Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro.

Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 µmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 µmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.

WGCNA and transcriptome profiling reveal hub genes for key development stage seed size/oil content between wild and cultivated soybean.

BACKGROUND: Soybean is one of the most important oil crops in the world. The domestication of wild soybean has resulted in significant changes in the seed oil content and seed size of cultivated soybeans. To better understand the molecular mechanisms of seed formation and oil content accumulation, WDD01514 (E1), ZYD00463 (E2), and two extreme progenies (E23 and E171) derived from RILs were used for weighted gene coexpression network analysis (WGCNA) combined with transcriptome analysis. RESULTS: In this study, both seed weight and oil content in E1 and E171 were significantly higher than those in E2 and E23, and 20 DAF and 30 DAF may be key stages of soybean seed oil content accumulation and weight increase. Pathways such as "Photosynthesis", "Carbon metabolism", and "Fatty acid metabolism", were involved in oil content accumulation and grain formation between wild and cultivated soybeans at 20 and 30 DAF according to RNA-seq analysis. A total of 121 oil content accumulation and 189 seed formation candidate genes were screened from differentially expressed genes. WGCNA identified six modules related to seed oil content and seed weight, and 76 candidate genes were screened from modules and network. Among them, 16 genes were used for qRT-PCR and tissue specific expression pattern analysis, and their expression-levels in 33-wild and 23-cultivated soybean varieties were subjected to correlation analysis; some key genes were verified as likely to be involved in oil content accumulation and grain formation. CONCLUSIONS: Overall, these results contribute to an understanding of seed lipid metabolism and seed size during seed development, and identify potential functional genes for improving soybean yield and seed oil quantity.

Size, number and phospholipid composition of milk fat globules are affected by dietary conjugated linoleic acid.

Milk fat globules (MFGs) surround the triacylglycerol core that composes milk fat. The aim of this study is to induce milk fat depression via dietary conjugated linoleic acid (CLA) supplementation to study MFG size parameters, number and glycerophospholipid composition. Eighteen Holstein dairy cows (136 ± 28 days in milk, 571 ± 37.9 kg body weight, 27.6 ± 2.1 kg milk/day) were selected and randomly assigned to a control or CLA group for a 14-day period. Cows were fed a basal diet (control, n = 8) or the control plus 400 g/day CLA (C18:2 cis-9, trans-11 38.1% and C18:2 trans-10, cis-12 36.8%) (n = 10) for 7 days after which the CLA group was switched to the basal diet for another 7 days along with the control group. Cow performance, milk composition, MFG size and numbers were measured daily. On the seventh day after the start of the experiment, milk samples were identified and the quantification of glycerophospholipid compounds, and RNA were isolated from milk fat samples for a real-time polymerase chain reaction. Compared with control, at Day 7 from the start of feeding, supplemental CLA did not affect milk production (28.09 vs. 28.50 kg/day), dry matter intake (14.9 vs. 15.4 kg/day), or milk protein (3.55/100 vs. 3.70 g/100 ml) and lactose contents (5.11/100 vs. 5.17 g/100 ml). However, although the specific surface area of MFG (2138 vs. 1815 m²/kg) was greater, CLA reduced milk fat content (1.95/100 vs 3.64 g/100 ml on Day 7) and particle size parameters of MFG. The number of MFG gradually decreased until Day 7 of feeding, and then increased by Day 14 (2.96 × 109 on Day 1, 1.63 × 109 on Day 7 and 2.28 × 109 on Day 14) in the CLA group. Compared with control, glycerophospholipid analysis revealed that concentrations of phosphatidylcholine (PC) (e.g., PC [16:0/18:1] 20322 vs. 29793 nmol/L), lysophosphatidylethanolamine (LPE) (e.g., LPE [18:1] 956 vs. 4610 nmol/L) and phosphatidylethanolamine (PE) (e.g., PE [16:0/18:1] 7000 vs. 9769 nmol/L) in milk lipids decreased during CLA feeding. In contrast, concentrations of phosphatidylinositol (PI) (e.g., PI [18:0/18:1] 4052 vs. 1799 nmol/L) and phosphatidylserine (PS) (e.g., PS [18:1/18:2] 9500 vs. 6843 nmol/L) increased. The messenger RNA abundance of fatty acid synthase, diacylglycerol O-acyltransferase 1, glycerol-3-phosphate acyltransferase 4 and phosphate cytidylyltransferase 1, choline, alpha (PCYT1A) were downregulated in the CLA group, confirming published data demonstrating a negative effect of CLA on lipogenesis in the mammary gland. Overall, these results provided evidence for the important role of lipogenic gene expression in the regulation of MFG size, number and glycerophospholipid composition.

Milk fat globule membrane proteins are involved in controlling the size of milk fat globules during conjugated linoleic acid-induced milk fat depression.

Milk fat globule membrane (MFGM) proteins surround the triacylglycerol core comprising milk fat globules (MFG). We previously detected a decrease in the size of fat globules during conjugated linoleic acid (CLA)-induced milk fat depression (MFD), and other studies have reported that some MFGM proteins play a central role in regulating mammary cellular lipid droplet size. However, little is known about the relationship between MFD, MFG size, and MFGM proteins in bovine milk. The aim of this study was to investigate the profile of MFGM proteins during MFD induced by CLA. Sixteen mid-lactating Holstein cows (145 ± 24 d in milk) with similar body condition and parity were divided into control and CLA groups over a 10-d period. Cows were fed a basal diet (control, n = 8) or control plus 15 g/kg of dry matter (DM) CLA (n = 8) to induce MFD. Cow performance, milk composition, and MFG size were measured daily. On d 10, MFGM proteins were extracted and identified by quantitative proteomic analysis, and western blotting was used to verify a subset of the identified MFGM proteins. Compared with controls, supplemental CLA did not affect milk production, DM intake, or milk protein and lactose contents. However, CLA reduced milk fat content (3.73 g/100 mL vs. 2.47 g/100 mL) and the size parameters volume-related diameter D[4,3] (3.72 µm vs. 3.35 µm) and surface area-related diameter D[3,2] (3.13 µm vs. 2.80 µm), but increased specific surface area of MFG (1,905 m2/kg vs. 2,188 m2/kg). In total, 177 differentially expressed proteins were detected in milk from cows with CLA-induced MFD, 60 of which were upregulated and 117 downregulated. Correlation analysis showed that MFG size was negatively correlated with various proteins, including XDH and FABP3, and positively correlated with MFG-E8, RAB19, and APOA1. The results provide evidence for an important role of MFGM proteins in regulating MFG diameter, and they facilitate a mechanistic understanding of diet-induced MFD.