Best Practice & Research Clinical Endocrinology & Metabolism
6MicroRNAs: Milk's epigenetic regulators
Introduction
Milk is the first postnatal nutritional environment of mammals, who physiologically wean off mother's milk to move into an adult diet. Milk is a highly specialized, complex and dynamic nutrient and signalling system developed by mammalian evolution to promote postnatal growth [1]. The environment in early life has important influence on epigenetic programming of far-reaching effects on the risk of developing common metabolic diseases in later life [2]. Intensive studies over the last years provided evidence that horizontal transfer of secreted miRNAs between cells plays a key role in intercellular communication [3], [4], [5]. miRNAs are short RNA sequences around 19–22 nucleotides with a high phylogenetic conservation. They can target their corresponding mRNAs, thereby inhibiting mRNA translation and protein synthesis [6]. Recently, miRNA-guided diagnostics are available as a powerful molecular approach for evaluating clinical samples through miRNA detection and/or visualization [7]. Milk, the secretory product of mammary gland epithelial cells (MECs), has been identified as the body fluid containing most abundant amounts of RNAs and miRNAs [8]. The majority of milk's miRNAs are transported and protected by the lipid bilayer of extracellular vesicles, predominantly exosomes of about 100 nm in diameter, which are secreted by MECs [9]. Exosomes play a crucial role in miRNA transport and uptake via endocytosis [10], *[11], [12]. Human and bovine milk exosomes thus provide natural means of genetic material transfer to human infants and human consumers of cow's milk, respectively.
In this review, we summarize recent evidence demonstrating that milk exosomal miRNAs are taken up by human intestinal cells, reach the systemic circulation of the milk recipient and modify gene expression of distant cells. We suggest that milk miRNA-mediated epigenetic regulation is a physiological signalling mechanism that unfortunately is absent in today's infant formula feeding. On the other hand, continued consumption of pasteurized cow's milk and efforts enhancing dairy milk yield apparently increase milk's miRNA content that contaminates the human food chain [13], *[14]. Persistent transfer of bovine milk miRNAs is of critical concern because it may disturb human epigenetic regulation inducing adverse long-term health effects such as obesity and type 2 diabetes mellitus.
Section snippets
Milk exosomes
Milk exosomes are regarded as most important signalosomes mediating cellular communication between the mother and her nursing infant [9], [15], [16], *[17]. For the first time, Admyre et al. [18] isolated and characterized exosomes from human colostrum and human milk. Thereafter, milk exosomes have been reported in colostrum and mature milk of humans, cows, buffalos, goats, pigs, marsupial tammar wallabies and rodents [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31].
Survivability of milk exosomes in the gastrointestinal tract
There is accumulating evidence that milk exosomes survive the degradative conditions in the gastrointestinal (GI) tract. Exosomal package of miRNAs serves as an important biological feature protecting and stabilizing miRNA transfer to the milk recipient. Compared with exogenous synthetic miRNAs, exosomal immune-related miRNAs of human milk are resistant to harsh degradative conditions [23]. Commercial bovine milk miRNAs are stable under acidic environments, RNase treatment, and freezing, but
Cellular uptake of milk exosomal miRNAs
Cells are able to take up exosomes by a variety of endocytic pathways, including clathrin-dependent endocytosis, clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft-mediated internalization [41]. Milk exosome uptake including their miRNAs has been demonstrated in macrophages and IECs [28], [36], [42], [43]. The intestinal uptake of bovine milk exosomes is mediated by endocytosis and depends on exosome surface glycoproteins [42], [43].
Systemic bioavailability of milk miRNAs
Bovine milk miRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow's milk and affect gene expression in peripheral blood mononuclear cells (PBMCs) of human volunteers [45]. Higher serum levels of immune-related miRNAs in porcine colostrum compared to mature porcine milk were also found in colostrum-only fed piglets compared to piglets fed mature milk, which further indicates a dose-dependent uptake of milk-derived miRNAs into the bloodstream of the
Milk miRNAs survive pasteurization
Pasteurization of milk (72–75 °C, 15–30 s) is commonly used to reduce the numbers of pathogenic microorganisms in commercial milk. Pasteurization does not affect the recovery of milk-derived miRNAs such as miRNA-148a *[34], *[35]. Golan-Gerstl et al. [35] reported a miRNA-148a-3p content of 23,3% in non-pasteurized bovine skim milk and 60.5% in milk fat. After pasteurization, the authors detected miRNA-148a-3p levels of 16.1% and 7.2%, respectively [35]. These data are in accordance with
Degradation of milk miRNAs by fermentation
Before the era of pasteurization and refrigeration, cow's milk was subject to immediate bacterial fermentation. Using scanning electron microscopy and dynamic light scattering, Yu et al. [51] compared the morphology and particle size distribution of exosomes from yogurt fermented with three different combinations of bacterial strains with those from raw milk. The protein content of exosomes was significantly reduced in fermented milk. The content of miRNA-29b and miRNA-21 was relatively high in
Increased miRNA-148a/152 levels in high performance dairy cows
Epigenetic regulation of MECs plays a key role in the biosynthesis pathways of lipid and protein components of milk. miRNA-148a targets key mediators involved in triacylglycerol and cholesterol homeostasis such as ABCA1, LDLR and CPT1A [52]. miRNA-148a, miRNA-148b, and miRNA-152 are members of the miRNA-148/152 family, which share substantial homology in their seed sequences [53]. Wang et al. [54] reported that miRNA-152 regulates DNMT1 and is involved in the development and lactation of
Infant formula is miRNA-deficient
The introduction of sterilized evaporated cow's milk for infant feeding in 1929 and the opinion at that time that milk is just food promoted the widespread use of artificial formula feeding in industrialized countries [61], [62]. Unfortunately, milk's function as a maternal–neonatal signalling system transferring miRNAs to the newborn infant had not been known. Current infant formulas are highly deficient in milk-derived miRNAs [25], *[35], [63], a deficit that may have negative effects on
Interspecies milk miRNA homology
Milk miRNA sequence and seed sequences exhibit high interspecies homology recently emphasized for miRNA-148a-3p pointing to a highly conserved, archaic miRNA signalling system of mammals [35]. Human and bovine miRNA-148a-3p share identical seed sequences with strong 7mer base pairing between miRNA-148a-3p and its target DNMT1 mRNA (Table 1). Low affinity miRNA-mRNA base pairing results in mRNA inhibition, whereas strong base pairing promotes target mRNA degradation [6]. It is thus conceivable
Milk exosomal miRNA-148a and DNMT1-regulated gene expression
DNA methylation has been regarded as a new guardian of the genome [68]. In general, DNA hypomethylation increases gene expression, whereas DNA methylation regulated by DNA methyltransferases suppresses transcription [69]. The maintenance DNA methyltransferase DNMT1 plays an essential role for mammalian development and growth control [69], [70]. DNMT1 is responsible for cytosine CpG methylation of DNA in mammals and has a role in gene silencing [69]. DNMT1 and its targeting miRNA-148a exhibit a
Milk miRNA-148a: epigenetic enhancer of food intake
One of milk's major anabolic functions is related to increased food intake, which has been related to overexpressed FTO [90]. Milk miRNA-148a-mediated DNMT1 suppression may result in increased FTO promoter demethylation stimulating FTO expression [90]. FTO erases m6A marks on a subset of mRNAs including ghrelin mRNA thereby enhancing ghrelin expression (Fig. 3) [90], [97]. FTO also enhances the activity of the dopaminergic midbrain circuitry critically involved in reward signalling from food
Milk miRNA-148a: epigenetic enhancer of adipogenesis
miRNA-148a directly controls several key target genes regulating lipid metabolism [52]. miR-148a suppresses salt-inducible kinase 1 (SIK1), a negative regulator of hepatic lipogenesis, which inhibits SREBP-1c activity by SIK1-mediated phosphorylation. Moreover, miRNA-148a suppresses PRKAA1, the catalytic unit of the heterotrimeric AMP-activated protein kinase (AMPK), a key energy stress sensor and negative regulator of mTORC1 (Fig. 4) [52]. Moreover, miRNA-148a regulates expression of carnitine
Summary
Milk is a program of mammalian evolution that provides archaic signalling information through highly conserved miRNAs which modify transcription and epigenetic regulation to promote infant's anabolism and growth. In analogy to a viral infection, milk provides virus-sized exosomes equipped with abundant miRNAs, that modify gene expression of receptor cells. Physiologically, milk's epigenetic signalling is restricted to the breastfeeding period (Fig. 5). Milk's most abundant miRNA is miRNA-148a
Conflict of interest statement
The authors declare no conflicts of interest.
Funding sources
There are no study sponsors in the collection, analysis, and interpretation of data.
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2022, Clinical NutritionCitation Excerpt :Infants have underdeveloped TJ protein integrity in the intestine [12], the formation of the same is essential for protection against the incidence of intestine-related diseases among them. Milk-derived miRNAs, miR-148a-3p (miR-148a) is abundant in human breast milk [7] Golan et al. showed that the treatment of breast milk exosomes increased the miR-148a expression and decreased DNA methyltransferase 1 (DNMT1), which is a direct target of miR-148a, in human normal colorectal cells [13]. He et al. reported that human breast milk-derived exosome administration restored intestinal TJ proteins such as ZO-1 in necrotizing enterocolitis-induced mouse pups [14].
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