We have evaluated the effects of three different omega-3 polyunsaturated fatty acids (ω-3 PUFAs) – docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) on fat accumulation and expression of adipogenic and inflammatory markers using both 3T3-L1 pre-adipocytes and differentiated 3T3-L1 adipocytes. Our results indicate that ω-3 PUFAs induce the degradation of fatty acid synthase through the ubiquitin-proteasome system, which is likely to have beneficial metabolic effect on adipose cells. Omega-3 PUFAs also increase overall levels of polyubiquitinated proteins, at least in part through decreasing the expression of proteasome subunits. Moreover, adipocytes are resistant to proteasome inhibition, which induces adipophilin while decreasing perilipin expression. On the other hand, ω-3 PUFAs decrease expression of SREBP1 while inducing expression of adipophilin and GLUT4. Moreover, all three ω-3 PUFAs appear to induce tumour necrosis factor-α without affecting NFκB levels. All three ω-3 PUFAs appear to have overall similar effects. Further research is needed to elucidate their mechanism of action.

Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development.

We applied quantitative bovine milk proteomic data as probes for the developing milk and mammary‐gland function (n = 5 cows with samples of colostrum at day 1 and milk at 1, 2, 3 and 6 months). 1913 protein were identified where 894 were quantified. The majority of proteins express constitutively during mature milk production. Main changes were observed between day 1 and 1 month with 66 and 139 upregulated (FDR‐adjusted p‐value < 0.05) in colostrum and milk, respectively. Proteolytically cleaved intercellular adhesion molecule (ICAM‐1) is upregulated in milk, suggesting that shedding of ICAM‐1 provide a means to suppress leukocyte recruitment into milk as lactation progresses. Shedding of E‐cadherin and catenin (adhesion complex) from mammary epithelium decreases in later milk, which suggests an increasing adherins junction barrier. Housekeeping proteins from blood serum are present at higher abundance in colostrum, which is consistent with the decreased mammary epithelial barrier during colostrum production. Abundance of enzymes in N‐glycoprotein biosynthesis decreases whereas abundance of enzymes in N‐glycoprotein degradation increases from colostrum to milk, indicating decreased N‐glycoprotein biosynthesis over lactation. Lysosomal enzymes are upregulated in milk (fold change > 3), implying higher lysosomal hydrolysis function during later milk production.

Improved understanding of temporal and geographical differences in the human milk proteome may provide novel insights into changes in milk composition and function across the globe during breastfeeding. We have compared milk proteomes among three populations (Shanghai, China, Mexico City, Mexico and Cincinnati, United States; n = 9 for each population) at two different stages of lactation (4 and 26 weeks), with all samples collected as part of the Global Exploration of Human Milk (GEHM) study. At each of the two lactation stages, the profiles of milk proteins are largely similar among the three populations. Differences were found to be greater with respect to lactation stage as opposed to population. Interestingly, proteins with significant abundance changes over time show patterns of regulation that are broadly similar among populations. Nevertheless, at each of the two lactation stages, immunoglobulins IgA2, IgG1, IgG2 and IgM are present at higher levels in milk from China and Mexico compared to those from the U.S. with fold changes between China and U.S. samples of 2.0, 1.6, 2.3 and 2.1, respectively, and fold changes between Mexico and U.S. samples of1.8, 1.3, 1.1 and 2.0, respectively. Similar observations can be made for the antimicrobial protein lactoferrin. Our findings suggest broad congruency in milk protein composition among the three populations. This study also suggests that a number of host‐defense proteins may be more abundant in human milk from developing countries. This research was supported by Mead Johnson Pediatric Nutrition Institute.