Glycomacropeptide is a prebiotic that reduces Desulfovibrio bacteria, increases cecal short-chain fatty acids, and is anti-inflammatory in mice - PubMed (original) (raw)

Glycomacropeptide is a prebiotic that reduces Desulfovibrio bacteria, increases cecal short-chain fatty acids, and is anti-inflammatory in mice

Emily A Sawin et al. Am J Physiol Gastrointest Liver Physiol. 2015.

Abstract

Glycomacropeptide (GMP) is a 64-amino acid (AA) glycophosphopeptide with application to the nutritional management of phenylketonuria (PKU), obesity, and inflammatory bowel disease (IBD). GMP is a putative prebiotic based on extensive glycosylation with sialic acid, galactose, and galactosamine. Our objective was to determine the prebiotic properties of GMP by characterizing cecal and fecal microbiota populations, short-chain fatty acids (SCFA), and immune responses. Weanling PKU (Pah(enu2)) and wild-type (WT) C57Bl/6 mice were fed isoenergetic AA, GMP, or casein diets for 8 wk. The cecal content and feces were collected for microbial DNA extraction to perform 16S microbiota analysis by Ion Torrent PGM sequencing. SCFA were determined by gas chromatography, plasma cytokines via a Bio-Plex Pro assay, and splenocyte T cell populations by flow cytometry. Changes in cecal and fecal microbiota are primarily diet dependent. The GMP diet resulted in a reduction from 30-35 to 7% in Proteobacteria, genera Desulfovibrio, in both WT and PKU mice with genotype-dependent changes in Bacteroidetes or Firmicutes. Cecal concentrations of the SCFA acetate, propionate, and butyrate were increased with GMP. The percentage of stimulated spleen cells producing interferon-γ (IFN-γ) was significantly reduced in mice fed GMP compared with casein. In summary, plasma concentrations of IFN-γ, TNF-α, IL-1β, and IL-2 were reduced in mice fed GMP. GMP is a prebiotic based on reduction in Desulfovibrio, increased SCFA, and lower indexes of inflammation compared with casein and AA diets in mice. Functional foods made with GMP may be beneficial in the management of PKU, obesity, and IBD.

Keywords: cytokines; inflammatory bowel disease; phenylketonuria; sialic acid; sulfate reducing bacteria.

Copyright © 2015 the American Physiological Society.

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Figures

Fig. 1.

Fig. 1.

A: glycomacropeptide (GMP) or caseinomacropeptide is a bioactive peptide released from one of the casein milk proteins (κ-casein) during cheesemaking. Rennet (chymosin) cleaves κ-casein between phe 105 and met 106, releasing GMP, a glycosylated peptide, into the whey. GMP constitutes 20–25% of nitrogen in most whey products. GMP is a unique peptide lacking aromatic acids and thus has been isolated from whey for use in medical foods needed for the management of phenylketonuria. It is an acidic, highly polar peptide (isoelectric point below 4.0) that is hydrophilic and heat stable, with a theoretical molecular mass between 7 and 11 kDa. B: bovine GMP represents a heterogenous group of 64 amino acid peptides due to genetic variance (variants A and B) and posttranslational modification, including phosphorylation at serine residues and glycosylation at threonine residues via OH linkages. The primary structure of bovine variant A is shown; the 2 sites corresponding to mutational differences in the B variant are indicated. Glycosylated forms of GMP include 5 different mucin-type carbohydrate chains containing _N_-acetylneuraminic acid (sialic acid), _N_-acetylgalactosamine, or galactose. Approximately 75% of glycosylated GMP molecules include trisaccharide and tetrasaccharide chains as shown.

Fig. 2.

Fig. 2.

Relative spleen mass in wild-type (WT) and phenylketonuria (PKU) mice fed casein, amino acid (AA), or GMP diets from weaning through 20–22 wk of age. Values shown are means + SE (n = 45–70) for significant main effects of genotype (A) and diet (B); there was no significant interaction of genotype and diet. Relative spleen mass was significantly greater in PKU compared with WT mice and lower with ingestion of the GMP diet compared with the casein and AA diets.

Fig. 3.

Fig. 3.

Intact jejunal wet mass (A and B) and jejunal mucosa dry mass (C and D) in WT and PKU mice fed casein, AA, or GMP diets from weaning through 20–22 wk of age. Values shown are means + SE (n = 45–70) for significant main effects of genotype and diet; there was no significant interaction of genotype and diet. Jejunal mass was significantly greater in PKU compared with WT mice and significantly lower with ingestion of the GMP diet compared with the casein and AA diets.

Fig. 4.

Fig. 4.

WT and PKU mice fed the GMP diet have altered bacterial populations from WT mice fed the casein (Cas) diet and PKU mice fed the AA diet from weaning through 8–9 wk of age. Relative bacterial phyla abundance was calculated compared with total bacteria in both the cecum (A) and feces (B). In cecal contents from both WT and PKU mice fed the GMP diet there was a significant reduction in the Proteobacteria phylum compared with WT mice fed the casein diet and PKU mice fed the AA diet, P = 0.002 and P = 0.001, respectively. In the feces, PKU mice fed the GMP diet had a significant increase in Bacteroidetes relative to PKU mice fed the AA diet, P = 0.047.

Fig. 5.

Fig. 5.

Relative bacterial genera abundance was calculated compared with total bacteria in both the cecum (A) and feces (B) in WT and PKU mice fed either the casein, AA, or GMP diets from weaning through 8–9 wk of age. In the cecal contents of WT mice fed the GMP diet there was a significant reduction in Desulfovibrio compared with WT mice fed the casein diet, P = 0.001. PKU mice fed the GMP diet had a significant reduction in Desulfovibrio compared with PKU mice fed the AA diet, P = 0.001.

Fig. 6.

Fig. 6.

Cecal bacterial communities clustered using principal coordinate analysis of the unweighted UniFrac distance. Each point corresponds to a cecal sample from an individual mouse colored according to genotype and diet. Principal coordinates 1 and 2 (PC1 and PC2) are plotted on _x_-and _y_-axes with percentage of variation explained in the parentheses. All 4 groups are circled to look at group effects of diet (A) and GMP diet separated from the casein and AA diets (B).

Fig. 7.

Fig. 7.

Cecal concentrations of the short-chain fatty acids (SCFA) acetate (A), propionate (B), and butyrate (C) in WT mice fed casein and GMP diets and in PKU mice fed AA and GMP diets from weaning through 8–9 wk of age. Values are means + SE, n = 6–8.

Fig. 8.

Fig. 8.

Plasma cytokine concentrations for interferon-γ (IFN-γ, A), tumor necrosis factor-α (TNF-α, B), interleukin-1α (IL-1α, C), interleukin-1β (IL-1β, D), interleukin-2 (IL-2, E), and interleukin-10 (IL-10, F) in PKU mice fed AA and GMP diets and in WT mice fed casein and GMP diets from weaning through 8–9 wk of age. Values are means + SE, n = 6–8.

Fig. 9.

Fig. 9.

Spleen cells were stimulated with PMA and ionomycin and were collected from both WT and PKU mice fed either the casein or GMP diet from weaning through 20–22 wk of age. A: cells were gated on CD8 and plots are representative of mice fed either the casein or GMP diets. Histogram shows means ± SE percentage of CD8+ CD44hiCD62Llo cells, n = 8–18. B: percentage of CD8+ cells producing IFN-γ detected by intracellular cytokine staining.

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