Caveolae-mediated endocytosis of VLDL-sized emulsion particles in macrophages requires NPC1 and STARD3 for further lysosomal processing

Triglyceride-rich lipoproteins and their remnants contribute to atherosclerosis, possibly by carrying remnant cholesterol and/or by exerting a pro-inflammatory effect on macrophages. Nevertheless, little is known about how macrophages process triglyceride-rich lipoproteins. We show that uptake by macrophages of VLDL-sized emulsion particles is dependent on the enzyme lipoprotein lipase via its C-terminal domain. Subsequent internalization of VLDL-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by hydrolysis by lysosomal acid lipase. STARD3 is required for the transfer of lysosomal fatty acids to the ER for lipid storage, while NPC1 likely is involved in promoting the extracellular efflux of fatty acids. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete internalized triglycerides as fatty acids. Overall design: Mouse RAW 246.7 macrophages were exposed to 1 mM artifical VLDL- (VLDLsep) and chylomicron-sized emulsion particles (CHYLsep) for 6 hrs, whereafter gene expression was profiled by RNA-sequencing

Caveolae-mediated endocytosis of VLDL-sized emulsion particles in macrophages requires NPC1 and STARD3 for further lysosomal processing

Triglyceride-rich lipoproteins and their remnants contribute to atherosclerosis, possibly by carrying remnant cholesterol and/or by exerting a pro-inflammatory effect on macrophages. Nevertheless, little is known about how macrophages process triglyceride-rich lipoproteins. We show that uptake by macrophages of VLDL-sized emulsion particles is dependent on the enzyme lipoprotein lipase via its C-terminal domain. Subsequent internalization of VLDL-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by hydrolysis by lysosomal acid lipase. STARD3 is required for the transfer of lysosomal fatty acids to the ER for lipid storage, while NPC1 likely is involved in promoting the extracellular efflux of fatty acids. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete internalized triglycerides as fatty acids. Overall design: Mouse RAW 246.7 macrophages were exposed to 1 mM artifical VLDL- (VLDLsep) and chylomicron-sized emulsion particles (CHYLsep) for 6 hrs, whereafter gene expression was profiled by RNA-sequencing

Comparison of bovine milk fat and vegetable fat for infant formula : Implications for infant health

Fat is an important component of human milk and infant formula (IF), delivering half of the energy a baby needs. Nowadays, mostly vegetable fats are used in IFs; however, the use of bovine milk fat in formulas is currently increasing. Bovine milk fat contains a composition of fatty acids and lipid components different from those of vegetable fats. We have compared the lipid profile of human and bovine milk with infant formulas with different fat sources. Furthermore, current knowledge of how infant digestion, absorption, metabolic responses, gut immunity, microbiota and/or cognition is affected by dietary fat is reviewed. The possible opportunities and drawbacks of the application of bovine milk fat in infant nutrition are described. Future perspectives for the development of IF containing bovine milk fat and future research directions are highlighted.</p

Macrophages take up VLDL-sized emulsion particles through caveolae-mediated endocytosis and excrete part of the internalized triglycerides as fatty acids

Triglycerides are carried in the bloodstream as part of very low-density lipoproteins (VLDLs) and chylomicrons, which represent the triglyceride-rich lipoproteins. Triglyceride-rich lipoproteins and their remnants contribute to atherosclerosis, possibly by carrying remnant cholesterol and/or by exerting a proinflammatory effect on macrophages. Nevertheless, little is known about how macrophages process triglyceride-rich lipoproteins. Here, using VLDL-sized triglyceride-rich emulsion particles, we aimed to study the mechanism by which VLDL triglycerides are taken up, processed, and stored in macrophages. Our results show that macrophage uptake of VLDL-sized emulsion particles is dependent on lipoprotein lipase (LPL) and requires the lipoprotein-binding C-terminal domain but not the catalytic N-terminal domain of LPL. Subsequent internalization of VLDL-sized emulsion particles by macrophages is carried out by caveolae-mediated endocytosis, followed by triglyceride hydrolysis catalyzed by lysosomal acid lipase. It is shown that STARD3 is required for the transfer of lysosomal fatty acids to the ER for subsequent storage as triglycerides, while NPC1 likely is involved in promoting the extracellular efflux of fatty acids from lysosomes. Our data provide novel insights into how macrophages process VLDL triglycerides and suggest that macrophages have the remarkable capacity to excrete part of the internalized triglycerides as fatty acids

Corrigendum to: “Comparison of bovine milk fat and vegetable fat for infant formula: Implications for infant health”

The authors regret that the cholesterol levels in current infant formulas quoted from Claumarchirant et al. (2015) on page 41, and additional calculations, were reported incorrectly. The average concentration of cholesterol in infant formulas based on vegetable fats should be 17.3 mg L−1, ranging from 14.6 to 22.2 mg L−1 (IFs 2, 3, 4, 7, 8, 11, 12, and 13 from Table 2 of Claumarchirant et al., 2015). The average concentration of cholesterol in infant formulas containing a fat blend of vegetable fats and bovine milk lipids should be 36.9 mg L−1, ranging from 20.3 to 51.0 mg L−1 (IFs 1, 5, 6, 9, and 10 from Table 2 of Claumarchirant et al., 2015). This has no consequences for the conclusions of the manuscript. Page 41 should read: “A recent study investigating sterol content of IFs showed that those based on vegetable fats contained, on average, 17.3 mg L−1 of cholesterol (Claumarchirant, Matencio, Sanchez-Siles, Alegría, & Lagarda, 2015). In line with the findings on phospholipids, the cholesterol present in IFs based on vegetable fats also mostly originates from the small amount of milk fat present in skimmed milk (Berger et al., 2000). Newer types of IF, containing a blend of vegetable fats and bovine milk fat, contain higher levels of cholesterol, on average 36.9 mg L−1 (Claumarchirant et al., 2015). Calculations based on literature values (NEVO online; RIVM, 2016) indicate that by replacing every 10% of vegetable fat by bovine milk fat in a fat blend for infant formula, about 10 mg L−1 of cholesterol could be added. The authors would like to apologise for any inconvenience caused.</p

Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles

BACKGROUND: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients' cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. OBJECTIVES: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. METHODS: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. RESULTS: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108-2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4-23.3 ± 10.0 mg/μL in processed milk, P < 0.05). CONCLUSIONS: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk

Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles

BACKGROUND: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients' cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. OBJECTIVES: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. METHODS: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. RESULTS: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108-2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4-23.3 ± 10.0 mg/μL in processed milk, P < 0.05). CONCLUSIONS: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk

Free fatty acid release from vegetable and bovine milk fat-based infant formulas and human milk during two-phase in vitro digestion

Background: Bovine milk fat is increasingly used in infant formula (IF). The triacylglycerol (TAG) structure of bovine milk fat might be beneficial for digestion and absorption. We investigated the release of fatty acids (FAs) of IF containing different fat blends and compared this to human milk. Methods: Fresh human milk was sampled and two IFs were produced; one containing 100% vegetable fat (IF1) and one with 67% bovine milk fat and 33% vegetable fat (IF2). Using a static in vitro infant digestion model, consisting of a gastric and duodenal phase, the time dependent release of individual free fatty acids (FFA) was studied and analysed using GC-MS, and residual TAG levels were determined by GC-FID. Results: Human milk and the IFs showed comparable total FA release. In the gastric phase, 4-11% of lipolysis occurred, and mainly short (SCFA)- and medium chain fatty acids (MCFA) were released. In the duodenal phase, lipolysis proceeded with release of C4:0 but was marked by a fast release of long-chain fatty acids (LCFA). The digestion of the IFs resulted in different FFA profiles during and at the end of digestion. IF2 gave more release of C4:0-C11:0, which reflects the FA composition of bovine milk. Conclusion: The addition of bovine milk fat to IF resulted in a total FA release comparable to an IF with only vegetable fat and human milk. However, it did lead to a different time-dependent release of individual FAs, which might result in differences in absorption and other health effects in vivo.</p

Correction: Free fatty acid release from vegetable and bovine milk fat-based infant formulas and human milk during two-phase: In vitro digestion

The authors regret that the lipid composition of IF1 was reported incorrectly. The percentage of C18:1n-9 should be 42.3%. Since the incorrect value was also used for some calculations, this also affects some of the results: it increases the total amount of fatty acids in the sample, and consequently the percentage of released FFA is lower. The FFA release (as a percentage of initial composition), both of total FFA and C18:1, is similar for both IFs. One small difference between IF1 and IF2 that was seen when using the incorrect value, i.e. a faster early duodenal digestion for IF1, was found to be no longer statistically significant. This has no consequences for the conclusions of the manuscript. Page 2106 should read " The human milk samples showed less release of FFA during the gastric phase compared to IF1 and IF2 (2.0 ± 0.2% vs. 4.3 ± 0.2% and 4.7 ± 0.1% respectively, p < 0.01). Compared to the amount of FFA released after the digestion, during the gastric phase 4% of FFA were released from human milk, about 10% from IF1, and about 11% from IF2. Except for 45 minutes (p = 0.04), i.e. 15 minutes after the start of the duodenal phase, no differences were found in FFA release between the IFs compared to the human milk samples during this phase. The total release of FAs at the end of the digestion, as percentage of initial composition, was found to be similar for the different samples (43.9 ± 2.0%, 42.2 ± 1.4%, and 52.3 ± 4.5% for IF1, IF2 and human milk respectively, p = 0.14)". The correspondingly updated Fig. 2B, Fig. 5K, Table 2 and Table 3 are as presented below.(Table Persented).</p

Bovine Milk-Derived Extracellular Vesicles Inhibit Catabolic and Inflammatory Processes in Cartilage from Osteoarthritis Patients

Scope: Data from the Osteoarthritis Initiative shows that females who drink milk regularly have less joint cartilage loss and OA progression, but the biologic mechanism is unclear. Bovine milk is a rich source of extracellular vesicles (EVs), which are small phospholipid bilayer bound structures that facilitate intercellular communication. In this study, the authors aim to evaluate whether these EVs may have the capacity to protect cartilage from osteoarthritis patients, ex vivo, by directly effecting chondrocytes. Methods and Results: Human cartilage explants are exposed to cow's milk-derived EVs (CMEVs), which results in reduced sulfated glycosaminoglycan release and inhibition of metalloproteinase-1 expression. Incubation of articular chondrocytes with CMEVs also effectively reduces expression of cartilage destructive enzymes (ADAMTS5, MMPs), which play key roles in the disease progression. In part, these findings are attributed to the presence of TGFβ on these vesicles, and in addition, a possible role is reserved for miR-148a, which is functionally transferred by CMEVs. Conclusion: These findings highlight the therapeutic potential of local CMEV delivery in osteoarthritic joints, where inflammatory and catabolic mediators are responsible for joint pathology. CMEVs are carriers of both TGFβ and miR-148a, two essential regulators for maintaining chondrocyte homeostasis and protection against cartilage destruction