Food-derived opioid peptides inhibit cysteine uptake with redox and epigenetic consequences

Abstract

Dietary interventions like gluten-free and casein-free diets have been reported to improve intestinal, autoimmune and neurological symptoms in patients with a variety of conditions; however, the underlying mechanism of benefit for such diets remains unclear. Epigenetic programming, including CpG methylation and histone modifications, occurring during early postnatal development can influence the risk of disease in later life, and such programming may be modulated by nutritional factors such as milk and wheat, especially during the transition from a solely milk-based diet to one that includes other forms of nutrition. The hydrolytic digestion of casein (a major milk protein) and gliadin (a wheat-derived protein) releases peptides with opioid activity, and in the present study, we demonstrate that these food-derived proline-rich opioid peptides modulate cysteine uptake in cultured human neuronal and gastrointestinal (GI) epithelial cells via activation of opioid receptors. Decreases in cysteine uptake were associated with changes in the intracellular antioxidant glutathione and the methyl donor S-adenosylmethionine. Bovine and human casein-derived opioid peptides increased genome-wide DNA methylation in the transcription start site region with a potency order similar to their inhibition of cysteine uptake. Altered expression of genes involved in redox and methylation homeostasis was also observed. These results illustrate the potential of milk- and wheat-derived peptides to exert antioxidant and epigenetic changes that may be particularly important during the postnatal transition from placental to GI nutrition. Differences between peptides derived from human and bovine milk may contribute to developmental differences between breastfed and formula-fed infants. Restricted antioxidant capacity, caused by wheat- and milk-derived opioid peptides, may predispose susceptible individuals to inflammation and systemic oxidation, partly explaining the benefits of gluten-free or casein-free diets.

Keywords: Autism spectrum disorder; Casomorphin; Celiac disease; Gliadin; Glutathione; Gluten-free/casein-free diet; Schizophrenia.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Food-derived opioid peptides and morphine inhibit cysteine uptake in SH-SY5Y cells

a, Beta casein from milk and alpha gliadin from wheat, barley and rye are hydrolysed in the intestine by pepsin, leucine aminopeptidase (LAP) and elastase to release peptides with opioid activity. The 7 aa proline-containing peptides from human and bovine milk, and that from wheat, have homologous sequences, including two or three proline residues. b, Radiolabeled cysteine uptake in SH-SY5Y human neuroblastoma and c, Caco2 colon carcinoma cell lines in the presence of increasing concentrations (0.1 nM to 1 µM) of morphine or opioid peptides. Cells were treated for 30 min. d, Time-dependent effects of opioid peptides and morphine on cysteine uptake by SH-SY5Y cells. SH-SY5Y cells were treated with 1 µM of morphine or opioid peptides for 0.5, 4, 24 and 48 h (n = 6). (*) p < 0.05 and (**) p < 0.01 vs. control.

Figure 2. Effect of opioids on GSH/GSSG and SAM/SAH ratios

Ratios for a, GSH / GSSG and b, SAM / SAH were calculated after treatment with opioid peptides and morphine for 0.5, 4 and 24 h, at a concentration of 1 µM (n = 4). (*) p < 0.05 and (**) p < 0.01vs. control.

Figure 3. Morphine and milk-derived opioid peptides increase genome-wide promoter methylation

SH-SY5Y cells were treated with 1 µM morphine, bBCM7 or hBCM7 for 4 h (n = 5) and genome-wide DNA methylation was analysed by MBD-seq. 53,561 genes were aligned at their transcription start site (TSS) and average methylation between −3000 bp and +3000 bp was computed and normalized to values at −3000 bp. Inset:Methylation of all genes was evaluated at the +80 bp peak for each group and expressed as a percentage of the control group level, yielding p-values for morphine, bBCM7 and hBCM7 of 1.8e-25, 0.016 and 3.2e-40, respectively.

Figure 4. Morphine and opioid peptides alter redox/methylation gene expression

SH-SY5Y cells were treated with opioid peptides or morphine (1 µM) for 4 h, RNA was isolated and mRNA levels were probed by qRT-PCR with primers designed for a panel of redox and methylation-linked genes. Values were normalized to β-actin levels and to control gene expression (n = 4). The Y-axis indicates fold changes in mRNA levels. (*) indicates significant difference (p

Figure 5. Summary diagram of redox-dependent epigenetic regulation by µ-opioid receptors