FoxO1 total protein expression (tFoxO1) (A) and the extent of phosphorylation of FoxO1 at serine 256 (pFoxO1) (B) in liver of cows.

<p>Data are shown in least squares means with standard errors in each group and at each time point. The results of type 3 test for the effects of time and diets are shown in the table under the diagrams. LC-CON (n = 5), HC-CON (n = 5), LC-NA (n = 5), HC-NA (n = 6): “CON or NA”: Dietary supplement of nicotinic acid (0 or 24 g/d) from d-42 to d24, “LC or HC”: 30 or 60% of concentrate proportion in the diet from d-42 to d0, increase in concentrate proportion in the diet after calving from 30 to 50% within 16 or 24 days. d: Days related to calving, N: Nicotinic acid, C: Concentrate proportion in the diet, d×N, d×C, N×C, d×N×C: Interaction effects of d, N, C, tFoxO1: Total protein expression of forkhead box protein O1, pFoxO1: Extent of phosphorylation of FoxO1 at serine 256.</p

Hepatic protein expression as affected by NA and adipose protein expression as affected by time.

<p>A: The ratios of pooled groups (pool “NA” = HC-NA, LC-NA and pool “CON” = HC-CON, LC-CON, 24 or 0 g/d nicotinic acid from d1 to d21) were tested for the influence of nicotinic acid supplementation on the hepatic protein expression of PI3K and GLUT2 by one-way ANOVA (n = 10/group). Hepatic GLUT2 expression decreased from d-21 to d21 (ratio < 1) in cows fed NA, and it was lower in cows fed NA (*p = 0.03) compared to control cows at d21. B, C: The ratios of the protein expression in subcutaneous (B, “SCAT”) and retroperitoneal (C, “RPAT”) adipose tissue from all the cows. Cows were pooled (HC-NA, LC-NA, HC-CON, LC-CON) and tested for the difference to 1 by one-sample t-test (n = 20). The expression of signaling proteins in RPAT was generally lower at d21 compared to at d-21 (ratio < 1; **p < 0.01). Ratio = protein expression d21/d-21; ratio > 1: increase, ratio < 1: decrease; The red line indicates the ratio of protein expression at d-21 (d-21/d-21 = 1). Data are shown as mean ± standard error. NA: nicotinic acid, PI3K: phosphatidylinositol-3-kinase, GLUT2: glucose transporter 2, INSR: insulin receptor, PKCζ = protein kinase Cζ.</p

Correlations between FoxO1-related variables and other investigated variables in the hepatic metabolic pathways related to gluconeogenesis.

<p>To demonstrate correlations graphically, the scheme was adopted to one published by Aschenbach et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146670#pone.0146670.ref011" target="_blank">11</a>]. Pearson’s correlation analyses were performed using the data set from d21 (A) and d100 (B) (N = 21 dairy cows for each). Variables with positive correlations were connected by green solid lines and those with negative correlations by red dotted lines. The level of significance was at least P ≤ 0.01. The metabolites connected by black arrows show the representative metabolic pathways related to gluconeogenesis (not investigated, OAA: Oxaloacetate, PEP: Phosphoenolpyruvate, Glucose-6-P: Glucose-6-phosphate). The variables other than DMI used in the correlation analysis are shown in boxes (blue, red, white for hepatic protein expression, hepatic mRNA expression, and blood metabolites, respectively). FoxO1: Forkhead box protein O1, tFoxO1: Total protein expression of FoxO1, pFoxO1: Extent of phosphorylation of FoxO1 at serine 256, pFo/tFo: pFoxO1-to-tFoxO1 ratio, PCCA: Propionyl CoA carboxylase A, PC: Pyruvate carboxylase, PCK1: Cytosolic phosphoenolpyruvate carboxykinase, G6P: Glucose-6-phosphatase, SLC2A2: Glucose transporter 2 (solute carrier family 2 (facilitated glucose transporter), member 2), IRA, IRB: Insulin receptor isoform A and B, PYGL: Glycogen phosphorylase, liver form, DMI: Dry matter intake, NEFA: Serum concentration of non-esterified fatty acid, Insulin: Insulin serum concentration.</p

Cell size in subcutaneous (SCAT) and retroperitoneal adipose tissue (RPAT) samples of dairy cows around parturition.

<p><b>(A)</b> The median of the measured cell size values was assigned to each sample, and plotted to visualize time-related and depot-related variation. Different superscripts indicate P < 0.05. <b>(B-C)</b> Relative frequency distribution of measured cell size values at 42 days prepartum (d-42), and at 1 day (d+1), 21 days (d+21), and 100 days (d+100) postpartum in <b>(B)</b> SCAT and in <b>(C)</b> RPAT. *P < 0.05, **P < 0.01, ***P < 0.001. Means ± SEM, n = 13.</p

Composition of subcutaneous (SCAT) and retroperitoneal adipose tissue (RPAT) samples of dairy cows around parturition.

<p><b>(A)</b> DNA content, <b>(B)</b> triacylglycerol (TAG) content, <b>(C)</b> total protein content, and <b>(D)</b> β-actin protein expression at 42 days prepartum (d-42), and at 1 day (d+1), 21 days (d+21), and 100 days (d+100) postpartum. Different superscripts indicate P < 0.05. Means ± SEM, n = 20.</p

Body condition score (BCS) of dairy cows corresponding to biopsy samplings.

<p>Body condition score was significantly affected by the time related to parturition (P < 0.01, ANOVA with repeated measures).</p><p>¹Related to parturition</p><p>²Mean ± SEM (n = 20)</p><p>^a,bP = 0.001 (Tukey’s post-test)</p><p>Body condition score (BCS) of dairy cows corresponding to biopsy samplings.</p