

Considering that obesity occurs when energy intake exceeds energy expenditure, we therefore evaluated whether oral administration of D3 could affect food intake and/or energy metabolism. Notably, both in the specific pathogen-free (SPF) mice and the GF mice, D3 treatment significantly reduced food intake over time, with a significant reduction in 24-hour food intake compared with vehicle-treated animals (p<0.05, Wilcoxon test) (online supplemental figure 1d and figure 2A,B). In addition, indirect calorimetry analysis was performed to address whether changes in energy expenditure contributed to the observed D3-mediated weight loss. As shown in figure 2C, there was almost no difference in energy expenditure between the two groups (p>0.05, Wilcoxon test), indicating that D3 treatment may affect energy intake rather than energy expenditure. Pair-feeding was performed to explore the effects of lower food overload. As a result, mice orally administered D3 caused a 10.04%±2.19% (p<0.01, Wilcoxon test) decrease in body weight gain compared with the HFD control mice over 8 weeks of HFD exposure (figure 2D). Additionally, mice in the pair-fed group caused a 6.02%±2.03% decrease in body weight gain. These results suggest that the physiological effects of D3 are largely based on the inhibitory on appetite in mice.

Figure 2

D3 treatment decreases food intake by upregulating uroguanylin (UGN) expression in the ileum. (A, B) Food intake was measured once a week. (C) Effect of D3 treatment on acute food intake, which was measured every other day for 1 week. For A and B, (A) (specific pathogen-free (SPF) mice, n=10–12), (B) (germ-free (GF) mice, n=6–8). (C) Energy expenditure of SPF mice measured for 20 hours (n=6 mice/group). (D) Grams of weight gain measured over time in mice of pair-feeding, starting at 4 weeks of age (n=6 mice per group). (E) Localisation of FITC-labelled D3 visualised by confocal microscopy. The cell nucleus was stained with DAPI (blue). Swiss rolls (above, scale bars: 500 µm) and intestinal cross sections (below, scale bars: 200 µm) of SPF mice were obtained after 3 hours of oral administration. The last column shows the localisation of FITC-D3 after 1 hour of coincubation with HCT cells. The magenta colour presented by FITC is a pseudocolour that was replaced later to distinguish it from the immunofluorescence staining of UGN. Images are representative of three independent experiments with similar results. (F) RNA sequencing and KEGG pathways of genes in the ileum. (G) Volcano plot showing relative gene expression in the D3-treated group versus the HFD control group. All genes were plotted, and each circle represents one gene. The diameter of the circle represents the value of FPKM, p<0.05 (green) and p<0.01 (red). The X-axis shows the log2-fold change, and the Y-axis shows the −log10 of the p values. (H) Relative Guca2b mRNA levels in the ileum of mice gavaged with D3 or phosphate buffered saline (PBS). (I) The concentration of UGN (Guca2b protein) (ng/mL) in the serum of mice was determined by ELISA. (J) Double immunofluorescence in ileal sections for UGN (green dots located at the base of intestinal epithelial cells) and nuclei (blue). The diffuse green thin layer refers to background noise. Scale bars: 10 µm. For A, B, H and I, p values were determined by a two-tailed Wilcoxon test, and data are presented as the means±SEM; *p<0.05; **p<0.01; ***p<0.001.

To further explore the mechanism of D3, FITC-labelled D3 (FITC-D3) was used to visualise its systemic distribution in mice. Three hours after oral administration of FITC-D3 or FITC, intense fluorescence was visible in the intestinal tract of FITC-D3-treated mice, especially in the distal small intestine, but not in the mice of the FITC group (figure 2E). No distribution of FITC-D3 was observed in other organs (heart, spleen, lungs and kidneys), except slightly in the liver (figure 2E and online supplemental figure 2a). After 24 hours of FITC-D3 treatment, no fluorescence was observed in any of the above organs, including the intestines. These findings suggest that the major target of D3 is the distal small intestine, and its half-life is short.

Rich in arginine and lysine residues, some small peptides have remarkable cellular uptake ability to bind intracellular targets or to deliver electrostatically bioactive agents into cells.23 To determine whether D3 can penetrate the cell membrane, HCT cells were coincubated with 10 µM FITC-D3/FITC for 2 hours. Confocal microscopy showed that FITC-D3 was homogeneously distributed in the cytosol of HCT cells and displayed a significantly higher fluorescence intensity than FITC (figure 2E and online supplemental figure 2b), implying enhanced cell uptake after conjugation with D3. The high efficiency of D3 in penetrating the membrane supports its potential to function as an intracellular regulator.

Subsequently, we investigated whether D3 could regulate the expression of genes in the distal small intestine of mice by using transcriptome analysis. Gene set enrichment analysis indicated that several KEGG pathways involved in metabolic and inflammatory responses, including the regulation of fat cell differentiation, positive regulation of catabolic process and negative regulation of I-kappaB kinase/NF-kappaB signalling, were the most significantly enriched pathways within D3-treated mice compared with HFD controls (figure 2F). Moreover, differential expression analysis identified 121 upregulated genes in the ileum of D3 mice (p<0.05, Wald test). Several genes involved in the regulation of lipolysis, including Zbtb1624 and Acyl-CoA Oxidase 2 (Acox2),25 and the inflammatory inhibition-related gene Zfp3626 were validated by real-time quantitative PCR, particularly the Guca2b gene (adjusted p<0.01) (online supplemental figure 3b-d and figure 2G). Similar results were also found in adipose tissue: D3 treatment increased the mRNA expression of markers of adipocyte differentiation (eg, CCAAT/enhancer-binding protein-α, encoded by Cebpa) and lipid oxidation (eg, Acox2) and downregulated lipogenesis markers (eg, fatty acid synthase, encoded by Fasn) (online supplemental figure 3b). Previous studies revealed that Guca2b can act as a regulator of body weight homeostasis by modulating food intake through the UGN-GUCY2C endocrine axis.27 We further verified the changes in UGN levels in serum by ELISA. Notably, D3 treatment significantly upregulated the expression of UGN in both SPF mice and GF mice (p<0.01, Wilcoxon test), as assessed by immunofluorescence (figure 2H–J and online supplemental figure 3e).

Intact leptin signalling contributes to the anti-obesity effect of D3

We sought to identify whether D3 treatment could protect against obesity in genetically obese (ob/ob) mice. As shown in figure 3A, no significant difference was observed in the body weight gain of the ob/ob mice between the D3 group and the HFD group after 6 weeks of oral administration. To validate that the anti-obesity effect of D3 was leptin associated, we next retrospectively examined the changes in leptin expression in SPF mice treated in previous experiments. Indeed, consistent with previous reports,28 feeding an HFD significantly upregulated the expression of leptin in both SPF and GF mice, which could be restored by oral administration of D3 (p<0.05, Wilcoxon test) (figure 3B). To further demonstrate that D3 can ameliorate the leptin resistance, we first investigated the response of body weight to leptin administration by using an acute method, in which WT mice were treated with leptin intraperitoneally for 3 days, and then their body weight change was determined. As shown in figure 3C,A, marked body weight reduction was observed in the mice that received D3 after exogenous leptin treatment (p<0.05, Wilcoxon test) but not in the mice of the HFD group. Second, we detected the leptin-stimulated cytokines, including signal transducer and activator of transcription 3 (STAT3) and phosphatidylinositol 3-kinase (PI3K) pathways.29 We found that within the negative regulators of leptin signalling, there was no significant difference in the mRNA expression level of PTB1B (online supplemental figure 4b) between the HFD and D3 groups, but SOCS-3 was significantly downregulated in D3-treated mice. The overexpression of SOCS-3 was found to inhibit leptin-induced pSTAT3 in vivo and consequently caused leptin resistance.30 In contrast, the expression level of PI3K, whose level is positively correlated with the total expression level and can reflect the leptin sensitivity,31 was significantly upregulated, which may represent an enhanced response to leptin (online supplemental figure 4b). Taken together, these results suggest that D3 treatment can restore leptin-induced signalling pathways in DIO mice and the leptin sensitivity.

Figure 3

D3 increases leptin sensitivity and ARC c-Fos expression in mice. (A) Percentage of weight gain measured over time (ob/ob mice, n=8 per group). (B) Leptin mRNA expression levels of epididymis fat in specific pathogen-free/germ-free (SPF/GF) mice gavaged with D3 or saline as controls fed a high-fat-diet (HFD) or normal chow (NC) diet (SPF mice, n=10–12; GF mice, n=6–8). (C) Body weight change after treatment with exogenous leptin or saline (wild-type (WT) mice, n=7–8). Lute diagrams of the same colour represent two equally divided groups of mice with the same treatment. (D) Percentage of weight gain in ob/ob mice on normal diet with different treatments (NC, n=3; D3, leptin and D3+leptin, n=4). (E) Guca2b mRNA expression levels in ob/ob or WT mice (ob/ob mice, n=3–4 per group; WT mice n=8–10 per group). (F) Double immunofluorescence in ileal sections for uroguanylin (UGN) (green dots located at the base of intestinal epithelial cells) and nuclei (blue). Scale bars: 10 µm. (G) AgRp and POMC mRNA expression levels in WT mice (n=3 per group). (H) Representative images showing ARC c-FOS (red) and POMC (green) in SPF mice. Scale bar: 100 µm; 3v, the third cerebral ventricle. (I) Colocalisation of c-Fos positive cells with POMC neurons. (J) Numbers of c-Fos positive cells/slide in ARC of mice. (K) Double immunofluorescence in ARC sections for POMC (green) and nuclei (blue). Scale bars: 100 µm; 3v, the third cerebral ventricle. For A–E, H and J, p values were determined by a two-tailed Wilcoxon test, and data are presented as the means±SEM; *p<0.05; **p<0.01; ***p<0.001.

We observed that leptin treatment caused a marked tendency towards a reduction in body weight in ob/ob mice, and leptin+D3 induced a more significant reduction than D3 alone (figure 3D), implying that supplementation with exogenous leptin can restore the function of D3 in ob/ob mice. Then, we quantified and compared the expression level of Guca2b in the ileum of ob/ob mice with that of WT mice. A 2.22-fold increase in the expression of Guca2b was observed after D3 treatment in ob/ob mice (OB-D3) compared with untreated ob/ob mice (OB-NC), whereas the WT mice of the SPF-D3 group achieved a 3.67-fold increase (figure 3E). Furthermore, ob/ob mice treated with leptin alone (OB-Lep) showed a 2.12-fold increase, which was lower than those treated with leptin+D3 (OB-Lep+D3, 3.46-fold increase) (figure 3E), indicating the synergistic effect of leptin and D3 on regulating the expression of UGN. The expression of UGN in the ileum of ob/ob mice treated with D3, leptin and D3+leptin was further visualised and verified by immunofluorescence (figure 3F). Taken together, these findings indicate that leptin may play a significant role in mediating the anorexigenic and anti-obesity effects of D3.

Some neurons in the hypothalamus associated with appetite are activated on fasting, which is accompanied by dramatic induction of c-Fos expression in these neurons.32 To observe the effect of D3 treatment on the appetite of mice, 8-week-old SPF mice were gavaged with D3 or vehicle (saline) at 10:00 a.m. Transcardial and c-Fos staining were subsequently performed every 1 hour for 3 hours. The expression of c-Fos increased significantly at 1 hour after D3 administration, and then gradually returned to the normal level (online supplemental figure 4c), which was confirmed by subsequent immunofluorescence staining (figure 3H,J). We further measured the expression of AgRP and POMC during this process to identify the population of hypothalamic neurons implicated in the observed effects. The expression of POMC was significantly improved at 2 and 3 hours after D3 administration (p<0.05, Wilcoxon test), but with no significant changes in AgRP (p>0.05, Wilcoxon test) (figure 3G,K). Moreover, the colocalisation of c-Fos and POMC expressing neurons was observed in arcuate nucleus (figure 3H,I). These results suggest that D3 administration may indirectly affect POMC neurons in arcuate nucleus, which signals satiety and reduces food intake, to achieve the effect of appetite suppression.

D3 treatment increases the abundance of A. muciniphila in the gut microbiota

The gut microbiota is an important contributor to metabolic function and has been linked with the development of human diseases,33 34 such as infantile autism35 and obesity.36 We next examined whether D3 protection against obesity was correlated with gut microbiota alteration. There was an obvious difference in the faecal microbiota composition between D3-treated and vehicle-treated mice (figure 4A,B and online supplemental figure 5a-c). The linear discriminant analysis showed that Oscillospira, Lawsonia and Desulfovibrio were significantly enriched in the vehicle-treated mice, whereas the genera Bacteroides, Bilophila and Akkermansia were significantly enriched in the D3-treated group (figure 4C).

Figure 4

D3 alters the gut microbiota in mice. (A) Faecal microbiota composition in high-fat-diet (HFD)-treated and D3-treated mice at the genus level. (B) Principal component analysis (PCoA) of faecal microbiota in HFD and D3 mice. The significance of two separated clusters was measured with the Adonis test. (C) LEfSe analysis showing bacterial taxa that were significantly different in abundance between HFD and D3. Taxa significantly enriched and depleted in D3-treated mice are shown in red and black, respectively. (D) Experimental workflow of faecal microbiota transplantation (FMT). Mice were first fed an HFD for 10 weeks to achieve dietary obesity. During the next 4 weeks, D3-treated mice were used as the donors. Their faeces were collected and then divided into an inactivated group and an active group and eventually transferred to receptor mice via FMT. In addition, mice in the “A. m” and “B. v” groups were orally gavaged with 1×109 CFU of Akkermansia muciniphila or Bacteroides vulgatus. Donor and receptor, n=6 per group, “A. m” and “B. vgroup, n=5 per group. (E–F) Percentage of weight gain measured over time (E, FMT; F, colonisation of exogenous bacteria), starting at the 14th week. (G) Relative abundance of A. muciniphila in the faecal samples of mice in figure 1D determined by quantitative PCR. (H–I) Periodic acid Schiff (PAS) and Alcian blue (AB) staining (H) and the thickness of the mucus layer (I) of colon sections from specific pathogen-free (SPF) and germ-free (GF) mice (figure 1D), as well as mice colonised by exogenous bacteria. The white dotted lines represent the boundaries of the mucus layer. (J) The growth curve of A. muciniphila exposed to different concentrations of D3 in vitro. Representative of three independent experiments. (K–I) IFNγ (K) and irgm1 (L) mRNA expression levels in ilea from SPF mice in figure 1D, Percentage of weight gain measured over time (n=6 mice per group). (N) CD36, Fasn and Acox2 (liver) and Adipoq (epididymis) mRNA expression levels of SPF mice fed a control diet (NC and HFD) or treated with D3 or gavaged with A. muciniphila (n = 6 mice per group). For E–G, I, and K–N, p values were determined by a two-tailed Wilcoxon test, and data are presented as the means±SEM; **p<0.01; ***p<0.001.

Faecal microbiota transplantation (FMT), which entails the transfer of gut microbial communities from healthy donors to patients, has emerged as a promising treatment option for a range of chronic disorders.37 Considering that compositional shifts in the gut microbiota may play a role during D3 treatment, we adopted FMT to determine whether the gut microbiota contributed to the rescue of obesity. The faecal microbiota from mice treated with D3 for 10 weeks were collected and orally given to the DIO mice. After 4 weeks, FMT promoted a 6.50%±2.91% weight reduction in the recipient mice compared with the HFD control mice, whereas the heat-inactivated faecal sample showed no effect (figure 4D,E). We wondered whether any particular faecal bacterial species exerted such protective effects. Therefore, we used two significantly enriched taxa (B. vulgatus and A. muciniphila) in the D3-treated DIO mice for 4 weeks. The colonisation of A. muciniphila led to a significant body weight reduction (p<0.05, Wilcoxon test), while the colonisation of B. vulgatus did not have such an effect (p>0.05, Wilcoxon test) (figure 4F). This finding suggests that individual members (eg, A. muciniphila) of the gut microbiota transferred to recipient mice may defer obesity development in DIO mice, which is in agreement with previous studies.38 We then returned to the initial experiment on the SPF mice and found that the abundance of A. muciniphila in the faeces of mice treated with D3 was approximately 100-fold higher than that in the HFD mice (figure 4G).

Recently, A. muciniphila was identified as a mucin-degrading bacterium that resides in the mucus layer.39 The specialisation of A. muciniphila in mucin degradation makes it a key player at the mucosal interface between the lumen and host cells. By staining the mucus of the colonic epithelium, we found that the thickness of the mucus layer was significantly decreased in the HFD-fed mice (SPF). Both D3 and A. muciniphila treatment counteracted this decrease (p<0.05, Wilcoxon test). Interestingly, this effect disappeared when using D3 to treat GF mice (figure 4H,I). Therefore, we speculate that the thickening of the mucus layer is due to the colonisation of A. muciniphila rather than the direct action of D3.

To further explore the mechanism for the increase in the abundance of A. muciniphila that occurs with D3 treatment, we first measured the growth curve of A. muciniphila associated with exposure to different concentrations of D3 in vitro. As shown in figure 4J, there was no significant change under different D3 concentrations, indicating that D3 cannot promote the growth of A. muciniphila in vitro. Previous studies found that A. muciniphila was significantly increased in IFNγ-deficient mice, and the restoration of IFNγ levels could reduce A. muciniphila abundance.40 IFNγ can induce the secretion of antimicrobial proteins from Paneth cells through Irgm1, a downstream molecule in its signalling cascade. Previous studies suggest that impaired production of antimicrobial peptides can lead to the overgrowth of A. muciniphila when the level of IFN or Irgm1 is reduced. In this study, we observed that D3 treatment significantly suppressed the expression of both IFNγ and Irgm1 in the ileum of mice relative to the HFD group (figure 4K,L). Therefore, we speculate that the increased abundance of A. muciniphila by D3 treatment may be attributed to the negative regulation of the IFNγ-Irgm1 axis.

To investigate whether A. muciniphila contributed to improving the phenotype of obese, we transplanted A. muciniphila into DIO mice for 8 weeks. Notably, A. muciniphila gavage with 1×109 CFU/mouse every 2 days led to a significant reduction (p<0.01, Wilcoxon test) in the body weight of mice, similar to the D3 treatment (figure 4M). We further compared the effects of A. muciniphila and D3 on lipid metabolism in DIO mice. We observed an increase in the expression of the Fasn and CD36 mRNAs, genes that regulate the synthesis and absorption of lipids,41 a decrease in the mRNA level of Adipoq that promotes lipid oxidation in epididymal fat, after the administration of D3 or A. muciniphila in the SPF mice but not in GF mice (online supplemental figure 5f), suggesting additional positive effects of the increased A. muciniphila. Interestingly, a dramatic upregulation of Acox2, which can increase fatty acid oxidation and further inhibit lipid accumulation in the liver, was observed only in D3-treated SPF mice (figure 4N and online supplemental figure 5f).42 These results indicate that the regulation of lipid metabolism should be a combined effect of D3 and A. muciniphila.

Recent studies showed that A. muciniphila could repair the thinning of the intestinal mucus caused by high-fat (HF) diet. Therefore, we further investigated how the administration of D3 or A. muciniphila affects the mucus in the colon. We found that the expression levels of MUC2 and IL4 were significantly increased after the administration of D3 or A. muciniphila in SPF mice (p<0.05, Wilcoxon test), but not in other markers of goblet cell development,43 such as Klf4 (p>0.05, Wilcoxon test) (online supplemental figure 5d-e). However, GF mice treated by D3 did not show the same results, suggesting that the thickening of the mucus layer may be attributed to the increased abundance of A. muciniphila rather than the direct action of D3.

The anti-obesity effect of D3 in other animal models

To investigate the anti-obesity effect of D3 in other animals, we further treated two additional DIO models, rats and macaques, with D3 (figure 5A,H). Notably, D3 treatment resulted in an 8.96%±3.11% decrease in body weight gain in rats compared with untreated controls over the 10 weeks of HFD exposure (p<0.05, Wilcoxon test) (figure 5B,C,I). As expected, we observed that D3 treatment significantly suppressed food intake in DIO rats compared with HFD control rats (p<0.05, Wilcoxon test) (figure 5D). The expression of Guca2b in the ileum was significantly increased (p<0.05, Wilcoxon test) and leptin in epididymal fat was significantly decreased (p<0.05, Wilcoxon test) (figure 5E,F). Similarly, the abundance of A. muciniphila was increased (p<0.05, Wilcoxon test) (figure 5G). For DIO macaques, the average increase in body weight gain in the D3 group was 7.71%±2.97% lower than that in the control group at the 6th week (figure 5j). (figure 5I). Moreover, we observed that the food intake of macaques in the D3 group was significantly decreased (p<0.05, Wilcoxon test) (figure 5J), with an increased abundance of A. muciniphila (p<0.05, Wilcoxon test) (figure 5K).

Figure 5

D3 protects rats and macaques from dietary obesity. (A) Experimental workflow in rats (n=8–10 per group). (B) Grams of weight gain of rats measured over time. (C) Percentage of weight gain of rats measured over time. The bracket represents a comparison between high-fat diet (HFD) and D3. (D) Food intake in rats measured every other day for 1 week. (E) Guca2b mRNA expression levels in the ileum of rats. (F) Leptin mRNA expression levels of epididymal fat in rats. (G) Relative abundance of Akkermansia muciniphila in the faecal samples of rats. (H) Experimental workflow in macaques fed a standard diet (normal chow, NC) or a HFD for 6 weeks (n=3 per group). (I) Percentage of weight gain of macaques measured over time. The bracket represents a comparison between HFD and D3. (J) Food intake in macaques measured every other day for 1 week. (K) Relative abundance of A. muciniphila in the faecal samples of macaques. (L) Summary of the role of D3 in treating obesity. As a leptin sensitiser, D3 can upregulate the expression of uroguanylin (UGN) in the ileum and suppress appetite by targeting the hypothalamus. D3 can also increase the abundance of A. muciniphila in the intestines, which in turn may influence lipid absorption and metabolism within the liver. For B–G and I–K, p values were determined by a two-tailed Wilcoxon test, and data are presented as the means±SEM; *p<0.05; **p<0.01.

Discussion

Although obesity management has improved significantly over the years, therapies that target its underlying processes are still lacking. There is a growing interest in drug therapies that can support weight loss.44 Currently, some endogenous peptides in humans have been used to treat type 2 diabetes and obesity, such as a K-casein-derived glycogenous peptide and casein.45 Moreover, some intestinal peptides (eg, α-defensin 5) were used to reverse dyslipidaemia and improve glucoregulatory capacity in DIO mice.21 We found that oral administration of D3 led to a 12.06%±2.35% decrease in body weight gain and ameliorated gut microbiota dysbiosis in DIO mice. We further demonstrated that D3 can upregulate the expression of UGN and leptin resistance, both of which can inhibit the appetite of mice. Our study provides a novel perspective and evidence that endogenous polypeptides derived from degraded fragments of protein precursors may play an important role in regulating obesity and other metabolic diseases (figure 5l).

To date, five medications (orlistat, phentermine plus topiramate, naltrexone plus bupropion, liraglutide and semaglutide), all of which have an anorexigenic effect, have been approved by the Food and Drug Administration (FDA) for long-term weight management in adults.46 In this study, we found that after D3 treatment, the food intake of both SPF mice and GF mice was significantly reduced, accompanied by an increased expression of UGN in the small intestine. UGN, a 16-amino-acid anorexic hormone secreted by enterochromaffin cells in the small intestine, has been shown to target the GUCY2C receptor of the hypothalamus and activate anorexigenic pathways through blood circulation.47 Intravenous injection of prouroguanylin could induce satiation in mice.47 Kim et al48 also found that transgenic UGN under a control of brain-specific promoter could produce physiological levels to induce durable anorexigenic responses opposing weight gain for at least 24 weeks without altering the metabolic rate in DIO mice. We found that D3 treatment could resist the onset and worsening of diet-induced obesity in mice by upregulating the expression of UGN and increasing its content in serum. Such a UGN (gut)-GUCY2C (hypothalamus) endocrine axis may have the potential to serve as a novel therapeutic target for appetite control and metabolic syndrome. Additionally, we found that D3 restored the compensatory leptin elevation caused by a HF diet and increased leptin sensitivity. Leptin can decrease appetite by inhibiting the activity of neurons in the ARC, the centre of appetite regulation in the hypothalamus.49 A dramatic induction of c-Fos expression occurs when neurons in the ARC are activated by starvation, which can be reversed by the administration of appetite inhibitors (eg, estradiol50 and leptin51) or feeding.32 Notably, the upregulated c-Fos expression and its colocalisation with POMC expressing neurons in mice after D3 administration, probably due to the combined effect of D3 promoting UGN expression and acting as a leptin sensitiser.

Currently, there is accumulating evidence of the role of gut microbiota in various metabolic disorders, and the manipulation of the gut microbiota might be a novel therapeutic option.52 53 In our study, we found that D3 treatment could reduce the abundance of Desulfovibrio and Ruminococcus. The administration of Desulfovibrio to GF mice led to an increase in body fat percentage and the expression of CD36,41 a critical regulator of lipid absorption and homeostasis within mammals.54 In addition, we observed a higher abundance of Bacteroides in D3-treated mice. Many observational and interventional studies have correlated leanness or weight loss to increased levels of Bacteroidetes, which can produce short-chain fatty acids.55 However, this relationship is controversial because observational studies have also found increased abundances of Bacteroides to be associated with Western diets, which are related to obesity.56 Here, we found that significantly enriched colonisation with the taxon B. vulgatus for 4 weeks did not lead to a significant change in the body weight of DIO mice. In contrast, the obesity phenotype was reversed by transplanting A. muciniphila in obese mice, which is consistent with previous studies.38 57 Notably, the difference between the percentage of body weight lost in GF mice (9.14%±2.93%) and SPF mice (12.06%±2.35%) treated with D3 further demonstrates the role of the gut microbiota in treating obesity. In fact, many drugs and chemicals that have been found to reduce body weight can affect the gut microbiota. For example, liraglutide changed the overall composition as well as the relative abundance of weight-relevant phylotypes, such as a reduction in Proteobacteria and an increase in A. muciniphila in treated DIO mice.58 Interestingly, D3 treatment increased the abundance of A. muciniphila by 100 times, and this effect was much stronger than that of the previous two drugs (three to five times).

A number of polypeptides have been used to act as signalling molecules in the regulation of the neuroendocrine system to prevent obesity, including liraglutide and semaglutide,44 glucagon-like peptide-1 (GLP-1) agonists and adrenomedullin 2.14 However, several reasons may explain the slow adoption of injectable GLP-1R agonists, including poor adherence of this patient population to injectable therapies and the general patient preference for oral medicines compared with injectable therapies.59 Interestingly, it is worth noting that in our study, oral administration of D3 significantly reduced the body weight of mice, indicating the potential of D3 as an effective and preferred treatment option for obesity. The use of medications for obesity has been limited by safety concerns. Among the drugs currently approved by the FDA for clinical use in the treatment of obesity, it seems that most of them have side effects. For example, GLP-1 analogues have been demonstrated to cause side effects, including transient nausea, GLP-1RA-related or insulin ‘gastrointestinal’ side effects60 acute pancreatitis,61 vomiting and a loss of appetite, which limit the dosage. Notably, no obvious side effects of D3 were observed in our study in mice, rats or macaques. Considering that our experiment was conducted only for 10–12 weeks, a longer experimental period is needed for verification of the results.

In summary, we report herein a small peptide, D3, that is not a GLP-1 analogue, can inhibit the development of obesity by suppressing appetite and regulating the gut microbiota. This study adds new evidence that the UGN-GUCY2G axis is a potential therapeutic target for the development of anti-obesity drugs and supportive evidence that D3 is a competitive candidate for counteracting diet-induced obesity with a favourable safety profile. The potential pleiotropic roles of this peptide in humans still need to be addressed in future research.

Methods

Rational optimisation of hydrophobic peptides from human defensin HD5

A hidden Markov model as implemented in TMHMM Server V.2.0 was used to evaluate the peptides’ abilities to penetrate cell membranes. The peptide secondary structure was modelled using SWISS-MODEL62 with human HD5 (1zmp) as the template. The amphipathic surface and the three-dimensional structures of D1-4 were modelled using PyMol V.2.3.2.63 D1–4 were synthesised using solid phase synthesis by Mimotopes (Wuxi, China).

Bacterial strains and administration and faecal microbial transplantation

Bacteroides vulgatus ATCC 8248 and Akkermansia muciniphila (ATCC BAA-835) were grown anaerobically at 37°C in Gifu anaerobic medium (HB8518-1, Hopepiol, Qingdao). Mice were treated with B. vulgatus and A. muciniphila by oral gavage at a dose of 1×109 CFU/0.2 mL suspended in sterile anaerobic phosphate buffered saline (PBS). A similar concentration of glycerol (2.5% vol/vol) was used as control. Treatments were continued for 4 weeks.

For the FMT experiment, 2–3 fresh faecal pellets were resuspended by vortexing in anaerobic PBS, and then the supernatant was harvested and mixed. Bacteria were harvested and resuspended in sterile and anaerobic PBS before FMT, and 100 µL of the suspension was administered to the mice by oral gavage two times per day for 4 weeks. Additionally, inactivated bacteria treated with high temperature were also administered to the mice by oral gavage two times per day for 4 weeks, which served as a control.

Animals and experiments

Operation of SPF mice and rats

All C57/Bl6J mice and Sprague Dawley (SD) rats (SPF Biotechnology, Beijing, China) and ob/ob mice (Hua Fukang Biotechnology, Beijing, China) were housed in groups of 4–6 animals per cage in a pathogen-free facility with standard bedding and enrichment in a temperature-controlled and humidity-controlled room on a 12-hour light/dark cycle with free access to food and water. All animals were fed a standard laboratory chow diet (growth and reproduction diet, SPFSLFZ003, SPF Biotechnology, Beijing, China) unless otherwise stated. HFD mice or rats were fed a HF diet (Rodent diet with 60 kcal% fat, D12492; Research diets, New Brunswick, New Jersey, USA). For rats, only D3 was used. After 1 week of recovery, animals were randomly assigned to treatment groups, and treatment continued for 10 weeks. Food intake and body weight were monitored weekly. Fresh excreta from animals were harvested and stored at −80°C for further analysis. All of the above experiments in mice and rats were started at 4 weeks of age, unless otherwise noted.

Operation of GF mice

GF C57Bl/6 mice obtained from the Department of Laboratory Animal Science of the Army Medical University (Chongqing, China) were used. Mice and rats were sacrificed by cervical dislocation. Relevant organs were collected and immediately immersed in liquid nitrogen and stored at −80°C for further analysis. Subcutaneous and visceral adipose deposits were precisely dissected and weighed.

Pair-feeding

The pair-feeding experiment was performed according to previously reported procedures.64 Pair feeding was accomplished by measuring the food intake of the ad libitum-fed D3-treated mice every 24 hours and presenting this amount of food to the pair-fed PBS-treated mice in two times (half within 12 hours). Blood samples were obtained, and tissues were harvested as described above.

Operation of macaques

The rhesus experiment was undertaken by Beijing Prima Biotech. Nine rhesus monkeys feeding on a standard chow diet (#2150230401, Prima Biotech, Beijing, China) were randomly assigned to three treatment groups: D3, NC and HFD.

Short-term food intake measurement

Food weights were recorded every other day over a period of 1 week, and the average intake per day for each 2-day period was determined and averaged over the week measurement period for each individual.

Antibiotic treatment

SPF WT mice were treated with combined antibiotics (ABX) containing 100 g/L neomycin (Sigma), 100 g/L penicillin (Sigma), 50 g/L vancomycin (Sigma) and 100 g/L metronidazole (Sigma) for 8 weeks.

Morphology and histology

Liver and adipose tissue samples were quickly collected and fixed in 4% paraformaldehyde (PFA) solution for 24 hours and embedded in paraffin. Sections of 10 µm were stained with H&E. ABPAS staining was performed using a previous method.65 A minimum of 10 different measurements were made perpendicular to the inner mucus layer per field and were manually delineated and analysed by ImageJ.

Localisation of D3 after oral administration

FITC-D3 is used to trace drugs in target organs and their action pathways. The Swiss rolls were made according to previously published methods,65 with minor modifications. Briefly, after oral administration of 0.8 mg/kg FITC-D3 for 3 hours, the mice were sacrificed, and the lumen of small intestine was cut open from one side of the intestine using spring scissors. Cotton swabs were used to roll up the intestines that had been unfolded. The intestines were gently rolled around the self-closing forceps evenly, ensuring that each successive roll neatly laid over the previous roll with edges flush. The roll was gently slid off the cotton swabs and placed into an appropriately labelled pot containing 10 times the tissue volume of formalin. Whole-tissue slide scans at ×40 magnification were performed under a microscope slide scanner (Pannoramic MIDI, 3DHISTECH). Images were analysed with ZEN Imaging Software by ZEISS. Pictures were obtained by using an LSM 880 (Zeiss) or a spinning disc confocal microscope (Olympus).

For the localisation of D3 uptake in cells, HCT116 cells were inoculated into confocal dishes (1×105 cells/well) and incubated for 18 hours at 37°C. Free FITC (10 µM) or FITC-labelled D3 (10 µM) was added to cultures for 1 hour. Following staining, the cells were washed twice with PBS and later observed under a spinning disc confocal microscope (Olympus) after the addition of fresh medium.

Immunofluorescence of the ileum and hypothalamus

Tissue segments were immediately removed, washed with PBS, mounted in embedding medium (Tissue-Te, O.C.T. Compound) and stored (−80°C) until use. Immunofluorescence was performed using an anti-UGN (1:200; MBS7605526; MyBioSource) antibody. Detection and labelling were performed with secondary antibodies conjugated to Alexa Fluor-488 donkey anti-rabbit IgG (H+L) (1/500, A21206, Invitrogen).

Neuronal identification and c-Fos staining

Twenty-four mice (male, 8 weeks old) feeding on a standard chow diet were equally divided into eight groups (NC-0h, NC-1h, NC-2h, NC-3h; D3-0h, D3-1h, D3-2h, D3-3h). For gene expression assays, the hypothalamus was harvested 0–3 hours following D3 administration (NC: saline), and the expression levels of c-Fos, AgRp and Pomc were quantified by quantitative RT-PCR.

The same experiment was carried out for the immunofluorescence. c-Fos staining was performed as described in a previous study.66 Briefly, mice were anaesthetised with a lethal dose of pentobarbital and transcardially perfused with PBS followed by 4% paraformaldehyde. Brains were removed, placed in 4% paraformaldehyde overnight and dehydrated in 30% sucrose for 1 week. Brains were cut into 25 mm sections. The sections were treated as described above and incubated overnight at room temperature in mouse anti-FOS (1:500; ab208942; Abcam) or rabbit anti-POMC (1:100; ab254257; Abcam). Detection and labelling were performed using secondary antibodies conjugated to Alexa Fluor-594 donkey anti-mouse IgG (H+L) (1/500, ab150108, Abcam) or Alexa Fluor-488 donkey anti-rabbit IgG (H+L) (1/500, ab150073, Abcam), and imaging was performed as described above. Images were pseudocoloured using Photoshop software (Adobe) or ImageJ (NIH).

Data availability statement

Data are available upon reasonable request.

Ethics statements

Patient consent for publication

Ethics approval

All animal studies were approved by the Institutional Animal Care and Use Committee of Institute of Zoology, Chinese Academy of Sciences (IOZ-IACUC-2020–091).

References

Supplementary materials

  • Supplementary Data

    This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.

Footnotes

  • ZL and BZ are joint first authors.

  • Twitter @Fangqing_Zhao

  • Contributors FZ is responsible for the overall content as the guarantor. FZ conceived and supervised the study. FZ and ZL designed the study, interpreted the results and wrote the manuscript. WH’s team fed the GF mice and recorded the experimental data. Ileal transcriptome and faecal flora diversity analyses were performed by BZ. ZL, HW, ZZ and NW performed all the other experiments and the data analysis and prepared the figures and tables.

  • Funding This work was supported by grants from the National Natural Science Foundation of China (32025009, 32001082), National Key R&D Project (2021YFA1301000, 2021YFC2301300) and the Strategic Priority Research Program of Chinese Academy of Sciences (XDB38020300). We thank Jingwei Wang, Xiuling Ma, You Yu and Yongxi Huang for their assistance in sample preprocessing.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

Linked Articles