Novel β-mannanase/GLP-1 fusion peptide high effectively ameliorates obesity in a mouse model by modifying balance of gut microbiota
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.
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These authors contributed equally to this study.