Whole and Isolated Protein Fractions Differentially Affect Gastrointestinal Integrity Markers in C57Bl/6 Mice Fed Diets with a Moderate-Fat Content

Abstract

Various proteins or protein fractions reportedly positively affect gastrointestinal integrity and inflammation in diets providing >45% energy as fat. This study tested whether benefits were seen in diets providing 30% of energy as fat. Purified diets (PD) with isolated soy protein (ISP), dried whole milk powder (DWMP), milk fat globule membrane (MFGM), or milk protein concentrate (MPC) as protein sources were fed to C57BL/6J mice (n = 15/diet group) for 13 weeks. MFGM-fed mice were heaviest (p < 0.005) but remained within breeder norms. Growth rates and gut motility were similar for all PD-fed mice. FITC-dextran assessed gut permeability was lowest in DWMP and MFGM (p = 0.054); overall, plasma endotoxin and unprovoked circulating cytokines indicated a non-inflammatory state for all PD-fed mice. Despite differences in cecal butyrate and intestinal gene expression, all PDs supported gastrointestinal health. Whole milk provided more positive effects compared to its fractions. However, ISP-fed mice showed a >370%, (p < 0.006) increase in colonic myeloperoxidase activity indicative of tissue neutrophil infiltration. Surprisingly, FITC-dextran and endotoxin outcomes were many folds better in PD-fed mice than mice (strain, vendor, age and sex matched) fed a "chow-type" nutritionally adequate non-PD. Additional variables within a diet's matrix appear to affect routine indicators or gastrointestinal health.

Keywords: bodyweight; dairy proteins; gastrointestinal permeability; intestinal health; isolated soy protein; myeloperoxidase; saturated fatty acids.

Figure 1

Representative sections from H&E-stained small intestine slides scanned at 20× magnification. Black bars represent distance measured for determination of villi length. ISP, isolated soy protein; DWMP, dried whole milk powder; MPC, milk protein concentrate; MFGM, milk fat globule membrane.

Figure 2

Intestinal gene expression: innate inflammatory markers. Gene expression data were from small intestines of n = 5/diet; fold increases were considered significant if at least 2-fold different from the ISP comparator and are denoted by the use of “*” TLR4 was increased 2.3-fold in MFGM compared to ISP; however, downstream responses (NFkB and TNFa) were modestly downregulated. Tight Junction Protein (ZO-1) was downregulated 2.4-fold in MPC compared to ISP.

Figure 3

Intestinal gene expression: cannabinoids and endocannabinoids. Gene expression data were from small intestines of n = 5/diet; fold increases were considered significant if at least 2-fold different from the ISP comparator and are denoted by the use of “*”. CB2 was increased 2.5-fold in intestines from milk fat globule membrane (MFGM)-fed mice compared to ISP. Other CB and eCB genes were unchanged.

Figure 4

Intestinal gene expression: adipocyte differentiation and lipogenesis. Gene expression data from small intestines of n = 5/diet; fold increases were considered significant if at least 2-fold different from the ISP comparator and are denoted by the use of “*”. AP2 was increased 2.75-fold and SREBP-1c increased 2-fold in intestines from MFGM-fed mice compared to ISP. Intestines of milk protein concentrate (MPC)-fed mice showed a 2-fold and 2.3-fold reduction in PPAR-γ and FASN, respectively, relative to ISP. Other adipocyte differentiation and lipogenesis genes were unchanged.

Figure 5

FITC-dextran permeability of chow-fed vs. purified diet-fed mice. Chow-fed mice (n = 10) averaged 18.9 ± 5.77 ug/mL FITC-d plasma fluorescence, a 118-fold increase over the average of all purified diet-fed mice. Purified diets (n = 60) averaged 0.16 ± 0.01 ug/mL FITC-d plasma fluorescence.

Figure 6

Plasma endotoxin concentrations of chow-fed vs. purified diet-fed mice. Plasma endotoxin content, measured by the limulus amebocyte lysate (LAL) assay, averaged 0.352 ± 0.011 EU/mL for chow-fed mice (n = 10) compared to average 0.109 ± 0.004 EU/mL for purified diet-fed mice (n = 60). Chow-fed mice showed a 322% increase in plasma compared to the average of purified diet-fed mice.