Protein- and zinc-deficient diets modulate the murine microbiome and metabolic phenotype

Protein- and zinc-deficient diets modulate the murine microbiome and metabolic phenotype1,2 Jordi Mayneris-Perxachs,3 David T Bolick,4 Joy Leng,6 Greg L Medlock,5 Glynis L Kolling,4 Jason A Papin,5 Jonathan R Swann,3* and Richard L Guerrant4 3 Division of Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College London, London, United Kingdom; 4University of Virginia Center for Global Health and 5Department of Biomedical Engineering, University of Virginia, Charlottesville, VA; and 6School of Veterinary Medicine, University of Surrey, Guildford, United Kingdom ABSTRACT Background: Environmental enteropathy, which is linked to undernutrition and chronic infections, affects the physical and mental growth of children in developing areas worldwide. Key to understanding how these factors combine to shape developmental outcomes is to first understand the effects of nutritional deficiencies on the mammalian system including the effect on the gut microbiota. Objective: We dissected the nutritional components of environmental enteropathy by analyzing the specific metabolic and gut-microbiota changes that occur in weaned-mouse models of zinc or protein deficiency compared with well-nourished controls. Design: With the use of a 1H nuclear magnetic resonance spectroscopy– based metabolic profiling approach with matching 16S microbiota analyses, the metabolic consequences and specific effects on the fecal microbiota of protein and zinc deficiency were probed independently in a murine model. Results: We showed considerable shifts within the intestinal microbiota 14–24 d postweaning in mice that were maintained on a normal diet (including increases in Proteobacteria and striking decreases in Bacterioidetes). Although the zinc-deficient microbiota were comparable to the age-matched, well-nourished profile, the protein-restricted microbiota remained closer in composition to the weaned enterotype with retention of Bacteroidetes. Striking increases in Verrucomicrobia (predominantly Akkermansia muciniphila) were observed in both wellnourished and protein-deficient mice 14 d postweaning. We showed that protein malnutrition impaired growth and had major metabolic consequences (much more than with zinc deficiency) that included altered energy, polyamine, and purine and pyrimidine metabolism. Consistent with major changes in the gut microbiota, reductions in microbial proteolysis and increases in microbial dietary choline processing were observed. Conclusions: These findings are consistent with metabolic alterations that we previously observed in malnourished children. The results show that we can model the metabolic consequences of malnutrition in the mouse to help dissect relevant pathways involved in the effects of undernutrition and their contribution to environmental enteric dysfunction. Am J Clin Nutr 2016;104:1253–62. Keywords: malnutrition, metabolome metabolic phenotype, metabolome metabonomics, microbiome microbiota, protein deficiency, undernutrition, zinc deficiency Am J Clin Nutr 2016;104:1253–62. Printed in USA. INTRODUCTION Many parts of the developing world are still struggling with malnutrition, inadequate clean-water supplies, and a lack of access to basic health care. These conditions lead to a vicious cycle of malnutrition, infection, and environmental enteric dysfunction that cause growth stunting and cognitive shortfalls. To better understand the impact of nutritional restrictions on the health and development of an individual, murine models have been developed to experimentally mimic human malnourishing diets. These models have previously been shown to replicate the characteristic side effects of malnutrition including reduced infant growth, delayed neurobehavioral development, and permanent alterations in macrophage function (1, 2). In this work, we have investigated 2 malnourishing mouse diets that reflected major nutritional deficiencies that are common in children who are living in impoverished areas. These diets included a protein-deficient diet (containing 2% protein; a normal diet typically contains w20% protein) that resulted in growth failure and a diet that was devoid of zinc that led to zinc deficiency compared with that shown in zinc-deficient children. These murine models provided a tool for dissecting the biochemical mechanisms through which specific nutritional restrictions can lead to phenotypic outcomes. Metabolic phenotyping (metabolomics and metabonomics) is a systems biology approach that enables the overall metabolic status of a biological system to be studied. The combination of metabolic phenotyping with specific models of undernutrition provides a top-down approach to illuminate the metabolic pathways that are modulated by such 1 Supported by the Bill & Melinda Gates Foundation [biomarker grant OPP1066140 entitled "Novel metabonomic biomarkers of gut function and health: modeling enteropathy (EE) and field validation”] and by the NIH (grant RO1 GM108501; to JAP). This is an open access article distributed under the CC-BY license (http://creativecommons.org/licenses/by/3.0/). 2 Supplemental Tables 1–3 and Supplemental Figures 1–3 are available from the “Online Supporting Material” link in the online posting of the article and from the same link in the online table of contents at http://ajcn. nutrition.org. *To whom correspondence should be addressed. E-mail: j.swann@imperial. ac.uk. Received February 2, 2016. Accepted for publication August 29, 2016. First published online October 12, 2016; doi: 10.3945/ajcn.116.131797. 1253 1254 MAYNERIS-PERXACHS ET AL. restrictions. A key variable that influences the metabolic fate of dietary inputs and, indeed, are shaped by the diet are the gut microbiota. Previous studies have shown that malnutrition can alter the structure and function of the bacterial populations that are present in the gut with subsequent alterations to the nutrient flow to the host (3–5). The importance of this biochemical interchange between the microbiota and host has been widely appreciated with known downstream consequences for host endogenous metabolism and, ultimately, host health. Therefore, to adequately resolve the biomolecular impact of a malnourishing diet and its influence on development, the bacterial-host supraorganism was studied including the diverse and complex trans-genomic metabolic interactions that occur (5, 6). To this extent, the urinary metabolic profiles and fecal bacterial populations of mice receiving either a protein-deficient or zinc-deficient diet have been characterized and compared with well-nourished control mice. Urinary metabolic profiles contain information relating to the host endogenous metabolism and also exogenous metabolic inputs such as those from the diet and the output from the gut microbiome. Through this parallel approach, the impact of malnourishing diets on the overall mammalian system can be resolved, thereby allowing the diet-associated mechanisms t (...truncated)