Adipocytes are now recognized as endocrine cells secreting adipocytokines, regulating multiple metabolic pathways. In this study, we addressed secretion of microvesicles by 3T3-L1 adipocytes. We found that MFG-E8, one of the exosomal proteins, was present in the microvesicles and was distributed in the sucrose density fractions with 1.13-1.20 g/ml, which has been reported for exosomes. Several integral, cytosolic, and nuclear proteins such as caveolin-1, c-Src kinase, and heat shock protein 70 were also found to be microvesicle components. Unexpectedly, adiponectin was also substantially distributed in the microvesicle fractions. Furthermore, proteomic analysis of the microvesicles revealed that many other proteins such as extracellular matrix-related proteins were also present. Microvesicles secreted by 3T3-L1 adipocytes exhibited heterogeneity in size and comprised both smaller exosome-like and larger membrane vesicles as revealed by electron microscopy. Milk fat globule-epidermal growth factor 8 (MFG-E8)-associated adiposomes exhibited binding activity toward phosphatidylserine and apoptotic cells. MFG-E8 in the microvesicles was reduced when cultured in the low-glucose medium or cultured in the high-glucose medium with antioxidant N-acetyl cysteine. Insulin and TNF-alpha also up-regulated MFG-E8 in the microvesicles. Moreover, MFG-E8 was strongly up-regulated in the hypertrophic adipose tissue, predominantly in adipocyte fractions, of diet-induced obese C57BL/6 mice, where increased oxidative stress is induced. Thus, it is suggested that microvesicles, especially MFG-E8-associated ones, modulate adipose functions under redox- and hormone-dependent regulation. Based on the above findings, the adipocyte-derived microvesicles were named adiposomes.

A milk membrane glycoprotein, MFG-E8 [milk fat globule-EGF (epidermal growth factor) factor 8], is expressed abundantly in lactating mammary glands in stage- and tissue-specific manners, and has been believed to be secreted in association with milk fat globules. In the present paper, we describe further up-regulation of MFG-E8 in involuting mammary glands, where the glands undergo a substantial increase in the rate of epithelial cell apoptosis, and a possible role of MFG-E8 in mediating recognition and engulfment of apoptotic cells through its specific binding to PS (phosphatidylserine). Immunoblotting and RNA blotting analyses revealed that both MFG-E8 protein and MFG-E8 mRNA were markedly increased in mammary tissue within 3 days of either natural or forced weaning (pup withdrawal) of lactating mice. Using immunohistochemical analysis of the mammary tissue cryosections, the MFG-E8 signal was detected around the epithelium of such involuting mammary glands, but was almost undetectable at early- and mid-lactation stages, although strong signals were obtained for milk fat globules stored in the alveolar lumen. Some signals double positive to a macrophage differentiation marker, CD68, and MFG-E8 were detected in the post-weaning mammary tissue, although such double-positive signals were much smaller in number than the MFG-E8 single-positive ones. Total MFG-E8 in milk was also increased in the post-weaning mammary glands and, furthermore, the free MFG-E8 content in the post-weaning milk, as measured by in vitro PS-binding and apoptotic HC11 cell-binding activities, was much higher than that of lactation. In addition, the post-weaning milk enhanced the binding of apoptotic HC11 cells to J774 macrophages. Sucrose density-gradient ultracentrifugation analyses revealed that such enhanced PS-binding activity of MFG-E8 was present in membrane vesicle fractions (density 1.05-1.13 g/ml), rather than milk fat globule fractions. The weaning-induced MFG-E8 might play an important role in the recognition and engulfment of apoptotic epithelial cells by the neighbouring phagocytic epithelial cells in involuting mammary glands.

Fructooligosaccharides (FOS) can selectively stimulate the growth of bifidobacteria. Here, we investigated the effect of maternal FOS ingestion on maternal and neonatal gut bifidobacteria. In a randomized, double-blind, placebo-controlled study, we administered 8 g/day of FOS or sucrose to 84 women from the 26th week of gestation to one month after delivery. The bifidobacteria count was detected using quantitative PCR in maternal (26 and 36 weeks of gestation) and neonatal (one month after delivery) stools. Maternal stool frequency was recorded from 24 to 36 weeks of gestation. The number of fecal Bifidobacterium spp. and Bifidobacterium longum in the FOS group was significantly higher than that in the placebo group at 36 weeks of gestation (2.7 × 1010/g vs. 1.1 × 1010/g and 2.3 × 1010/g vs. 9.7 × 109/g). In their neonates, these numbers did not differ between the groups. Also, stool frequency in the FOS group was slightly higher than that in the placebo group two weeks after the intervention (1.0 vs. 0.8 times/day), suggesting a potential constipation alleviation effect. In conclusion, the maternal FOS ingestion showed a bifidogenic effect in pregnant women but not in their neonates.

To examine the effect of dietary supplementation with 1-kestose on the IgA levels in milk, BALB/c mice were fed diets with or without 5% 1-kestose during pregnancy and lactation. The total and specific IgA levels in the milk were measured at 7 and 14 days after delivery. A two-way ANOVA with repeated measures resulted in a significant effect of 1-kestose-supplementation on total IgA concentrations (p < 0.05) and the level of anti-Bacteroides IgA (p < 0.05). A significant positive correlation was found between the mean count of Bacteroides spp. in maternal feces and the total IgA concentration in maternal milk (r = 0.55, p < 0.05), suggesting a potential link between the gut and mammary gland immune system. In conclusion, this study demonstrated the effects of dietary prebiotics on milk IgA production.

Bifidobacterium bifidum OLB6378 (OLB6378) was selected as a strain that enhances the production of secretory immunoglobulin A (IgA) in vitro. This ability of non-live OLB6378 has been shown by a clinical trial in preterm infants. In the present study, we examined whether non-live OLB6378 also enhances the production of secretory IgA, even in full-term infants. One hundred full-term infants were allocated to receive formula with (BbF group, 49 infants) or without non-live OLB6378 (PF group, 51 infants). Breastfeeding was prioritised, so infant formula was used for infants with breastfeeding difficulties. The intervention was initiated by five days of age. The faecal IgA concentration and OLB6378 level were determined at one, two, four, and eight weeks of age.Faecal IgA in the BbF group (1.04 ± 0.47 mg/g of faeces, n = 45) was significantly higher than that in the PF group (0.85 ± 0.42 mg/g of faeces, n = 49) at four weeks of age (p = 0.047). OLB6378 was not detected in faeces at any age. This indicated that production of secretory IgA in full-term infants may also be enhanced by non-live OLB6378 intake. IntroductionNon-live Bifidobacterium is a safer and more convenient food material than live Bifidobacterium. Moreover, non-live Bifidobacterium cells stimulate the gut immune system in vitro and in vivo [1,2]. However, those effects have not been studied sufficiently in a clinical setting, especially in infants. We selected Bifidobacterium bifidum OLB6378 (OLB6378), which enhances the production of secretory immunoglobulin A (IgA) [3], and confirmed the effect of non-live OLB6378 on enhancement of faecal IgA in low-birth-weight infants [4]. From the standpoint of trying to extend the versatility of non-live OLB6378, we hypothesised that non-live OLB6378 enhances the production of secretory IgA, even in full-term infants.However, the effect observed in low-birth-weight infants may not be observed in full-term infants. Preterm infants have lower body weights, heights, head circumferences, muscle and fat cross-sectional areas, and bone mineral densities than term-born infants [5]. With regard to nutrient digestion and absorption, premature infants do not have levels of intestinal lactase activity and gastric protein digestion capacities comparable to those of mature infants [6][7][8]. They also have immature immune systems and higher levels of intestinal permeability than term infants [9, 10]. Moreover, low birth weight is negatively associated with exclusive breastfeeding [11]. Given these differences between preterm and full-term infants, we considered that the effects of non-live OLB6378 should be demonstrated again in full-term infants.As a pilot study, we evaluated the effects of non-live OLB6378 administration on the enhancement of faecal IgA in full-term infants in this study. Materials and Methods Study Design and Ethical ApprovalThis was a double-blind, randomised, placebo-controlled study conducted among 100 newborn infants and their mothers. The protocol was approved by the Ethics Committee of Chiba Univers...

We evaluated the protein and energy intakes of infants fed commercial infant Formula A (protein, 2.2 g/100 kcal; energy, 68 kcal/100 mL) and examined whether changes in feeding intervals are involved in constant energy intake. Daily nutritional intake of 378 Formula A-fed infants was assessed using reference values and compared to that of infants fed Formulas B (protein: 2.3 g/100 kcal, energy: 68 kcal/100 mL) and C (protein: 2.4 g/100kcal, energy: 70 kcal/100 mL). From 15 to 149 days of age, the mean formula volume and protein intake were 758–887 mL/day and 11.4–13.3 g/day, respectively, higher than the protein intake of breast-fed infants. Daily energy intake (86–129 kcal/kg/day) was comparable to the estimated energy requirements. Feeding intervals were shorter in infants fed Formulas A and B than in those fed Formula C, whereas energy intake was similar. The protein intake of infants decreased as the protein concentration per energy in infant formula was reduced, and accordingly the protein intake of Formula A-fed infants was significantly lower than that of Formula C-fed infants. In conclusion, the new composition of Formula A is suitable in protein and energy intake of infants, and daily energy intake remains constant by shortening in feeding intervals when the energy concentration in infant formula is reduced. Clinical Trial Registration: UMIN000023110

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To evaluate the suitability of the new nutritional composition of renewed commercial Formula A (protein reduced to 2.2 g/100 kcal, arachidonic acid increased to 13.2 mg/100 kcal, and docosahexaenoic acid maintained at 20 mg/100 kcal), we examined whether the growth of Formula A-fed infants was equivalent to that of breastfed infants. In this observational study, 1,053 infants were followed-up to 12 months. Growth, stool consistency, and the health condition of 99 infants fed with Formula A and 295 breastfed infants were compared. Body weight, body mass index, and head circumference of Formula A-fed infants were similar to those of breastfed infants. Additionally, there were no differences in the stool consistency and the health condition (infection and allergy prevalence) between the two groups. Formula A-fed infants grew as well as breastfed infants, suggesting the appropriate nutritional composition of Formula A. The findings may contribute to further improvements in infant formulas.