Novel β-mannanase/GLP-1 fusion peptide high effectively ameliorates obesity in a mouse model by modifying balance of gut microbiota

https://doi.org/10.1016/j.ijbiomac.2021.09.150Get rights and content

Highlights

  • MGLP_1 shows ability to ameliorate obesity.

  • Oral administration of MGLP_1 ameliorated lipid and glucose metabolism.

  • MGLP_1 modifying balance of gut microbiota and metabolism.

  • The mechanism of weight loss of MGLP_1 is similar to MOS but different.

Abstract

We constructed a novel β-mannanase/GLP-1 fusion peptide, termed MGLP_1, and evaluated its ability to ameliorate obesity in a high-fat/high-sugar diet (HFSD)-induced mouse model. Eight-wk MGLP_1 treatment notably reduced obesity, as reflected by significant changes of body weight, serum triglyceride level, fatty liver and adipose tissue distribution. Amelioration of HFSD-induced gut dysbiosis by MGLP_1 was evidenced by reduced abundance ratio of bacterial phyla Firmicutes to Bacteroidetes, enhanced abundance of beneficial probiotic genera (Bifidobacterium, Lachnospiraceae, Ileibacterium), and reduced abundance of harmful genera (Clostridium, Romboutsia). Mechanisms of weight loss were investigated by comparing effects of treatment with MGLP_1 vs. prebiotics manno-oligosaccharides (MOS). MGLP_1 ameliorated gut microbiota imbalance by enhancing carbohydrate catabolism, whereas MOS promoted glycan synthesis and metabolism. Our findings, taken together, indicate that MGLP_1 fusion peptide has strong potential for amelioration of obesity by modifying relationships between gut microbiota and lipid and glucose metabolism.

Introduction

Obesity is a worldwide health issue, associated with economic progress, modern lifestyles and overconsumption. Roughly one third of world's people are obese or overweight, and the incidence of associated metabolic diseases has increased sharply during recent decades [1]. World Health Organization (WHO) data indicate that among adults >20 years old, ~35% can be considered overweight (body mass index, BMI >25 kg/m2), and ~11% obese (BMI >30 kg/m2) – a total of ~2.5 billion people worldwide [2]. Clinical characteristics associated with obesity include excessive accumulation of body fat, dyslipidemia, insulin resistance, and low-grade inflammation [3], [4]. Obesity is a high-risk factor for a variety of chronic diseases, including type 2 diabetes, nonalcoholic fatty liver, cardiovascular disease, and certain cancers [5], [6], and often leads to serious disruption of lipid, glucose, and/or amino acid metabolism through dysfunction of essential signaling pathways [7], [8], [9].

Environmental and genetic factors obviously play crucial roles in obesity development, and contribute to high-calorie consumption habits and reduced physical activity. However, pathological and molecular mechanisms underlying obesity development remain largely unclear. Gut microbiota are involved in obesity pathogenesis through a wide range of mechanisms that include promotion of energy absorption from diet, altered production of intestinal hormones, and induction of insulin resistance by obesity-induced inflammation. Our knowledge of health-related roles of gut microorganisms has increased greatly in recent decades. The gut microbiota, considered as an environmental factor, plays a major role in initiation and development of obesity [9], and in various aspects of host metabolism [10], [11], [12]. Numerous studies indicate that alterations of gut microbiota are closely related to lipid, glucose or amino acid metabolism, insulin resistance, energy metabolism, and immune system function [13], [14]. On the bacterial phylum level, gut microbiota composition shows higher abundance of Firmicutes (gram-positive) and lower abundance of Bacteroidetes (gram-negative) associated with obesity [15], [16], [17]. Certain bacteria are able to alter metabolic signaling pathways via their secondary metabolites. Studies as above, taken together, clearly demonstrate that changes in gut microbiota and their secondary metabolites are closely associated with development of metabolic diseases [18].

Glucagon-like peptide-1 (GLP-1) plays an important role in weight loss, glucose homeostasis, and nutrient metabolism [19], [20]. Under normal physiological conditions, GLP-1 is digested by the enzyme dipeptidyl peptidase-IV (DPP-IV) and then rapid filtered by glomeruli, which restricts its clinical scope [21]. Several incretin-based hypoglycemic and weight loss agents (e.g., semaglutide, liraglutide) have been utilized widely for control of glycemia and body weight control. However, there is persistent demand for longer-lasting and more multifunctional drugs, particularly for oral administration and improvement of gut microbiota function [22], [23], [24]. Development of long-term oral GLP-1 analogues is highly desirable, but is very difficult because of protease degradation and low pH in the digestive tract.

We constructed a novel, long-acting GLP-1 fusion peptide (termed MGLP_1), consisting of two domains: (i) β-mannanase with enhanced protease-resistance and gastric acid stability, and the ability to degrade mannan to yield prebiotic manno-oligosaccharides (MOS) [25], [26]; (ii) a mutated GLP-1 (Arg34Lys) fragment that displays pharmacological effects similar to those of GLP-1. Using a C57BL/6 J mouse obesity model, we investigated in vivo pharmacodynamic properties of MGLP_1, its effects on gut microbiota, and its potential application for weight loss and regulation of blood lipid and glucose metabolism.

Section snippets

Strains and reagents

Pichia pastoris recombinant strain X-33 and pPICZαA expression vector are maintained in our laboratory. T4 DNA ligase and restriction enzymes EcoRI and XbaI were from New England Biolabs (Ipswich, MA, USA). PCR reagents, DNA markers, zeocin, and purification kits were from Invitrogen/Thermo Fisher (Waltham, MA, USA). Streptozotocin (STZ), d-glucose, and D-mannose were from Sigma-Aldrich (St. Louis, MO, USA). Orlistat (a weight loss drug; lipase inhibitor) was from Pharscin Pharmaceutical Co.

Fermentation and purification of MGLP_1 fusion peptide

After 200 h fermentation in 7.5-L bioreactor, cell density reached OD600 193 (Fig. 1A). After 120 h fermentation in 50-L bioreactor, cell density reached OD600 311. MGLP_1 yield increased as a function of cell density, and reached 2.01 g/L (Fig. 1B).

Methanol is a key component in control of fermentation process, as both carbon source and inducer. Methanol accumulation strongly affects P. pastoris metabolism; lack of methanol leads to cell death, whereas excessive methanol accumulation inhibits

Discussion and conclusions

This study addressed the mechanism whereby MGLP_1 fusion peptide improves metabolic health. Our findings demonstrated the importance of changes in gut microbiota in effective amelioration of obesity and associated phenotypes, particularly lipid metabolism and glucose metabolism. The mechanism for regulatory effect of MGLP_1 on intestinal microorganisms is similar to that of prebiotics such as MOS and mannose. These treatments all cause alteration of gut microbiota at the phylum level, with

CRediT authorship contribution statement

Yan Wang: Formal analysis, Investigation, Writing - original draft. Nuraliya Ablimit: Formal analysis, Investigation. Yunpeng Zhang: Formal analysis, Investigation. Jifu Li: Formal analysis, Investigation. Xinrui Wang: Formal analysis, Investigation. Ting Miao: Formal analysis, Investigation. Lei Wu: Formal analysis, Investigation. Junquan Liu: Formal analysis, Investigation. Zegnli Wang: Formal analysis, Supervision. Huiqiang Lou: Formal analysis, Supervision. Wei Jiang: Conceptualization,

Declaration of competing interest

The authors declare no conflict of interest for this work.

Acknowledgments

This work was supported by The National Key Research and Development Program of China (Program 2019YFA0904700) and National Natural Science Foundation of China (Program 31770084, 31630005, and 31570067) and Opening Project of the State Key Laboratory of Microbial Resources. The authors thank Professor Bing Zhang for his convenience in keeping C57BL/6J mice during the experiment and Professor Chong Xu and Zhongliang Zhu for the help in MGLP_1 3D structure prediction and the analysis sections.

References (61)

  • G. Xu et al.

    Activation of pluripotent genes in hepatic progenitor cells in the transition of nonalcoholic steatohepatitis to pre-malignant lesions

    Lab. Investig.

    (2017)
  • B. Zhang et al.

    Intestinal pharmacokinetics of resveratrol and regulatory effects of resveratrol metabolites on gut barrier and gut microbiota

    Food Chem.

    (2021)
  • C. Zhao et al.

    Monascus ruber fermented Panax ginseng ameliorates lipid metabolism disorders and modulate gut microbiota in rats fed a high-fat diet

    J. Ethnopharmacol.

    (2021)
  • V. Sharma et al.

    Mannose alters gut microbiome, prevents diet-induced obesity, and improves host metabolism

    Cell Rep.

    (2018)
  • N.R. Shin et al.

    Proteobacteria: microbial signature of dysbiosis in gut microbiota

    Trends Biotechnol.

    (2015)
  • H. Zeng et al.

    Colonic inflammation accompanies an increase of β-catenin signaling and Lachnospiraceae/Streptococcaceae bacteria in the hind gut of high-fat diet-fed mice

    J. Nutr. Biochem.

    (2016)
  • C. Kong et al.

    Probiotics improve gut microbiota dysbiosis in obese mice fed a high-fat or high-sucrose diet

    Nutrition

    (2019)
  • L.J. den Hartigh et al.

    Obese mice losing weight due to trans-10, cis-12 conjugated linoleic acid supplementation or food restriction harbor distinct gut microbiota

    J. Nutr.

    (2018)
  • W. Guo et al.

    Hypoglycemic and hypolipidemic activities of Grifola frondosa polysaccharides and their relationships with the modulation of intestinal microflora in diabetic mice induced by high-fat diet and streptozotocin

    Int. J. Biol. Macromol.

    (2020)
  • A. Koh et al.

    From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolite

    Cell

    (2016)
  • T.D. Müller et al.

    Glucagon-like peptide 1 (GLP-1)

    Mol. Metab.

    (2019)
  • C.T. Damsgaard et al.

    Trends in adult body-mass index in 200 countries from 1975 to 2014: a pooled analysis of 1698 population-based measurement studies with 19.2 million participants

    Lancet

    (2016)
  • C.L. Boulangé et al.

    Impact of the gut microbiota on inflammation, obesity, and metabolic disease

    Genome Med.

    (2016)
  • O. Osborn et al.

    The cellular and signaling networks linking the immune system and metabolism in disease

    Nat. Med.

    (2012)
  • T.F. Lüscher

    Nutrition, obesity, diabetes, and cardiovascular outcomes: a deadly association

    Eur. Heart J.

    (2020)
  • M.J. Khandekar et al.

    Molecular mechanisms of cancer development in obesity

    Nat. Rev. Cancer

    (2011)
  • D.S. Ghorpade et al.

    Hepatocyte-secreted DPP4 in obesity promotes adipose inflammation and insulin resistance

    Nature

    (2018)
  • P.D. Cani

    Human gut microbiome: hopes, threats and promises

    Gut

    (2018)
  • C.A. Thaiss et al.

    The microbiome and innate immunity

    Nature

    (2016)
  • P.D. Cani

    The gut microbiota manages host metabolism

    Nat. Rev. Endocrinol.

    (2014)
  • Cited by (21)

    View all citing articles on Scopus
    1

    These authors contributed equally to this study.

    View full text