Dietary intake of a MFGM/EV-rich concentrate promotes accretion of very long odd-chain sphingolipids and increases lipid metabolic turnover at the whole-body level.

Lipids from cow milk fat globule membranes (MFGMs) and extracellular vesicles (EVs) are considered beneficial for neurodevelopment, cognitive maintenance and human health in general. Nevertheless, it is largely unknown whether intake of infant formulas and medical nutrition products rich in these particles promote accretion of specific lipids and whether this affects metabolic homeostasis. To address this, we carried out a 16-week dietary intervention study where mice were supplemented with a MFGM/EV-rich concentrate, a control diet supplemented with a whey protein concentrate and devoid of milk lipids, or regular chow. Assessment of commonly used markers of metabolic health, including body weight, glucose intolerance and liver microanatomy, demonstrated no differences across the dietary regimes. In contrast, in-depth lipidomic analysis revealed accretion of milk-derived very long odd-chain sphingomyelins and ceramides in blood plasma and multiple tissues of mice fed the MFGM/EV diet. Furthermore, lipidomic flux analysis uncovered that mice fed the MFGM/EV diet have increased lipid metabolic turnover at the whole-body level. These findings help fill a long-lasting knowledge gap between the intake of MFGM/EV-containing foods and the health-promoting effects of their lipid constituents. In addition, the findings suggest that dietary sphingomyelins or ceramide-breakdown products with very long-chains can be used as structural components of cellular membranes, lipoprotein particles and signaling molecules that modulate metabolic homeostasis and health.

The Effect of Human and Bovine Milk Osteopontin on Intestinal Caco-2 Cells: A Transcriptome Comparison.

Osteopontin (OPN) is a multifunctional protein abundantly present in human milk, whereas the concentration is significantly lower in bovine milk. Human and bovine milk OPN are structurally similar and both proteins resist gastric digestion and reach the intestines in a bioactive form. Intervention studies have indicated the beneficial effects of supplementing infant formula with bovine milk OPN and several in vivo and in vitro studies have shown that bovine milk OPN positively influences intestinal development. To investigate the functional relationship, we compared the effect of simulated gastrointestinal digested human and bovine milk OPN on gene expression in Caco-2 cells. After incubation, total RNA was extracted and sequenced and transcripts were mapped to the human genome. Human and bovine milk OPN regulated the expression of 239 and 322 genes, respectively. A total of 131 genes were similarly regulated by the OPNs. As a control, a whey protein fraction with a high content of alpha-lactalbumin had a very limited transcriptional impact on the cells. Enrichment data analysis showed that biological processes related to the ubiquitin system, DNA binding, and genes associated with transcription and transcription control pathways were affected by the OPNs. Collectively, this study shows that human and bovine milk OPN have a significant and highly comparable effect on the intestinal transcriptome.

Naturally Occurring N-Terminal Fragments of Bovine Milk Osteopontin Are Transported across Models of the Intestinal Barrier.

Osteopontin (OPN) is a bioactive integrin-binding protein found in high concentrations in milk, where it is present both as a full-length protein and as several N-terminally derived fragments. OPN resists gastric digestion, and via interaction with receptors in the gut or by crossing the intestinal barrier into circulation, ingested milk OPN may influence physiological processes. The aim of this study was to investigate OPN interaction with intestinal cells and its transport across models of the intestinal barrier. Immunodetection of OPN incubated with Caco-2 cells at 4 °C and 37 °C showed that OPN binds to the intestinal cells, but it is not internalised. Transepithelial transport was studied using mono- and co-cultures of Caco-2 cells and mucus-producing HT29-MTX cells in transwell membranes. OPN was shown to cross the barrier models in a time-, temperature-, and energy-dependent process inhibited by wortmannin, indicating that the transport takes place via the transcytosis pathway. Analyses of the naturally occurring milk mixture of full-length and N-terminal fragments showed that the N-terminal fragments of OPN bound intestinal cells most effectively and that the fragments were transported across the intestinal membrane models. This suggests that proteolytic processing of OPN increases its biological activity after ingestion.

Lipidomic Characterization of Whey Concentrates Rich in Milk Fat Globule Membranes and Extracellular Vesicles.

Lipids from milk fat globule membranes (MFGMs) and extracellular vesicles (EVs) are considered beneficial for cognitive development and human health. Milk-derived whey concentrates rich in these lipids are therefore used as ingredients in infant formulas to mimic human milk and in medical nutrition products to improve the metabolic fitness of adults and elderly people. In spite of this, there is no consensus resource detailing the multitude of lipid molecules in whey concentrates. To bridge this knowledge gap, we report a comprehensive and quantitative lipidomic resource of different whey concentrates. In-depth lipidomic analysis of acid, sweet, and buttermilk whey concentrates identified 5714 lipid molecules belonging to 23 lipid classes. The data show that the buttermilk whey concentrate has the highest level of fat globule-derived triacylglycerols and that the acid and sweet whey concentrates have the highest proportions of MFGM- and EV-derived membrane lipids. Interestingly, the acid whey concentrate has a higher level of cholesterol whereas sweet whey concentrate has higher levels of lactosylceramides. Altogether, we report a detailed lipid molecular compendium of whey concentrates and lay the groundwork for using in-depth lipidomic technology to profile the nutritional value of milk products and functional foods containing dairy-based concentrates.

Dairy-Derived Emulsifiers in Infant Formula Show Marginal Effects on the Plasma Lipid Profile and Brain Structure in Preterm Piglets Relative to Soy Lecithin.

Breastfed infants have higher intestinal lipid absorption and neurodevelopmental outcomes compared to formula-fed infants, which may relate to a different surface layer structure of fat globules in infant formula. This study investigated if dairy-derived emulsifiers increased lipid absorption and neurodevelopment relative to soy lecithin in newborn preterm piglets. Piglets received a formula diet containing soy lecithin (SL) or whey protein concentrate enriched in extracellular vesicles (WPC-A-EV) or phospholipids (WPC-PL) for 19 days. Both WPC-A-EV and WPC-PL emulsions, but not the intact diets, increased in vitro lipolysis compared to SL. The main differences of plasma lipidomics analysis were increased levels of some sphingolipids, and lipid molecules with odd-chain (17:1, 19:1, 19:3) as well as mono- and polyunsaturated fatty acyl chains (16:1, 20:1, 20:3) in the WPC-A-EV and WPC-PL groups and increased 18:2 fatty acyls in the SL group. Indirect monitoring of intestinal triacylglycerol absorption showed no differences between groups. Diffusor tensor imaging measurements of mean diffusivity in the hippocampus were lower for WPC-A-EV and WPC-PL groups compared to SL indicating improved hippocampal maturation. No differences in hippocampal lipid composition or short-term memory were observed between groups. In conclusion, emulsification of fat globules in infant formula with dairy-derived emulsifiers altered the plasma lipid profile and hippocampal tissue diffusivity but had limited effects on other absorptive and learning abilities relative to SL in preterm piglets.

Effects of Prolonged Whey Protein Supplementation and Resistance Training on Biomarkers of Vitamin B12 Status: A 1-Year Randomized Intervention in Healthy Older Adults (the CALM Study).

We investigated the effect of long-term whey supplementation on biomarkers of B12 status in healthy older adults subjected to different schemes of supplements and exercise. The total study population examined at baseline consisted of 167 healthy older adults (age ≥ 65 year) who were randomized to 1-y intervention with two daily supplements of (1) whey protein (3.1 µg B12/day) (WHEY-ALL), (2) collagen (1.3 µg B12/day) (COLL), or (3) maltodextrin (0.3 µg B12/day) (CARB). WHEY-ALL was comprised of three groups, who performed heavy resistance training (HRTW), light resistance training (LITW), or no training (WHEY). Dietary intake was assessed through 3-d dietary records. For the longitudinal part of the study, we included only the participants (n = 110), who met the criteria of ≥ 50% compliance to the nutritional intervention and ≥ 66% and ≥ 75% compliance to the heavy and light training, respectively. Fasting blood samples collected at baseline and 12 months and non-fasting samples collected at 6 and 18 months were examined for methylmalonic acid, B12 and holotranscobalamin. At baseline, the study population (n = 167) had an overall adequate dietary B12 intake of median (range) 5.3 (0.7-65) µg/day and median B12 biomarker values within reference intervals. The whey intervention (WHEY-ALL) caused an increase in B12 (P < 0.0001) and holotranscobalamin (P < 0.0001). In addition, methylmalonic acid decreased in the LITW group (P = 0.04). No change in B12 biomarkers was observed during the intervention with collagen or carbohydrate, and the training schedules induced no changes. In conclusion, longer-term daily whey intake increased plasma B12 and holotranscobalamin in older individuals. No effect of intervention with collagen or carbohydrate or different training regimes was observed. Interestingly, the biomarkers of B12 status appeared to be affected by fasting vs. non-fasting conditions during sample collection.

Cyano-B12 or Whey Powder with Endogenous Hydroxo-B12 for Supplementation in B12 Deficient Lactovegetarians.

Lactovegetarians (n = 35) with low vitamin B12 (B12) status were intervened for eight weeks capsules containing cyano-B12 (CN-B12), (2 × 2.8 µg/day), or equivalent doses of endogenous B12 (mainly hydroxo-B12 (HO-B12)) in whey powder. Blood samples were examined at baseline, every second week during the intervention, and two weeks post-intervention. The groups did not differ at baseline in [global median (min/max)] plasma B12 [112(61/185)] pmol/L, holotranscobalamin [20(4/99)] pmol/L, folate [13(11/16)], the metabolites total homocysteine [18(9/52)] µmol/L and methylmalonic acid [0.90(0.28/2.5)] µmol/L, and the combined indicator of B12 status (4cB12) [-1.7(-3.0/-0.33)]. Both supplements caused significant effects, though none of the biomarkers returned to normal values. Total plasma B12 showed a higher increase in the capsule group compared to the whey powder group (p = 0.02). However, the increase of plasma holotranscobalamin (p = 0.06) and the lowering of the metabolites (p > 0.07) were alike in both groups. Thereby, the high total plasma B12 in the capsule group was not mirrored in enhanced B12 metabolism, possibly because the B12 surplus was mainly accumulated on an "inert" carrier haptocorrin, considered to be of marginal importance for tissue delivery of B12. In conclusion, we demonstrate that administration of whey powder (HO-B12) or capsules (CN-B12) equivalent to 5.6 µg of B12 daily for eight weeks similarly improves B12 status but does not normalize it. We document that the results for plasma B12 should be interpreted with caution following administration of CN-B12, since the change is disproportionately high compared to the responses of complementary biomarkers.

Comparative analysis of discrete exosome fractions obtained by differential centrifugation.

BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines.

Functional screening identifies miRNAs influencing apoptosis and proliferation in colorectal cancer.

MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.

Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase.

Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome-destabilizing experimental anticancer agent siramesine inhibits ASM by interfering with the binding of ASM to its essential lysosomal cofactor, bis(monoacylglycero)phosphate. Like siramesine, several clinically relevant ASM inhibitors trigger cancer-specific lysosomal cell death, reduce tumor growth in vivo, and revert multidrug resistance. Their cancer selectivity is associated with transformation-associated reduction in ASM expression and subsequent failure to maintain sphingomyelin hydrolysis during drug exposure. Taken together, these data identify ASM as an attractive target for cancer therapy.

MiRNA-362-3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan(®) Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR-362-3p, miR-570, miR-148 a* and miR-944) were expressed at a higher level in tumors from patients with no recurrence (p<0.015), compared with tumors from patients with recurrence. A significant association with increased disease free survival was confirmed for miR-362-3p in a second independent cohort of 43 CRC patients, using single TaqMan(®) microRNA assays. In vitro functional analysis showed that over-expression of miR-362-3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR-362-3p targets by mRNA profiling of HCT116 cells over-expressing miR-362-3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR-362-3p over-expression. We conclude that miR-362-3p may be a novel prognostic marker in CRC, and hypothesize that the positive effects of augmented miR-362-3p expression may in part be mediated through the targets E2F1, USF2 and PTPN1.

Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs.

BACKGROUND: microRNAs (miRNA) are short, endogenous transcripts that negatively regulate the expression of specific mRNA targets. miRNAs are found both in tissues and body fluids such as plasma. A major perspective for the use of miRNAs in the clinical setting is as diagnostic plasma markers for neoplasia. While miRNAs are abundant in tissues, they are often scarce in plasma. For quantification of miRNA in plasma it is therefore of importance to use a platform with high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the performance of three commonly used commercial miRNA quantification platforms: GeneChip miRNA 2.0 Array, miRCURY Ready-to-Use PCR, Human panel I+II V1.M, and TaqMan Human MicroRNA Array v3.0. RESULTS: Using synthetic miRNA samples and plasma RNA samples spiked with different ratios of 174 synthetic miRNAs we assessed the performance characteristics reproducibility, recovery, specificity, sensitivity and linearity. It was found that while the qRT-PCR based platforms were sufficiently sensitive to reproducibly detect miRNAs at the abundance levels found in human plasma, the array based platform was not. At high miRNA levels both qRT-PCR based platforms performed well in terms of specificity, reproducibility and recovery. At low miRNA levels, as in plasma, the miRCURY platform showed better sensitivity and linearity than the TaqMan platform. CONCLUSION: For profiling clinical samples with low miRNA abundance, such as plasma samples, the miRCURY platform with its better sensitivity and linearity would probably be superior.

Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer.

MicroRNAs (miRNA) are small noncoding RNAs commonly deregulated in cancer. The miR-200 family (miR-200a, -200b, -200c, -141 and -429) and miR-205 are frequently silenced in advanced cancer and have been implicated in epithelial to mesenchymal transition (EMT) and tumor invasion by targeting the transcriptional repressors of E-cadherin, ZEB1 and ZEB2. ZEB1 is also known to repress miR-200c-141 transcription in a negative feedback loop, but otherwise little is known about the transcriptional regulation of the miR-200 family and miR-205. Recently, miR-200 silencing was also reported in cancer stem cells, implying that miR-200 deregulation is a key event in multiple levels of tumor biology. However, what prevents miR-200 expression remains largely unanswered. Here we report concerted transcriptional regulation of the miR-200 and miR-205 loci in bladder tumors and bladder cell lines. Using a combination of miRNA expression arrays, qPCR assays and mass spectrometry DNA methylation analyses, we show that the miR-200 and miR-205 loci are specifically silenced and gain promoter hypermethylation and repressive chromatin marks in muscle invasive bladder tumors and undifferentiated bladder cell lines. Moreover, we report that miR-200c expression is significantly correlated with early stage T1 bladder tumor progression, and propose miR-200 and miR-205 silencing and DNA hypermethylation as possible prognostic markers in bladder cancer. In addition, we observe that the mesoderm transcription factor TWIST1 and miR-200 expression are inversely correlated in bladder tumor samples and cell lines. TWIST1 associates directly with the miR-200 and miR-205 promoters, and may act as a repressor of miR-200 and miR-205 expression.

Genomic profiling of microRNAs in bladder cancer: miR-129 is associated with poor outcome and promotes cell death in vitro.

microRNAs (miRNA) are involved in cancer development and progression, acting as tumor suppressors or oncogenes. Here, we profiled the expression of 290 unique human miRNAs in 11 normal and 106 bladder tumor samples using spotted locked nucleic acid-based oligonucleotide microarrays. We identified several differentially expressed miRNAs between normal urothelium and cancer and between the different disease stages. miR-145 was found to be the most down-regulated in cancer compared with normal, and miR-21 was the most up-regulated in cancer. Furthermore, we identified miRNAs that significantly correlated to the presence of concomitant carcinoma in situ. We identified several miRNAs with prognostic potential for predicting disease progression (e.g., miR-129, miR-133b, and miR-518c*). We localized the expression of miR-145, miR-21, and miR-129 to urothelium by in situ hybridization. We then focused on miR-129 that exerted significant growth inhibition and induced cell death upon transfection with a miR-129 precursor in bladder carcinoma cell lines T24 and SW780 cells. Microarray analysis of T24 cells after transfection showed significant miR-129 target down-regulation (P = 0.0002) and pathway analysis indicated that targets were involved in cell death processes. By analyzing gene expression data from clinical tumor samples, we identified significant expression changes of target mRNA molecules related to the miRNA expression. Using luciferase assays, we documented a direct link between miR-129 and the two putative targets GALNT1 and SOX4. The findings reported here indicate that several miRNAs are differentially regulated in bladder cancer and may form a basis for clinical development of new biomarkers for bladder cancer.

Diagnostic and prognostic microRNAs in stage II colon cancer.

MicroRNAs (miRNA) are a class of small noncoding RNAs with important posttranscriptional regulatory functions. Recent data suggest that miRNAs are aberrantly expressed in many human cancers and that they may play significant roles in carcinogenesis. Here, we used microarrays to profile the expression of 315 human miRNAs in 10 normal mucosa samples and 49 stage II colon cancers differing with regard to microsatellite status and recurrence of disease. Several miRNAs were differentially expressed between normal tissue and tumor microsatellite subtypes, with miR-145 showing the lowest expression in cancer relative to normal tissue. Microsatellite status for the majority of cancers could be correctly predicted based on miRNA expression profiles. Furthermore, a biomarker based on miRNA expression profiles could predict recurrence of disease with an overall performance accuracy of 81%, indicating a potential role of miRNAs in determining tumor aggressiveness. The expression levels of miR-320 and miR-498, both included in the predictive biomarker, correlated with the probability of recurrence-free survival by multivariate analysis. We successfully verified the expression of selected miRNAs using real-time reverse transcription-PCR assays for mature miRNAs, whereas in situ hybridization was used to detect the accumulation of miR-145 and miR-320 in normal epithelial cells and adenocarcinoma cells. Functional studies showed that miR-145 potently suppressed growth of three different colon carcinoma cell lines. In conclusion, our results suggest that perturbed expression of numerous miRNAs in colon cancer may have a functional effect on tumor cell behavior, and, furthermore, that some miRNAs with prognostic potential could be of clinical importance.