Palmitate and pyruvate carbon flux in response to choline and methionine in bovine neonatal hepatocytes.

Choline and methionine may serve unique functions to alter hepatic energy metabolism. Our objective was to trace carbon flux through pathways of oxidation and glucose metabolism in bovine hepatocytes exposed to increasing concentrations of choline chloride (CC) and D,L-methionine (DLM). Primary hepatocytes were isolated from 4 Holstein calves and maintained for 24 h before treatment with CC (0, 10, 100, 1000 µmol/L) and DLM (0, 100, 300 µmol/L) in a factorial design. After 21 h, [1-14C]C16:0 or [2-14C]pyruvate was added to measure complete and incomplete oxidation, and cellular glycogen. Reactive oxygen species (ROS), cellular triglyceride (TG), and glucose and ß-hydroxybutyrate (BHB) export were quantified. Exported very-low density lipoprotein particles were isolated for untargeted lipidomics and to quantify TG. Interactions between CC and DLM, and contrasts for CC (0 vs. [10, 100, 1000 µmol/L] and linear and quadratic contrast 10, 100, 1000 µmol/L) and DLM (0 vs. [100, 300 µmol/L] and 100 vs. 300 µmol/L) were evaluated. Presence of CC increased complete oxidation of [1-14C]C16:0 and decreased BHB export. Glucose export was decreased, but cellular glycogen was increased by the presence of CC and increasing CC. Presence of CC decreased ROS and marginally decreased cellular TG. No interactions between CC and DLM were detected for these outcomes. These data suggest a hepato-protective role for CC to limit ROS and cellular TG accumulation, and to alter hepatic energy metabolism to support complete oxidation of FA and glycogen storage regardless of Met supply.

The effects of prepartum energy intake and peripartum rumen-protected choline supplementation on hepatic genes involved in glucose and lipid metabolism.

Nutritional interventions, either by controlling dietary energy (DE) or supplementing rumen-protected choline (RPC) or both, may mitigate negative postpartum metabolic health outcomes. A companion paper previously reported the effects of DE density and RPC supplementation on production and health outcomes. The objective of this study was to examine the effects of DE and RPC supplementation on the expression of hepatic oxidative, gluconeogenic, and lipid transport genes during the periparturient period. At 47 ± 6 d relative to calving (DRTC), 93 multiparous Holstein cows were randomly assigned in groups to dietary treatments in a 2 × 2 factorial of (1) excess energy (EXE) without RPC supplementation (1.63 Mcal of NEL/kg of dry matter; EXE-RPC); (2) maintenance energy (MNE) without RPC supplementation (1.40 Mcal of NEL/kg dry matter; MNE-RPC); (3) EXE with RPC supplementation (EXE+RPC); and (4) MNE with RPC supplementation (MNE+RPC). To achieve the objective of this research, liver biopsy samples were collected at -14, +7, +14, and +21 DRTC and analyzed for mRNA expression (n = 16/treatment). The interaction of DE × RPC decreased glucose-6-phosphatase and increased peroxisome proliferator-activated receptor α in MNE+RPC cows. Expression of cytosolic phosphoenolpyruvate carboxykinase was altered by the interaction of dietary treatments with reduced expression in EXE+RPC cows. A dietary treatment interaction was detected for expression of pyruvate carboxylase although means were not separated. Dietary treatment interactions did not alter expression of carnitine palmitoyltransferase 1A or microsomal triglyceride transfer protein. The 3-way interaction of DE × RPC × DRTC affected expression of carnitine palmitoyltransferase 1A, glucose-6-phosphatase, and peroxisome proliferator-activated receptor α and tended to affect cytosolic phosphoenolpyruvate carboxykinase. Despite previously reported independent effects of DE and RPC on production variables, treatments interacted to influence hepatic metabolism through altered gene expression.

Postpartum supplementation with fermented ammoniated condensed whey altered nutrient partitioning to support hepatic metabolism.

Our previously published paper demonstrated that fermented ammoniated condensed whey (FACW) supplementation improved feed efficiency and metabolic profile in postpartum dairy cows. The objective of this study was to further explore the effects of FACW supplementation on liver triglyceride content, hepatic gene expression and protein abundance, and plasma biomarkers related to liver function, inflammation, and damage. Individually fed multiparous Holstein cows were blocked by calving date and randomly assigned to postpartum (1 to 45 d in milk, DIM) isonitrogenous treatments: control diet (n = 20) or diet supplemented with FACW (2.9% dry matter of diet as GlucoBoost; Fermented Nutrition, Luxemburg, WI, replacing soybean meal; n = 19). Liver biopsies were performed at 14 and 28 DIM for analysis of mRNA expression, protein abundance, and liver triglyceride content. There was marginal evidence for a reduction in liver triglyceride content at 14 DIM in FACW-supplemented cows compared with the control group. Cows supplemented with FACW had greater mRNA expression of glucose-6-phosphatase at 14 DIM relative to control. Supplementation with FACW increased mRNA expression of pyruvate carboxylase (PC), but did not alter cytosolic phosphoenolpyruvate carboxykinase (PCK1), resulting in a 2.4-fold greater PC:PCK1 ratio for FACW-supplemented cows compared with control. There was no evidence for a FACW effect on mRNA expression of propionyl-CoA carboxylase nor on mRNA expression or protein abundance of lactate dehydrogenase A or B. Cows supplemented with FACW had lower plasma urea nitrogen compared with control. Plasma l-lactate was greater for FACW-supplemented cows compared with control at 2 h before feeding time at 21 DIM. There was no evidence for altered expression of IL1B or IL10, or blood biomarkers related to liver function and damage. Greater glucose-6-phosphatase and PC gene expression, together with greater blood glucose and similar milk lactose output, suggests that FACW increased the supply of glucose precursors, resulting in greater gluconeogenesis between 3 and 14 DIM. Greater hepatic PC:PCK1 ratio, together with previously reported decreased plasma ß-hydroxybutyrate and the marginal evidence for lower liver triglyceride content at 14 DIM, suggests greater hepatic capacity for complete oxidation of fatty acids in FACW-supplemented cows compared with control. Overall, improvements in metabolite profile and feed efficiency observed with postpartum supplementation of FACW may be attributed to increased gluconeogenic and anaplerotic precursors, most likely propionate, due to modulated rumen fermentation.

Short communication: Effect of manipulating fatty acid profile on gluconeogenic gene expression in bovine primary hepatocytes.

During the peripartum period, dairy cows experience both an increase in circulating fatty acid (FA) profile and a change in circulating FA profile, which have been shown to alter regulation of gluconeogenic genes. The objective was to quantify gene expression of key enzymes involved in gluconeogenesis and FA transport into the mitochondria in primary hepatocytes in response to exposure to an FA mixture mimicking what is circulating in a transition dairy cow with or without enrichment of C16:0, C18:0, and C18:1. Primary hepatocytes were isolated from 4 Holstein bull calves 3 d of age (± standard deviation 2 d) and cultured. Twenty-four hours after plating, treatments were applied to the cells for 24-h incubation. Treatments consisted of (1) control (1% BSA), (2) 0.75 mM FA cocktail (3% C14:0, 27% C16:0, 23% C18:0, 31% C18:1, 8% C18:2, and 8% C18:3 to mimic the FA profile of dairy cattle at calving), (3) 0.90 mM FA cocktail, (4) 0.75 mM FA cocktail + 0.15 mM C16:0, (5) 0.75 mM FA cocktail + 0.15 mM C18:0, and (6) 0.75 mM FA cocktail + 0.15 mM C18:1. After harvest in Trizol (Life Technologies, Carlsbad, CA), samples were stored at -80°C until RNA extraction, purification, and reverse transcription. Abundance of mRNA was measured using quantitative real-time PCR. Expression of genes of interest [carnitine palmitoyltransferase 1A, pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase] was calculated relative to the average abundance of 2 reference genes (ribosomal protein L32 and glyceraldehyde 3-phosphate dehydrogenase), which were the most stable out of 3 tested. Data were analyzed using PROC MIXED (SAS version 9.4; SAS Institute, Cary, NC) with the fixed effect of treatment and calf in the random statement. The addition of FA compared with the 1% BSA treatment increased the expression of carnitine palmitoyltransferase 1A and cytosolic PCK1. Enrichment with individual FA did not further regulate pyruvate carboxylase or PCK1 beyond that achieved by the basal profile. These results suggest that shifts in circulating FA profile within a biological range, without a difference in the total FA concentration, have minimal effects on transcriptional regulation of hepatic gluconeogenic genes in primary bovine hepatocytes.

Technical note: Validation of the BHBCheck blood ß-hydroxybutyrate meter as a diagnostic tool for hyperketonemia in dairy cows.

Accurate cow-side blood ß-hydroxybutyrate (BHB) detection meters are valuable tools for rapid diagnosis of hyperketonemia. The main objective of this study was to compare the blood BHB measured in whole blood by the BHBCheck meter (PortaCheck, Moorestown, NJ) to a previously validated meter, Precision Xtra meter (Abbott Laboratories, Abbott Park, IL) and a colorimetric laboratory assay. Samples (n = 426) were collected from postpartum primiparous and multiparous Holstein cows (n = 79 cows) enrolled in 1 of 2 experiments (Exp) with different sampling schedules (Exp 1: n = 39 cows, 58 samples; Exp 2: n = 40 cows, 368 samples). In both Exp, whole-blood samples were collected from the coccygeal vessels after morning milking, before morning feeding. Blood samples were used immediately for BHB quantification via the BHBCheck meter and the Precision Xtra meter. Blood was also collected into evacuated tubes containing no additive (Exp 1) or potassium oxalate/sodium fluoride (Exp 2), which were centrifuged for serum or plasma separation and stored at -20°C for subsequent analysis. Laboratory quantification of BHB concentration was done by the BHB LiquiColor Assay (EKF Diagnostics-Stanbio, Boerne, TX; certified for serum and plasma). Data were analyzed by UNIVARIATE, CORR, FREQ, REG, and LOGISTIC procedures of SAS 9.4 (SAS Institute Inc., Cary, NC). Within this sample set, average parity was 3.3 lactations and DIM was 14 d. The proportion of samples classified as hyperketonemia (BHB ≥1.2 mmol/L) was 25, 28, and 31% as determined by the colorimetric assay, BHBCheck meter, and Precision Xtra meter, respectively. The correlation for BHBCheck meter BHB concentration compared with the colorimetric assay concentrations was r = 0.96, with a sensitivity of 91% and specificity of 93%. Correlation, sensitivity, and specificity of the Precision Xtra meter concentrations were 0.97, 98%, and 92%, respectively. Bland-Altman plots demonstrated minimal bias for both meters. Area under the receiver operator characteristic curve suggests adequate diagnostic accuracy of both meters. Overall, accuracy, sensitivity, and specificity of the BHBCheck meter was similar to the Precision Xtra meter and laboratory assay, indicating the BHBCheck meter is appropriate for use as a cow-side diagnostic test for hyperketonemia in dairy cows.