Incorporation of dairy lipids and Lactobacillus fermentum in infant formulas: long-term effects on intestinal permeability, mucosal immunity and metabolism in a minipig model

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Incorporation of dairy lipids and Lactobacillus fermentum in infant formulas: long-term effects on

intestinal permeability, mucosal immunity and metabolism in a minipig model

Marion Lemaire, Gaëlle Boudry, Stéphanie Ferret-Bernard, Isabelle Nogret, Laurence Le Normand, Gwenaëlle Randuineau, Michele Formal, Sylvie

Guerin, Armelle Cahu, Véronique Rome, et al.

To cite this version:

Marion Lemaire, Gaëlle Boudry, Stéphanie Ferret-Bernard, Isabelle Nogret, Laurence Le Normand, et al.. Incorporation of dairy lipids and Lactobacillus fermentum in infant formulas: long-term effects on intestinal permeability, mucosal immunity and metabolism in a minipig model. 4. International Conference on Nutrition & Growth Conference, Mar 2017, Amsterdam, Netherlands. �hal-01530771�

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TEM-EXP1 EXP1-EXP2 TEM-EXP2

Cont ributio n to discri mi nation OTU's discrimination Fusobacteriaceae Coriobacteriaceae Actinomycetaceae Pasteurellaceae Enterobacteriaceae Bacteroidaceae Rikenellaceae Bacteroidales S24-7 group Prevotellaceae Porphyromonadaceae Peptococcaceae Peptostreptococcaceae Family XI Family XIII Lactobacillaceae Christensenellaceae Lachnospiraceae Ruminococcaceae

Incorporation of dairy lipids and Lactobacillus fermentum in infant formulas:

long-term effects on intestinal permeability, mucosal immunity

and metabolism in a minipig model

Incorporation of dairy lipids (DL) in infant formulas (IF) has been associated with short-term benefits on digestion, gut physiology and microbiota in a piglet model. Clinical studies showed that the probiotic Lactobacillus fermentum CECT 5716 (Lf) might prevent infections in infants.

The objective of this study was to investigate the long-term effects of addition of both DL and Lf in IF on growth, intestinal barrier function and metabolic and immunologic status of adult Yucatan minipigs. We hypothesized that gut microbiota could be a key factor of this programming.

This study highlights a long-term programming effect of early consumption of DL+Lf on intestinal epithelial barrier and endocrine function and of DL (± Lf) on immune orientation. These long-term effects might be mediated by changes in gut microbiota (composition and metabolism), which are different with DL alone or DL+Lf early consumption. In-depth analysis of changes in microbiota composition along with analysis of the potential mechanisms leading to the observed long-term effects are ongoing.

This work was founded by Lactalis Group. The authors acknowledge all the members of the porcine experimental facilities of the INRA (Saint-Gilles) and all the technical staff of the NGB team for their expert assistance and help. The authors wish to thank the Nutrition, Chemical Food Safety and Consumer Behaviour research division of the INRA for financing the metabolomics study and the analytical platform for metabolomics and toxicology (MetaToul-AXIOM, INRA, UMR1331 Toxalim, Toulouse, France) for their help in interpreting these results.

M. Lemaire1-2_{, G. Boudry}1_{, S. Ferret-Bernard}1_{, I. Nogret}1_{, L. Le Normand}1_{, G. Randuineau}1_{, M. Formal}1_{, S. Guérin}1_{, A. Cahu}1_{, V. Romé}1_,

P. Le Ruyet2, I. Cuinet2, C. Baudry2, S. Blat1, I. Le Huërou-Luron1

1 _{INRA, INSERM, Univ Rennes 1, Univ Bretagne Loire, Nutrition Metabolisms and Cancer (NuMeCan), Rennes, France,} 2 _{Lactalis R&D, 35240 Retiers, France}

Conclusion

Context and objective

Methods

26 piglets received from postnatal day (PND) 2 to 28 a formula containing as lipids: - only plant lipids (PL)

- a half-half mixture of PL and DL (DL)

- a half-half mixture of PL and DL supplemented with Lf (DL+Lf). Pigs were subsequently fed:

- a standard diet for 1 month

- then challenged with a hyperenergetic diet (HE) for 3 months - euthanized at PND140.

PL (n=9)

DL (n=8)

DL+Lf (n=9)

 Analyses at PND140:

- Intestinal permeability (Ussing chambers)

- Mucosal immunity (cytokine secretion of ileal explants challenged with LPS)

- Entero-insular axis (density of GLP-1 secreting cells, meal test, pancreas anatomy)

- Metabolism (lipid profile, intravenous glucose tolerance test (IVGTT))

- Gut microbiota metabolism (faecal metabolome)

- Gut microbiota composition (16S RNA sequencing)

 Statistics:

- ANOVA testing diet, gender and replication factors followed by post-hoc tests -

_*

- Data are presented as boxplot or mean ± SEM IVGTT PND140 Birth Weaning PND28 Standard diet 1 month HE diet 3 months PND2 PL DL DL + Lf

Results

Gut microbiota metabolism and composition at the operational taxonomic unit (OTU) level were different between groups. It might explain the long-term effects observed on gut physiology. Basal and meal-stimulated GLP-1 secretory capacities were increased in DL+Lf.

GLP-1 secreting cells density and pancreas anatomy (data not shown) were similar between groups. 0 200 400 600 800 Caecum GLP-1 PL DL DL+ Lf 0 20 40 60 80 p m o l/ g t iss u e

1. Intestinal permeability

5. Gut microbiota metabolism and composition

2. In vitro secretion of LPS-stimulated ileal explants

4. Host metabolism

The IF composition modulated gut barrier function.

Ileal trans- and paracellular permeabilities were increased in DL ± Lf compared to PL, though jejunal LPS passage was decreased in DL+Lf compared to PL.

Pro-inflammatory cytokine (TNFα and IL-8) secretions of LPS-stimulated ileal explants were decreased in DL ± Lf LPS-responders compared to PL.

Growth, food intake and metabolic dysfunctions (dyslipidemia and insulin resistance) induced by the HE diet were similar between groups.

3. Entero-insular axis

Secretory response to meal-stimulation PL DL DL+Lf 0 10 20 30 p m o l/l GLP-1-secreting cells PL DL DL+Lf 0 2 4 6 De n si ty n b /m m 2 Transcellular permeability Ileum PL DL DL+Lf 0 100 200 300 400 HRP (ng /m l) * Paracellular permeability Ileum PL DL DL+Lf 0 10 20 30 40 Con du c ta nc e (m S .c m 2 ) * LPS passage Jejunum PL DL DL+L f 0 50 100 150 200 L P S -F (n g /m l) * 0 2 4 6 8 10 p g /m g t issu e

IL-1β TNFα IL-10 _IL-8

# * * * # * Plasma GLP-1 Secretory response to meal stimulation

FFA: free fatty acids, HOMA-IR: homeostasis model assessment of insulin resistance Adiposity gain

under the HE diet

PL DL DL+Lf 0 200 400 600 % HOMA-IR PL DL DL+ Lf 0 5 10

Plasma lipid profile

Plasma lipid profile

0 1 2 3 4 m m o l/ l FFA Cholesterol HOMA-IR Adiposity gain

under the HE diet

Triglycerides 0 5 10 15 20 -15 5 25 45 65 mm ol /L Time (min) Glycemia 0 100 200 -15 5 25 45 65 µUI/ mL Time (min) Insulinemia Faecal metabolome PL DL DL+Lf PL DL DL+Lf

Fu: Fusobacteria, A: Actinobacteria, P: Proteobacteria, B: Bacteroidetes, Fi: Firmicutes

1 _{Collaboration with P. Gérard and M. Rhimi (INRA, Micalis, Jouy-en-Josas)}

Fu P Main families Fi B Phylum 1 LPS passage Jejunum, 120 min Transcellular permeability Ileum, 90 min Paracellular permeability Ileum, 90 min GLP-1-secreting cells Caecum Caecum GLP-1 A