The objective of the present study was to describe plasma hormonal and metabolite profile and mRNA expression levels and activities of the enzymes pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and acetyl-coenzyme A (CoA) carboxylase in the liver of male Holstein calves before (1 and 3 wk of age) and after (8, 13, and 19 wk of age) weaning at 6 wk of age. The mean plasma concentration of acetate and beta-hydroxybutyrate increased, and that of plasma lactate and nonesterified fatty acids decreased with week, particularly after weaning. Plasma glucose concentration was lowest at 8 wk of age. The mean plasma concentration of insulin and glucagon did not change with time, and that of cortisol was greatest at 1 wk of age. In the liver, enzyme activity of PC was greatest at 1 wk of age and decreased with time. There was a significant relationship between the activity and the mRNA level for PC. Activity of PEPCK also decreased with week. Acetyl-CoA carboxylase activity tended to decrease with week, and activity at 13 wk of age was lower than that at other times. Expression of PC mRNA, but not that of PEPCK and acetyl-CoA carboxylase alpha, decreased with week. We conclude that the hepatic gluconeogenic enzymes and acetyl-CoA carboxylase activities tend to decrease with age, reflecting changes in plasma metabolites in early weaning production systems.

Levels of alpha-tocopherol (α-Toc) decline gradually in blood throughout prepartum, reaching lowest levels (hypovitaminosis E) around calving. Despite numerous reports about the disease risk in hypovitaminosis E and the effect of α-Toc supplementation on the health of transition dairy cows, its risk and supplemental effects are controversial. Here, we present some novel data about the disease risk of hypovitaminosis E and the effects of α-Toc supplementation in transition dairy cows. These data strongly demonstrate that hypovitaminosis E is a risk factor for the occurrence of peripartum disease. Furthermore, a study on the effectiveness of using serum vitamin levels as biomarkers to predict disease in dairy cows was reported, and a rapid field test for measuring vitamin levels was developed. By contrast, evidence for how hypovitaminosis E occurred during the transition period was scarce until the 2010s. Pioneering studies conducted with humans and rodents have identified and characterised some α-Toc-related proteins, molecular players involved in α-Toc regulation followed by a study in ruminants from the 2010s. Based on recent literature, the six physiological factors: (1) the decline in α-Toc intake from the close-up period; (2) changes in the digestive and absorptive functions of α-Toc; (3) the decline in plasma high-density lipoprotein as an α-Toc carrier; (4) increasing oxidative stress and consumption of α-Toc; (5) decreasing hepatic α-Toc transfer to circulation; and (6) increasing mammary α-Toc transfer from blood to colostrum, may be involved in α-Toc deficiency during the transition period. However, the mechanisms and pathways are poorly understood, and further studies are needed to understand the physiological role of α-Toc-related molecules in cattle. Understanding the molecular mechanisms underlying hypovitaminosis E will contribute to the prevention of peripartum disease and high performance in dairy cows.

Objective: Endoplasmic reticulum (ER) stress engages the unfolded protein response (UPR) that serves as an important mechanism for modulating hepatic fatty acid oxidation and lipogenesis. Chronic fasting in mice induced the UPR activation to regulate lipid metabolism. However, there is no direct evidence of whether negative energy balance (NEB) induces ER stress in the liver of cows. This study aimed to elucidate the relationship between the NEB attributed to feed deprivation and ER stress in bovine hepatocytes.Methods: Blood samples and liver biopsy tissues were collected from 6 non-lactating cows before and after their starvation for 48 h. The blood non-esterified fatty acids (NEFA), β-hydroxybutyric acid (BHBA) and glucose level were analyzed. Real-time quantitative polymerase chain reaction and Western blotting were used to explore the regulation of genes associated with UPR and lipid metabolism.Results: The starvation increased the plasma BHBA and NEFA levels and decreased the glucose level. Additionally, the starvation caused significant increases in the mRNA expression level of spliced X-box binding protein 1 (XBP1s) and the protein level of phosphorylated inositol-requiring kinase 1 alpha (p-IRE1α; an upstream protein of XBP1) in the liver. The mRNA expression levels of peroxisome proliferator-activated receptor alpha and its target fatty acid oxidation- and ketogenesis-related genes were significantly upregulated by the starvation-mediated NEB. Furthermore, we found that the mRNA expression levels of lipogenic genes were not significantly changed after starvation.Conclusion: These findings suggest that in the initial stage of NEB in dairy cows, the liver coordinates an adaptive response by activating the IRE1 arm of the UPR to enhance ketogenesis, thereby avoiding a fatty liver status.

Blood alpha a-tocopherol (alpha-Toc) concentrations decline gradually throughout the prepartum period, reaching the nadir after calving in dairy cows. The 6 alpha-Toc-related molecules [alpha-Toc transfer protein (TTPA); afamin; scavenger receptor class B, Type I; ATP-binding cassette transporter Al; tocopherol-associated protein (SEC14L2); and cytochrome P450 family 4, subfamily F, polypeptide 2 (CYP4F2)] are expressed in liver and other peripheral tissues. These molecules could regulate alpha-Toc transport, blood concentrations, and metabolism of alpha-Toc. Therefore, the aim of this study was to evaluate the changes in the expression of alpha-Toc related genes in liver and mammary gland tissues of dairy cows around calving, which have remained elusive until now. In experiment (Exp.) 1, 28 multiparous Holstein cows were used (from -5 to 6 wk relative to parturition) to monitor the changes in dietary alpha-Toc intake, blood concentrations of alpha-Toc, and lipoproteins; in Exp. 2, 7 peripartum Holstein cows were used (from -4 to 4 wk relative to parturition) for liver tissue biopsy; and in Exp. 3, 10 peripartum Holstein cows were used (from -8 to 6 wk relative to parturition) to carry out the mammary gland tissue biopsy and milk sampling. In Exp. 1, the serum alpha-Toc concentrations declined gradually with decreasing amount of alpha-Toc intake and plasma high-density lipoprotein concentrations toward calving time. However, in the early lactation period after calving, serum alpha-Toc concentrations remained at a lower concentration despite the recovery of alpha-Toc intake and plasma high-density lipoprotein concentrations. In Exp. 2, just after calving, the TTPA, SEC14L2, afamin, and albumin mRNA expression levels in the liver were temporarily downregulated, and the hepatic mRNA levels of endoplasmic reticulum stress-induced unfolded protein response markers and acute-phase response marker increased at calving. In Exp. 3, the concentrations of alpha-Toc in colostrum were greater than those in precolostrum (samples were collected at wk -1 relative to parturition) and mature milk. The expression of TTPA, SEC14L2, arid CYP4F2 mRNA in bovine mammary gland tissue was detected. However, TTPA and SEC14L2 mRNA expressions showed the opposite trends: the expression levels of TTPA mRNA peaked whereas SEC14L2 mRNA reached a nadir at calving. These results indicate that the expression of alpha-Toc-related genes involved in specific alpha-Toc transfer and metabolism in the liver and mammary gland are altered during calving. Moreover, these changes might be associated with the maintenance of lower serum alpha-Toc concentrations after calving.

Insulin-like growth factor-1 (IGF-1) plays a key role in proliferation and galactopoiesis in mammary epithelial cells (MEC), but its definitive functions on endoplasmic reticulum (ER) during protein synthesis remain unknown. The present study aimed to elucidate the effects of IGF-1 on ER biogenesis in MEC in vitro and examined the expression of ER biogenesis-associated genes in the mammary gland during early lactation. We treated mammary alveolar cells-large T antigen cells (immortalized bovine MEC line established via stable transfection with simian virus-40 large T-antigen) with IGF-1 and examined ER biogenesis using the fluorescence intensity of an ER tracker and quantitative real-time PCR. We found IGF-1 significantly increased ER tracker staining and upregulated mRNA levels of ER biogenesis-related genes, such as CHKA (choline kinase α), PCYT1A (choline-phosphate cytidylyltransferase A), and SURF4 (surfeit locus protein 4). We focused on unfolded protein response to explore molecular mechanisms by which IGF-1 induces ER biogenesis. We found IGF-1 significantly increased mRNA levels of the XBP1 splicing form (XBP1s). Based on western blot analysis, IGF-1 induced the expression of (inositol-requiring kinase 1 α) protein, upstream of XBP1s, and phosphorylated-IRE1α. The inhibition of IRE1 endoribonuclease activity with 4-methylumbelliferone 8-carbaldehyde (4μ8C) significantly suppressed the increase in ER tracker fluorescence and ER biogenesis-related gene expression induced by IGF-1. Also, IGF-1-induced XBP1s and ER biogenesis-associated gene expression was inhibited by rapamycin, an inhibitor of mTORC1 (mammalian target of rapamycin complex 1), indicating that IRE1-XBP1 activation by IGF-1 is mediated by mTORC1. Moreover, to clarify the expression of XBP1s and ER biogenesis-associated genes expression under normal physiological conditions, mammary gland tissue from biopsies of dairy cows during late gestation and lactation were analyzed. In vivo data highlighted the significant increases in the mRNA levels of XBP1s and ER biogenesis-related genes in mammary gland tissue immediately after calving through 6 wk of lactation. The mRNA levels of IGF1R (IGF-1 receptor) in mammary glands increased during 6 wk of lactation. Therefore, the present study indicated for the first time that IGF-1 induces ER biogenesis by activating the IRE1-XBP1 axis under the regulation of mTORC1 in bovine MEC line.