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The Journal of Nutrition logoLink to The Journal of Nutrition
. 2023 Feb 18;152(4):961–970. doi: 10.1093/jn/nxab452

Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-Defined Rodent Diet

Sonal Sukreet 1, Camila Pereira Braga 2, Thuy T An 3, Jiri Adamec 2, Juan Cui 3, Janos Zempleni 1,*
PMCID: PMC8970979  PMID: 34982830

Abstract

Background

Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids, and microRNAs. Consumption of an exosome- and RNA-depleted (ERD) diet elicited phenotypes compared with controls fed an exosome- and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and nonultrasonicated milk, respectively, for casein in the AIN-93G formulation.

Objectives

The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content.

Methods

Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation [ultrasonicated exosomes (USEs)]; controls were not ultrasonicated [nonultrasonicated exosomes (NSEs)]. Exosome count, size, and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids, and proteins were analyzed by RNA sequencing and MS. Intestinal transport, bioavailability, and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells, and C57BL/6J mice (n = 3; age: 6–8 wk).

Results

The exosome count was 76% ± 22% lower in USEs than in NSEs (P < 0.05). Ultrasonication caused a degradation of ≤100% of microRNAs. USEs and NSEs contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected a total of 525 and 484 proteins in USEs and NSEs, respectively. The uptake of USEs decreased by 46% ± 30% and 40% ± 27% compared with NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05).

Conclusions

Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells, and C57BL/6J mice and causes a near-complete degradation of microRNA cargos.

Key words: bovine milk exosomes, lactose intolerance, lipids, microRNA, proteins, rodent diet, ultrasonication

Introduction

Species in the animal, plant, and bacteria kingdoms use exosomes and exosome-like particles to transmit information from donor cells to recipient cells including the transmission of signals between distant tissues in eukaryotes (1). Communication may include cell-to-cell communication by exosomes in animals and exosome-like nanovesicles in plants and communication with the environment through exosome-like nanovesicles in bacteria. In animals, information may be transmitted by the binding of exosomes to receptors on the surface of recipient cells or by the internalization of exosomes and their RNA, lipid, and protein cargos by recipient cells (1, 2). Among exosome cargos with regulatory function, microRNAs are of particular importance, because microRNAs regulate >60% of human genes and loss of microRNA maturation is embryonically lethal in Dicer knockout mice (3, 4).

We demonstrated that exosomes and their cargos not only originate in endogenous synthesis but may also be obtained from dietary sources such as milk. Human intestinal and vascular endothelial cells and rat intestinal cells transport bovine milk exosomes (BMEs) by endocytosis and release microRNA cargos across the basal membrane in cell cultures, and humans absorb microRNAs from bovine milk (5., 6., 7.). BMEs and their microRNA cargos accumulate primarily in the small intestinal mucosa, liver, spleen, kidneys, and brain after administration by oral gavage in mice (7). Encapsulation in exosomes protects labile compounds such as RNA against degradation under harsh conditions as present in the gastrointestinal tract and dairy plants (8., 9., 10.). Ten laboratories have independently confirmed that microRNAs, delivered by milk exosomes, are bioavailable in whole organisms and cell culture models (6, 7, 11., 12., 13., 14., 15., 16., 17., 18., 19., 20., 21., 22.). Four reports raised concerns that the mass of microRNAs absorbed from milk may be insufficient to elicit biological effects. The limitations of these studies were discussed in a recent review and include lack of microRNA encapsulation in exosomes, compromised sample integrity, first-passage elimination in mucosa and liver, and biased bioinformatics analysis (23).

Evidence is accumulating that endogenous synthesis of microRNAs cannot compensate for dietary depletion of exosomes and their microRNA cargos. The concentrations of microRNAs were ≤60% lower in the plasma, liver, skeletal muscle, intestinal mucosa, and placenta in mice fed an exosome- and RNA-depleted (ERD) diet than in controls fed an exosome- and RNA-sufficient (ERS) diet (5, 24., 25., 26., 27.). The ERD and ERS diets used in these studies were based on the AIN-93G formulation, modified by substituting ultrasonicated milk (ERD diet) or nonultrasonicated milk (ERS diet) for dairy protein (casein) in the AIN-93G formulation before pelleting (see the Methods). The depletion of tissue microRNAs in mice fed ERD was associated with phenotypes such as altered purine metabolism, changes in bacterial communities in the gut, a moderate loss of muscle grip strength, increased severity of symptoms of inflammatory bowel disease, and loss of fecundity and postnatal survival compared with ERS controls (24., 25., 26., 27., 28., 29.).

To date, ERD and ERS diets have not been characterized regarding exosome and cargo content and bioavailability. In light of recent discoveries regarding the biological activities of milk exosomes and their microRNA cargos in nutrition, we recognized the importance of a comprehensive characterization of ERD and ERS diets. The objective of this study was to assess the effect of ultrasonication of milk on BME count, size, morphology, and bioavailability, and the content of microRNAs, proteins, and lipids in BMEs. Adult mice are lactose intolerant (30). Therefore, we also assessed whether lactose in ERD and ERS diets and the AIN-93G formulation elicits phenotypes in mice. Any such phenotypes would complicate the interpretation of studies that used the diets to assess nutritional activities of milk exosomes and their cargos.

Methods

ERD and ERS diets

ERD and ERS diets are based on the AIN-93G formulation, modified for their content of BMEs and BME cargos (Table 1) (31). For preparing the ERD diet, fat-free (skim) milk was ultrasonicated by using an ultrasonic water bath (Branson CPXH 5800) at the highest energy setting (185 W, 40 kHz) for 90 min followed by a 60-min incubation at 37°C before lyophilization of milk and pelleting of diets. The milk used for preparing the ERS diet was not ultrasonicated. Lyophilized milk contributes ∼54.4 g protein/kg diet, which is less than the amount of casein in the AIN-93G formulation (Supplemental Table 1) (32); the difference was made up by adding soy protein (Bob's Red Mill, Inc.) to avoid introducing BMEs through dietary casein. Ingredients were thoroughly mixed starting with the quantitatively smallest ingredients and dried at 40°C overnight before cutting the sheets into pieces of sizes similar to commercial mouse diet pellets. Other than milk, none of the ingredients were ultrasonicated and their content was identical in the ERD and ERS diets. Ingredients were chemically defined (MP Biomedicals) except for milk and soy protein. The diets provided mice with an amount of milk equivalent to 0.5 L/d consumed by an adult human, adjusted by body weight.

Table 1.

Composition of AIN-93G-based ERD and ERS diets1

ERD g/kg ERS g/kg AIN-93G g/kg
Cornstarch 330 Cornstarch 330 Cornstarch 398
Soy protein 163 Soy protein 163 Casein (>85% protein) 200
Dextrinized cornstarch 132 Dextrinized cornstarch 132 Dextrinized cornstarch 132
Milk powder (ultrasonicated exosomes), containing ∼5% lactose 105 Milk powder (exosomes), containing ∼5% lactose 105 Sucrose 100
Sucrose 100 Sucrose 100 Soybean oil (no additives) 70
Soybean oil (no additives) 70 Soybean oil (no additives) 70 Fiber 50
Fiber 50 Fiber 50 Mineral mix (AIN-93G-MX) 35
Mineral mix (AIN-93G-MX) 35 Mineral mix (AIN-93G-MX) 35 Vitamin mix (AIN-93-VX) 10
Vitamin mix (AIN-93-VX) 10 Vitamin mix (AIN-93-VX) 10 l-Cystine 3
l-Cystine 3 l-Cystine 3 Choline bitartrate (41.1% choline) 2.5
Choline bitartrate (41.1% choline) 2.5 Choline bitartrate (41.1% choline) 2.5 Tert-butylhydroquinone 0.014
Tert-butylhydroquinone 0.014 Tert-butylhydroquinone 0.014
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1

ERD, exosome- and RNA-depleted; ERS, exosome- and RNA-sufficient; MX, mineral mixture; VX, vitamin mixture.

Experiment 1: exosome isolation and authentication

Ultrasonicated exosomes (USEs) and nonultrasonicated exosomes (NSEs, control) were isolated from ultrasonicated and nonultrasonicated skim bovine milk, respectively. Exosomes were isolated by ultracentrifugation at 120,000 × g and 4°C for 60 min using a Fiberlite-F37L-8 × 100 rotor (Thermo-Scientific), as previously described with minor modifications (6, 11). The protocol has been deposited in the EV-Track database under accession ID EV200174 (33). Exosomes were authenticated using Nanosight NS300 (Malvern, Inc.), immunoblot analysis, and electron microscopy (Hitachi H7500 and S4700) as previously described (34).

Experiment 2: microRNA sequencing and data analysis

Total microRNA was isolated from an equal number of NSEs and USEs using the miRNeasy Serum/Plasma Kit (Qiagen, Inc.) and shipped on dry ice to the Genomics Sequencing Core Facility in the University of Nebraska Medical Center (Omaha, NE). The quantity of microRNAs was assessed by using a Qubit microRNA Assay Kit (Thermo-Fisher). Ten nanograms of microRNAs were used for small RNA library preparation for each sample using the NEXTflex Small RNA Seq Kit v3 (Bioo Scientific Corp.). Libraries were sequenced using the Illumina NextSeq 500 platform in single-read mode. Raw sequencing data were deposited in the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) database under ID PRJNA773492. FastQC was used to assess the quality of sequencing reads, and cutadapt was used to remove adapter sequences, ambiguous bases, and bases with quality scores of <10 (35, 36). With no mismatches allowed, reads between 18 and 40 nucleotides were mapped to the bovine reference genome [Bos taurus (bta), University of Maryland, UMD 3.1]. Mature microRNAs were considered real if ≥10 raw counts were detected in ≥1 of the 3 biological repeats of a sample. The abundance of microRNAs in USEs and NSEs was analyzed by using miRDeep2 (37). When comparing the abundance of microRNAs in USEs and NSEs we included only microRNAs that were expressed in all biological replicates with a minimum of 10 raw counts. A hierarchical clustering analysis was used to organize microRNAs by abundance in USEs and NSEs. Principal component analysis (PCA) was conducted to assess whether microRNAs in USEs clustered discretely from microRNAs in NSEs.

Experiment 3: qRT-PCR

Select microRNAs were analyzed by RT-qPCR to confirm the findings from the sequencing analysis. MicroRNA was purified from an equal number of USEs and NSEs by using the miRNeasy Serum/Plasma Kit (Qiagen, Inc.). Reverse transcription was performed by using the miScript-II RT Kit (Qiagen, Inc.). RT-qPCR was performed by using miScript SYBR Green (Qiagen, Inc.), the universal reverse primer provided in the kit, and sequence-specific forward primers (Supplemental Table 2). Data were analyzed as previously described (17).

Experiment 4: lipidomics

Lipids were analyzed by LC coupled to Fourier transform ion cyclotron resonance MS. Total lipids were extracted from ∼1 × 1012 NSEs and USEs using 660 μL methanol/chloroform (2:1; vol:vol; n = 3 independent samples, each in triplicate). Samples were incubated for 30 min followed by centrifugation at 7800 × g for 10 min at 4°C. The lipid fraction in the chloroform phase was collected and dried under a nitrogen stream followed by resuspension in 50 μL of 100% methanol. Samples were analyzed in random order. Lipid analysis was carried out using an Agilent 1200 Series HPLC system coupled to a high-resolution Fourier-transform ion cyclotron resonance 7.05T mass spectrometer. Five microliters were injected and separated using an ACE5 C8-300 column (2.1 × 100 mm, Advanced Chromatography Technologies Ltd). Lipids were separated by using a linear gradient of the following phases and a flow rate of 0.1 mL/min. The mobile phases consisted of 20 mmol/L formic acid and 10 mmol/L ammonium acetate in Milli-Q water (solvent A) and 20 mmol/L formic acid and 10 mmol/L ammonium acetate in acetonitrile/isopropanol (50/50; vol:vol; solvent B). The following gradient was used: t = 0 min: 30% B; t = 1 min: 30% B; t = 25 min: 100% B; t = 45 min: 100% B; t = 47 min: 30% B; and t = 60 min: 30% B (column re-equilibration). Spectra were acquired in positive mode over the scan range of 244–1800 m/z. Raw data files were formatted and converted using CompassXport (Bruker, Inc.) and analyzed (peak alignment and normalization) by using MZmine 2.0 (38). The output data represented the relative abundance of compounds represented by chromatographic peaks (lipid signatures). Any signature was considered real if the corresponding peak was detected in ≥5 out of the 9 samples that were analyzed, i.e., the threshold used was ∼55%. The missing 1–4 values were calculated and imputed by k-nearest neighbor, and normalization was achieved by autoscaling of raw data. Univariate and multivariate protocols in MetaboAnalyst 4.0 were used for final data analysis (39). The Human Metabolome Database was used to identify the class and subclass of a lipid with a minimal concentration of 10 ppm and within an m/z tolerance of 0.01 Daltons in positive ion mode [(M + H)+ and (M + NH4)+] (40).

Experiment 5: proteomics

Proteins were analyzed by LC-tandem MS and immunoblot analysis. For proteome analysis 5.0 × 1012 USEs and NSEs were suspended in ∼0.2 mL PBS, mixed with 3 volumes of cold acetone, and incubated at −20°C for 30 min (n = 6 independent replicates). Samples were centrifuged for 2 min at 15,000 × g and 4°C to pellet proteins and supernatants were discarded. MS analysis of trypsin digests was performed, and proteins were identified by using a multidimensional protocol as previously described (41, 42). Exosome-specific marker proteins were identified by using the ExoCarta database as reference (43). For immunoblot analysis, protein from ∼1 × 1010 USEs and NSEs was loaded per lane on 4%–12% gradient Bis-Tris gels (NPO322, ThermoFisher) (44). Membranes were probed using mouse anti-CD9 (Abcam), mouse anti-bovine CD63 (Bio-Rad), rabbit anti-CD81 (Antibodies Online), goat anti-Alix (Santa Cruz Biotechnology), and Tumor Susceptibility Gene 101 (TSG101) (Abcam) as markers for exosomes (45), anti-ɑ-lactalbumin (Abcam) as a marker for whey protein (46), anti-β-integrin (Abcam) as a marker for microvesicles (47), and goat anti-bovine histone H3 (Santa Cruz Biotechnology) as a negative control (45). Tetraspanins were analyzed under nonreducing conditions (45). All primary antibodies were diluted 1000-fold and bands were visualized using 50,000-fold dilution of infrared dye (IRDye) 800CW–labeled secondary antibodies (LI-COR) in an Odyssey® CLx infrared imaging system (LI-COR Biosciences).

Experiment 6: BME transport

Human colon carcinoma Caco-2 cells and fetal human small intestinal FHs 74 Int cells were purchased from American Type Culture Collection (ATCC) and used at passages 39–42 and 2–5, respectively. Cells were maintained following the vendor's recommendations. For studies of BME uptake, the FBS in cell culture media was depleted of exosomes by ultracentrifugation at 120,000 × g and 4°C for 18 h (48). Caco-2 and FHs 74 Int cells were seeded at a density of 15,000 cells/well in 96-well plates and allowed to adhere to plastic surfaces for 48–72 h. Transport studies were conducted by using fluorophore (FM4-64)-labeled NSEs and USEs (70 μg BME protein/well) as previously described (6).

Experiment 7: bioavailability and distribution

We loaded USEs and NSEs with synthetic, fluorophore (IRDye)-labeled miR-320a (IDTDNA, Inc.) as previously described (7). Negative controls were prepared by mixing exosomes with IRDye-labeled miR-320a in the absence of transfection reagent, i.e., the microRNAs did not enter the exosomes (7). Bioavailability and distribution were assessed in C57BL/6J mice (Jackson Laboratory, stock number 000664), age 6–8 wk. MicroRNA-loaded exosomes or control mixtures were administered by oral gavage using a dose of 1 × 1012 USEs or NSEs per gram body weight. The dose was chosen based on previous dose-response studies in mice (7). After BME administration, mice were deprived of food with free access to water for 12 h, after which tissues were collected. This time was chosen because a previous study suggested that the accumulation of BMEs in the liver reaches a plateau 12 h after oral gavage (7). Excised tissues were rinsed twice with cold PBS to remove blood; the small and large gastrointestinal tract was flushed 3 times to remove unabsorbed BMEs. The fluorescence intensity in tissues was quantified as previously described. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Nebraska-Lincoln (protocol 1713).

Experiment 8: lactose content in milk powder and casein

The milk powder used to prepare the ERD and ERS diets and the casein in the AIN-93G diet were analyzed for lactose by dissolving milk powder and casein (MP Biomedicals) in PBS containing 100 mM NaOH at 40°C with constant stirring for 60 min. Lactose was analyzed by using an enzyme-coupled colorimetric assay following the manufacturer's recommendations (Lactose Assay Kit; EnzyChromTM, Bioassay Systems).

Experiment 9: lactose intolerance in mice

Lactose intolerance was assessed by challenging mice with lactose administered by oral gavage. We used a hypothetical reference mouse (age: 12 wk; weight: 28 g) and delivered 30 mg lactose dissolved in 0.1 mL PBS. The lactose dose was adjusted based on body weight and the volume of PBS was kept constant. Mice aged 3 and 12 wk were challenged with lactose on 2 consecutive nights; controls received PBS. The lactose dose was chosen based on the observation that mice at age 12 wk consumed ∼5.8 g of Envigo rodent diet per day, containing 30 mg lactose (49). The ERD and ERS diets contained ∼5.3 g lactose/kg diet (5, 32). After lactose administration, mice were housed individually for 12 h with no access to food and free access to water. Feces were collected for macroscopic inspection for softness and diarrhea.

For the analysis of lactase expression, intestinal tissues were dissected and flushed with cold PBS to remove luminal contents and stored at −80°C in RNA Later solution (ThermoFisher Scientific). Ceca were weighed because lactose led to an increase in ceca weight in lactose-intolerant adult mice (50). Total RNA was extracted and reverse transcribed; lactase mRNA expression was quantified via RT-qPCR using lactase-specific primers and normalized for the expression of GAPDH mRNA (Supplemental Table 3) as previously described (27). For assessment of lactase activity, the entire small intestine was cut open along its longitudinal axis and separated into 3 sections, approximately representing duodenum, jejunum, and ileum. Mucosal cells were collected by scraping the luminal side of tissues with a glass slide using an equal volume of 0.5 mL cold PBS. Cell suspensions were diluted with cold lysate buffer (Qiagen, Inc.; 1:4 dilution by volume) and homogenized by using a bead homogenizer. The homogenate was centrifuged at 2000 × g and 4°C for 10 min to remove debris. Lactase activity was assessed in the supernatant by measuring the amount of glucose released from 10 mmol/L lactose (final concentration) in the media at 37°C in 30 min (51). Glucose was measured colorimetrically by using a glucose detection kit (Abcam, Inc.). Lactase activity is expressed in units of nmol glucose × 30 min−1 × mL supernatant−1.

Statistical analysis

The F test was used to assess the homogeneity of variances (52). Some variances were heterogeneous, e.g., BME size. Log transformation of heterogeneous data resulted in homogeneous data variation. Normality of distribution was assessed by using the Shapiro–Wilk test. Paired and unpaired t tests were used when comparing paired and unpaired parametric data, respectively. Wilcoxon's signed rank test and the Mann–Whitney U test were used when comparing paired and unpaired nonparametric data, respectively. One-factor ANOVA was used in the statistical analysis of lactase activity and lactase mRNA expression. Sidak's post hoc test was used when comparing treatments with a designated control, whereas Tukey's post hoc test was used when comparing all groups. Sexes were not compared. Prism 8.0 was used for statistical calculations (GraphPad Software Inc.). P < 0.05 was considered statistically significant. Data are reported as mean ± SD.

Results

Experiment 1: BME size, count, and morphology

Ultrasonication of milk altered the size, count, and morphology of BMEs compared with nonultrasonicated controls. The diameter and count of BMEs were larger and lower, respectively, in USEs than in NSEs (218 nm ± 99 nm compared with 117 nm ± 44 nm, and 1.4 × 1011 ± 2.2 × 1010 compared with 5.9 × 1011 ± 6.5 × 1010 exosomes/mL; P < 0.05; n = 6). USEs were irregularly shaped, whereas NSEs showed the spherical shape expected for exosomes (Figure 1A–F). For example, many USEs showed an irregular surface topology, some USEs had a thickened membrane, and there were incidents in which BME-sized vesicles were encapsulated in larger vesicles (Figure 1A–C). In USEs, BME-sized vesicles had multiple bilayer membranes and there were many fusion and aggregation events (Figure 1E, F). We did not formally assess whether the membrane orientation changed in USEs, i.e., whether the inside-facing surface was oriented toward the outer surface after ultrasonication.

Figure 1.

Figure 1

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Characterization of USE and NSE preparations. (A–D) Morphology and aggregation of USEs and NSEs assessed by using transmission electron microscopy. (A) Aggregated USEs, (B) BME-sized vesicles were encapsulated in larger vesicles, (C) USEs noted with a fused and thickened membrane, and (D) morphology of NSEs. White arrows indicate extracellular vesicles ≤ 150 nm and white triangles indicate larger vesicles > 200 nm in diameter. Large fields = 20,000-fold magnification; inserts = 50,000-fold magnification of sections from the large fields. (E) Morphology and aggregation of USEs, and (F) morphology of NSEs assessed by using scanning electron microscopy. NSE, nonultrasonicated exosome; USE, ultrasonicated exosome.

Experiments 2 and 3: microRNA cargos

Ultrasonication of milk caused a substantial loss in the diversity and abundance of microRNA cargos in exosomes. Each sample yielded ∼20 million raw reads in RNA-sequencing analysis. After low-quality reads were removed, a mean of 7.6 and 11.8 million reads remained in USEs and NSEs, respectively, for mapping to the bovine genome (Supplemental Table 4). The number of reads mapped to the bovine genome was smaller for USEs than for NSEs (P < 0.05; n = 3). When accepting as real any microRNA that was detected in ≥1 biological replicate, USEs and NSEs expressed 75 and 201 microRNAs, respectively (Supplemental Table 5). When accepting as real only microRNAs that were detected in all biological replicates, USEs and NSEs expressed 25 and 114 microRNAs, respectively. The 10 most abundant microRNAs in NSEs accounted for 71% of total microRNAs (Table 2). The same microRNAs were detected in USEs, albeit at a lower cumulative abundance (48% of total microRNAs). We did not formally investigate features that conveyed resistance to ultrasonication. However, it appeared that ultrasonication had a stronger effect on the degradation of some microRNAs than others; ultrasonication-dependent degradation varied between 36% and 100% among microRNAs (Supplemental Table 6, Figure 2A, B). Out of the 75 microRNAs detected in USEs, 10 microRNAs were not detected in NSEs, and 6 were more abundant in USEs than in NSEs (Supplemental Table 6). Sequencing results were corroborated by RT-qPCR analysis of 6 select microRNAs; ultrasonication caused a 74.0% ± 18.0% to 99.0 ± 3.0% loss of Bos taurus (bta)-lethal (let)-7a-5p, bta-miR-320a, bta-miR-200c, bta-miR-30d, bta-miR-423-5p, and bta-miR-148a. For bta-miR-30d and bta-miR-423-5p, the Ct values in USEs were below the detection limit of RT-qPCR under the conditions used here (Supplemental Table 7) (17).

Table 2.

Cumulative abundance of the 10 most abundant miRs in USEs compared with NSEs1

Preparations
MiRs USE NSE
bta-let-7a-5p 5.1 17.5
bta-miR-320a 28.8 34.4
bta-miR-200c 32.2 44.8
bta-miR-92a 34.4 49.6
bta-miR-30d 38.6 54.0
bta-miR-21-5p 41.2 57.5
bta-miR-423-5p 42.0 61.0
bta-miR-200b 43.5 64.5
bta-miR-30a-5p 46.1 67.9
bta-miR-26a 48.3 71.0
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2

Values are cumulative percentages, calculated based on raw counts. NSE was used to identify the 10 most abundant miRs. bta, Bos taurus; let, lethal; miR, microRNA.

Figure 2.

Figure 2

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Mature miRs in USE and NSE preparations. Heatmaps of (A) all and (B) the 30 most abundant miRs in NSEs compared with USEs. bta, Bos taurus; let, lethal; miR, microRNA; NSE, nonultrasonicated exosome; USE, ultrasonicated exosome.

Experiment 4: lipid cargos

Ultrasonication of milk caused a change in lipid profiles in BMEs. Nontargeted lipidomics analysis of BMEs detected 2428 and 2586 lipid signatures in USEs and NSEs, respectively (Supplemental Table 8). USEs and NSEs had 2252 lipid signatures in common, whereas 176 and 334 signatures were unique to USEs and NSEs, respectively. The abundance of 1716 lipid signatures was significantly different in USEs and NSEs and subsequent analyses focused on these lipids (Figure 3A, Supplemental Table 8). One hundred and forty-five lipid signatures were detected only in USEs and the concentrations of another 63 lipids were higher in USEs than in NSEs. All 25 lipid signatures for which the concentration differences reached the highest level of statistical significance were significantly lower in USEs than in NSEs (Figure 3B). PCA suggested that lipid profiles were similar, yet clustered discretely when comparing USEs and NSEs (Supplemental Figure 1). Note the apparently greater variation among replicates of USEs than of NSEs.

Figure 3.

Figure 3

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Lipid profiles in USE and NSE preparations. (A) Volcano plot of lipid signatures in USEs and NSEs (≥1.5-fold change; P < 0.05; Q < 0.05). (B) Heatmap of the 25 lipid signatures for which P was the smallest when comparing USEs with NSEs (0.001 ≤ P ≤ 0.02; n = 3 independent samples). NSE, nonultrasonicated exosome; USE, ultrasonicated exosome.

We identified 194 lipids in the pool of 1716 lipid signatures by comparing m/z values and retention times to authentic standards. Of the lipids, 137 were sphingolipids and eicosanoids in both USEs and NSEs combined (Supplemental Table 8); the abundance of 70 sphingolipids and 21 eicosanoids was lower in USEs than in NSEs, and 20 sphingolipids were not detectable in USEs.

Experiment 5: protein cargos

Ultrasonication had a modest effect on the number of proteins detected in USEs and NSEs. Five hundred and twenty-five and 484 proteins were identified in USEs and NSEs, respectively, and the 2 preparations had 127 proteins in common (Supplemental Table 9). Sequence coverage and confidence scores of the 127 shared proteins typically were lower in USEs than in NSEs. Twenty-five widely recognized exosome marker proteins were detected in NSEs (Supplemental Table 9); 15 of the markers, including CD9, CD81, TSG101, and HSP70, were not detected in USEs by using LC-MS/MS. Immunoblot analysis detected CD9, CD63, CD81, α-lactalbumin, TSG101, and Alix in NSEs, whereas Alix and CD81 were not detectable in USEs (Supplemental Figure 2). Note that markers for extracellular vesicles other than exosomes, microvesicles, were identified in USEs but not in NSEs, e.g., mitofilin (MIC60), actin filament associated protein 1 (AFAP1), ADAM Metallopeptidase With Thrombospondin Type 1 Motif 13 (ADAMTS13), and histone H4 (Supplemental Table 9).

Experiments 6 and 7: BME transport and bioavailability

Ultrasonication led to a decrease in BME uptake in human intestinal cell cultures and mice. The uptake of USEs was 46% ± 30% and 40% ± 27% lower than NSE controls in Caco-2 and FHs 74 Int cells, respectively (Supplemental Figure 3). In mice, the accumulation of USEs loaded with IRDye-labeled miR-320a was 48% ± 28% and 41% ± 27% lower than NSE controls for liver and spleen, respectively (Figure 4) (P < 0.05; n = 3).

Figure 4.

Figure 4

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Bioavailability and distribution of USEs and NSEs loaded with IRDye-labeled miR-320a in C57BL/6J mice. (A) Fluorescence in excised gastrointestinal tract from mice 12 h after administration of USEs, loaded with IRDye-labeled miR-320a, by oral gavage and (B) fluorescence in tissues from mice 12 h after administration of USEs, loaded with IRDye-labeled miR-320a, by oral gavage. (C) Fluorescence in excised gastrointestinal tract from mice 12 h after administration of NSEs, loaded with IRDye-labeled miR-320a, by oral gavage and (D) fluorescence in tissues from mice 12 h after administration of NSEs, loaded with IRDye-labeled miR-320a, by oral gavage. (E–I) Densitometry analysis and comparison of (E) excised liver, (F) spleen, (G) heart, (H) brain, and (I) lungs 12 h after administration of USEs and NSEs, loaded with IRDye-labeled miR-320a, by oral gavage. Data are reported as mean ± SD (*P < 0.05 compared with NSE; n = 3). B, brain; IRDye, infrared dye; K, kidneys; L, liver; Lu, lungs; NSE, nonultrasonicated exosome; S, spleen; USE, ultrasonicated exosome.

Experiments 8 and 9: digestion of dietary lactose

All diets contained detectable amounts of lactose. The lyophilized milk powder used to prepare the ERD and ERS diets contained 90 ± 2.7 mg/kg and 87 ± 2.7 mg/kg lactose, respectively. The diets contained 105 g lyophilized powder per kilogram (5), i.e., the lactose content was 9.4 ± 0.3 mg/kg and 9.2 ± 0.3 mg/kg in the ERD and ERS diets, respectively. The casein used in the AIN-93G diet contained 7.5 ± 3.0 mg/kg lactose. The diet contained 200 g casein/kg (31), i.e., the lactose content was 1.5 ± 0.6 mg/kg.

Dietary lactose had no frank adverse effects in mice. The size of ceca was not significantly different in lactose-fed mice and PBS-fed controls, although the feces of lactose-fed mice appeared softer than in PBS controls (Supplemental Figure 4A, B, Supplemental Table 10). The expression of lactase mRNA and lactase activity were highest in the jejunum and lowest in the ileum (Supplemental Table 11) and lactase activity was not significantly different in mice at ages 3 and 12 wk (Supplemental Figure 5).

Discussion

This article is the first that we know of to provide a comprehensive characterization of ERD and ERS rodent diets, which have been used extensively in the identification of phenotypes caused by dietary depletion of milk exosomes and their cargos (24., 25., 26., 27., 28., 29.). These phenotypes suggest that milk exosomes meet the definition of bioactive food compounds by the National Cancer Institute, which is “a type of chemical found in small amounts in plants and certain foods […]. Bioactive compounds have actions in the body that may promote good health. They are being studied in the prevention of […] diseases” (53). A caveat of previous phenotyping studies is that the exact composition of ERD and ERS was not known, which has slowed progress toward establishing cause-and-effect relations. This article suggests that the amount of bioavailable milk exosomes is 85% lower in the ERD diet than in the ERS diet: 76% of the exosomes were lost during ultrasonication, and the uptake of the remaining exosomes was 40% less efficient for USEs than for NSEs in intestinal cell cultures. These estimates are consistent with our observation that USEs, loaded with IRDye-labeled miR-320a, produced a fluorescence signal in tissues that was 1 order of magnitude greater than the signal produced by NSEs loaded with IRDye-labeled miR-320a in major sites of miR-320a accumulation, i.e., liver and spleen. When considering that ultrasonication of milk caused an extensive degradation of microRNAs in addition to depleting bioavailable exosomes, it is plausible to suggest that the phenotypes caused by ERD feeding were caused by microRNA depletion. This conclusion is consistent with the ≤60% decrease in microRNA concentration in plasma, liver, skeletal muscle, intestinal mucosa, and placenta in mice fed the ERD diet compared with ERS controls (5, 24., 25., 26., 27.). We cannot exclude with 100% certainty the possibility that a decrease in BME binding to exosome receptors on recipient cells or the quantitatively modest changes in lipid or protein cargos might also have contributed to the phenotypes observed in ERD-fed mice. Finally, BMEs might have an indirect effect. For example, we demonstrated that bacterial communities in ceca were different in mice fed the ERD diet compared with the ERS control (28). However, this is an uncertainty inherent to most, if not all, feeding studies published to date.

So, what are the strengths of the ERD and ERS rodent diets? First, the diets are based on the AIN-93G formulation, which allows for optimal growth and development of mice (31) with a few limitations as discussed in a recent opinion article (54). Second, milk is the only dietary ingredient that is ultrasonicated when preparing the ERD diet; all other ingredients are chemically defined and identical in the ERD and ERS diets. Third, the amount of milk used is nutritionally relevant (see the Methods). Fourth, the diets can be prepared in nonspecialty laboratories; the only equipment needed is a water bath ultrasonicator. Fifth, if researchers were to pursue dose response studies, graded levels of ultrasonicated milk could be blended with control milk.

One could raise the following concerns regarding ERD and ERS diets. First, the milk used in the preparation of the diets is not chemically defined. We argue that this is no different from casein in the widely used AIN-93G diet. Second, the diets do not allow the establishment of cause-and-effect between the depletion of individual microRNAs and phenotypes of disease or poor development. We agree that the best possible use of the diets is in combination with genetics approaches such as the knockout of individual microRNAs in dams nursing wild-type mice. Third, it is unknown why the uptake of USEs is lower than that of NSEs in intestinal cells. We speculate that ultrasonication of milk caused an exchange of membrane domains containing lipid and protein features between BMEs and other milk components, e.g., fat globules or microvesicles, and the loss of these features impaired BME uptake by target cells. This theory is consistent with a previous report suggesting that homogenization of milk causes an exchange of membrane lipids among particulate materials in milk (55). Fourth, the lactose content in ERD and ERS diets is higher than in the AIN-93G diet. However, we did not observe frank symptoms of lactose intolerance in mice fed ERD and ERS diets and the expression and activity of lactase were not different between juvenile mice and adult mice.

In conclusion, this article lays the groundwork for assessing phenotypes of BME and RNA depletion in rodents and, if scaled up, in large animals. Previously, we referred to ERD and ERS diets as ExoMinus and ExoPlus diets, respectively (5). In that initial report, the content of miR-29b and miR-200c in ultrasonicated milk was used as a marker for successful manipulation of exosome cargos by ultrasonication. Now that the diets have been comprehensively characterized, we propose using the new names ERD and ERS when referring to the exosome- and RNA-defined diets.

Acknowledgments

We acknowledge the use of the Biomedical and Obesity Research Core in the Nebraska Center for the Prevention of Obesity Disease through Dietary Molecules (supported by NIH grant 1P20GM104320) and the Holland Computing Center in the University of Nebraska-Lincoln, as well as the DNA Sequencing Core in the University of Nebraska Medical Center (supported by NIH grants P20GM103427, 1P30GM110768, and P30CA036727). We thank You Zhou and Julia Russ in the Nebraska Center for Integrated Biomolecular Communication (supported by National Institute of General Medical Sciences grant P20 GM113126) in the University of Nebraska-Lincoln for their help with electron microscopy imaging. The authors’ responsibilities were as follows—SS, JA, JC, and JZ: contributed to the experimental design; SS, CPB, and TTA: performed the experiments; SS, CPB, TTA, JA, and JC: analyzed the data; SS: performed the statistical analysis and wrote the draft version of the manuscript; JA and JC: interpreted the data and performed manuscript revision; JZ: wrote the manuscript and took responsibility for the final content; and all authors: read and approved the final manuscript.

Conflict of interest

JZ is a consultant for PureTech, Inc. and a member of the journal's Editorial Board. All other authors report no conflicts of interest.

Footnotes

Supported by USDA National Institute of Food and Agriculture awards 2016-67001-25301 and 2020-67017-30834, NIH grant 1P20GM104320, the University of Nebraska Agricultural Research Division (Hatch Act; grant NEB-36-087), and USDA multistate group W4002 (all to JZ). The granting agencies had no influence on the study design; the collection, analysis, and interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication.

Author disclosures: JZ is a consultant for PureTech, Inc. and a member of the journal's Editorial Board. All other authors report no conflicts of interest.

Supplemental Figures 1–5 and Supplemental Tables 1–11 are available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/.

Supplemental Material

nxab452_Supplemental_File

Lipid signatures in USE and NSE. (A) Venn diagram of lipid signatures in USE and NSE. Percentages represent percent of lipid signatures in USE and NSE. (B) Principal component analysis of lipid signatures in USE and NSE. n = 3 independent samples; analyzed 3 times in both panels A and B. NSE, non-ultrasonicated exosome preparation; USE, ultrasonicated exosome preparation.

mmc1-sup1-supinfo.zip (3MB, zip)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

nxab452_Supplemental_File

Lipid signatures in USE and NSE. (A) Venn diagram of lipid signatures in USE and NSE. Percentages represent percent of lipid signatures in USE and NSE. (B) Principal component analysis of lipid signatures in USE and NSE. n = 3 independent samples; analyzed 3 times in both panels A and B. NSE, non-ultrasonicated exosome preparation; USE, ultrasonicated exosome preparation.

mmc1-sup1-supinfo.zip (3MB, zip)

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