Colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi

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Highlights

  • After first colostrum feed ER-resident chaperone BiP is strongly decreased.

  • BiP decreased as expected, but surprisingly, p-eIf2a increased.

  • Inflammation inhibitor IkB strongly increased after colostrum.

  • Colostrum oxytocin contributed to activation of the autophagy marker LC3A.

  • LC3A level correlates with the increase in NF-kB inhibitor, IkB.

Abstract

Little is known about the role of oxytocin (OT) in colostrum during early gut colonization. We previously showed that transient OT receptor (OTR) expression on newborn rat enterocytes coincides with the milk-suckling period, and that OT activates endoplasmic reticulum stress sensors in cultured enterocytes. Here, we explored whether colostrum-OT attenuates stress in newborn villi primed and unprimed by colostrum by measuring levels of stress markers including BiP (an ER chaperone), eIF2a (translation initiation factor), and pPKR (eIF2a kinase). We also measured two inflammation-signaling proteins NF-κB and its inhibitor IκB. To test the impact of colostrum on autophagy, we measured a marker of autophagy initiation, LC3A. Colostrum increased inactive p-eIF2a, p-PKR and IκB and reduced p-IκB, BiP and LC3A. LPS increased and OT decreased p-IkB. BiP (GRP78) was higher in unprimed than primed villi. Together, these data suggest that colostrum OT attenuates the impact of inflammation on postnatal gut villi and that OT enhances autophagy to protect against amino acid insufficiency-induced stress during the interval between birth and the first feeding.

Introduction

The presence of the neuropeptide oxytocin (OT) in milk and its role in milk letdown are well known. Less known is the role of OT delivered in mothers' milk on newborn gut development. Recent in vivo studies in rodent gut have demonstrated multiple important effects of OT/OT receptor (OTR) signaling on enteric neurons (1). Other experiments have shown that OT in combination with secretin is anti-inflammatory in animal models of colitis (2) and OTR deficient mice have altered gastrointestinal structure, motility, macromolecular permeability, mucosal maintenance and inflammatory responses [1]. OT appears to play an important role in early gut function and development. For instance, OTR expression is developmentally regulated during the milk-suckling period, and toward the end of the suckling period, OTR expression appears to migrate toward stem cells in the crypts [4].

There are several stressors to the newborn gut. These include temporary starvation, particularly of essential amino acids prior to first feed [2]. The first feeding of colostrum exposes the gut to high concentrations of foreign microbiota [3], [4], as well as the anti-inflammatory OT [5], [6], [7]. Another stressor includes early colonization by microbiota. In vitro experiments utilizing lipopolysaccharide (LPS) to mimic exposure of the newborn gut to bacterial endotoxin have shown that inflammatory signaling in enterocytes may be attenuated by OT to reduce cellular stress [ [8]. Other studies in gut cells support this hypothesis. OT downregulates the PI3K/Akt/mTORC protein synthesis pathway [9], a pathway dysregulated in autism and linked to mRNA translation and [12]. OT also modulates central sensors of the unfolded protein response (UPR) [10], a transcriptional program that strictly limits mRNA translation and clears unfolded proteins to resolve endoplasmic reticulum (ER) stress [14]. LPS activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB), which is a transcription factor that regulates the expression of ∼150 genes, including pro-inflammatory proteins that are normally downregulated by NF-KB inhibitor, IKB [11]. Phosphorylated IKB (pIKB) releases NF-KB, which translocates to the nucleus to serve as an active transcription factor [12], [13]. Collectively, these studies show that exposure to OT suppresses activation of the inflammatory pathway and stimulates the UPR.

In the present study, we tested the impact of colostrum OT on markers of cellular stress and inflammation signaling in early postnatal villus tissue. We also measured LC3A, a marker of autophagy, to test whether amino acid insufficiency stress stimulates or inhibits inflammation during the interval between birth and first feeding before villus tissue is exposed to colostrum [14]. We examined protein expression in harvested villi that were unprimed (prior to first feed) and in vivo primed (subsequent to first feed). We exposed in vivo unprimed and colostrum primed tissue at a subsequent ex vivo stage to vehicle alone, LPS, exogenous OT or OTR antagonist (OTA). In a second protocol, we compared ex vivo the effects of vehicle versus colostrum in the presence and absence of OTA on villi obtained from unprimed rats using extracted colostrum. Our results suggest that colostrum OT plays a pivotal anti-inflammatory role that includes autophagy in the newborn rat gut villi.

Section snippets

Reagents

Oxytocin (OT; Phoenix Pharmaceuticals Inc., Burlingame, CA) and the oxytocin receptor antagonist (OTA; desGly-NH2-d(CH2)5[D-Tyr2,Thr4]OVT (ST-11-61)) were donated by Dr. Maurice Manning, University of Toledo, OH; OTA is 102-fold more efficient than OT in its interaction with OTR [15]. LPS-EB standard, derived from E coli 0IIIB4 and TLR4 ligand were obtained from InvivoGen (San Diego, CA).

Antibodies

The following antibodies were used: anti-rabbit IgG horseradish peroxidase (HRP) conjugate and anti-mouse

Colostrum suppresses the UPR marker BiP via OT receptor

We tested the impact of exogenous OT (ex vivo stimulation) on the ER-resident chaperone BiP, an important marker of cellular stress and inflammation signaling, including the unfolded protein response (UPR) to ER stress and nutrient insufficiency [2]. Our antibody recognized two families of BiP; the HSP70 family at 78 kDa (Fast BiP, GRP78), and the HSP90 family at 94 kDa (Slow BiP, GRP94). We measured BiP levels under two different protocols (Fig. S1).

Under Protocol A, we measured Slow and Fast

Summary

Our findings show that colostrum OT attenuates the impact of inflammation on postnatal gut villi during the colonization period. In addition, OT enhances autophagy to protect against amino acid insufficiency-induced stress during the interval between birth and the first feeding.

Acknowledgments

We thank Muhammad Anwar for animal rearing and surgery. We also thank Michael M. Myers for editing. Finally, we thank David Hirschberg for his technical advice. Funding for this project was provided by the Einhorn Family Charitable Trust and the Fleur Fairman Family to the BrainGut Initiative at Columbia University Medical Center. Funding in part was also provided by Columbia University's CTSA from NCRR/NIH (UL1RR024156). The study sponsors had no role in the conduct of the study, the

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