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Abomasal carnitine infusion during acute feed restriction increases hepatic fatty acid oxidation and decreases liver lipid in dairy cows. Eight mid-lactation Holstein cows were used in a replicated 4×4 Latin square design with 14-d periods. A 2×2 factorial arrangement was used to determine the effects of water infusion+ad libitum dry matter intake (DMI), water infusion+restricted DMI (50% of previous 5-d average), l-carnitine infusion (20 g/d)+ad libitum DMI, or l-carnitine infusion+restricted DMI. Liver RNA from 7 healthy cows was used for transcriptome profiling using a bovine microarray. An ANOVA with a false discovery rate was used to identify treatment and interaction effects. A substantial transcriptome change was observed only with DMI restriction, resulting in 312 (155 downregulated, 157 upregulated) differentially expressed genes. Quantitative PCR was performed to verify microarray data and measure expression of additional genes not present on the microarray. The quantitative PCR data confirmed the effect of feed restriction but not of l-carnitine treatment. Feed restriction increased expression of GPX3 and of genes associated with gluconeogenesis (PC, PDK4), inflammation (SAA3), and signaling (ADIPOR2). In contrast, feed restriction downregulated BBOX, a key for l-carnitine biosynthesis, and the transcription factor HNF4A. The bioinformatics functional analysis of genes affected by DMI restriction uncovered biosynthesis of cholesterol and energy generation by mitochondrial respiration as the most relevant and inhibited functions. The data also indicated an increase of flux toward gluconeogenesis. We interpreted those results as a likely response of the liver to spare energy and provide glucose for the lactating mammary gland during feed deprivation., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Selection of appropriate housekeeping genes (HKG) for normalization of quantitative PCR data for genes of interest is critical for interpretation of results. Ideally, copy number of the chosen HKG mRNA will not vary with experimental treatments or physiological state in the tissue studied, which improves accuracy in detecting changes in genes of interest. Because of the liver's dynamic role in metabolism, physiological state or dietary treatments could alter mRNA expression of commonly used HKG. Therefore, the objective of this study was to evaluate stability of mRNA expression for a number of candidate HKG in bovine liver across different physiological and dietary experimental conditions during the periparturient period. A publicly available program (geNorm) was used to evaluate expression stability of 8 HKG (beta-actin, glyceraldehyde 3-phosphate dehydrogenase, beta-glucuronidase, peptidylprolyl isomerase A, polyubiquitin, ribosomal protein S9, ribosomal protein L32, and 18S ribosomal RNA) in 91 liver RNA samples. Screened samples included liver from cows in 3 groups: 1) cows receiving a dietary supplement pre- and postpartum (n = 10); 2) cows with clinical or subclinical ketosis (n = 7); and 3) cows consuming different amounts of energy prepartum (n = 74). In group 3, samples from d -65, -30, -14, 1, 14, 28, and 49 relative to parturition were included to enable characterization of HKG mRNA expression across different physiological states. Initial analyses indicated that mRNA for ribosomal protein S9 (RPS9) was one of the most stably expressed across different experiment types. To determine the best gene, 200 bootstrap replications of the original data set were performed to determine if the ranking of RPS9 was superior to the other 7 genes evaluated. Average ranks and estimated standard errors for the top 3 genes were 1.64 +/- 0.06, 3.27 +/- 0.10, and 3.71 +/- 0.12 for RPS9, GAPDH, and beta-actin, respectively. Ribosomal protein S9 was ranked first 59% of the time and was never ranked lower than fifth. The lowest-ranked gene was polyubiquitin, ranked last 46.5% of the time (average rank = 6.85 +/- 0.10). In this study, physiological state, amount of intake, or dietary treatment influenced the mRNA expression of commonly used HKG in bovine liver. Ideally, expression stability should be tested before collection of data in all experiments; however, we have shown that RPS9 mRNA is stable across several physiological and diet-related experimental conditions for dairy cows, making it a good HKG in liver quantitative PCR experiments.

Indole-3-carbinol (I3C), a compound found in Brassica vegetables has been widely studied for its chemopreventive properties. I3C has been shown to block tumor initiation and promotion; however, it also acts as a tumor promoter. I3C and some of its acid condensation products, particularly 3,3'-diindolylmethane (I33'), have exhibited antiestrogenic properties. We report that I33' acts as an estrogen in the rainbow trout liver in vitro and in vivo by inducing vitellogenin (Vg), a well-characterized biomarker for estrogens. Precision-cut liver slices from male rainbow trout, Oncorhynchus mykiss, were incubated at 14 degrees C for 96 h in media containing I3C, I33', or a mixture of I3C acid condensation products (RXN) (0-250 microM). I33' and RXN increased Vg levels in rainbow trout liver slices by over 300- and 20-fold, respectively, vs vehicle. The efficacy of I33' induction of Vg was comparable to 17 beta-estradiol (E(2)) with 2500-fold less potency. I33' and E(2) cotreatment resulted in additive Vg induction. Tamoxifen completely inhibited I33'-induced Vg induction, suggesting that Vg induction by I33' is entirely through the estrogen receptor. In vivo, juvenile male rainbow trout were fed I3C, RXN (0-2000 mg/kg), or I33' (0-250 mg/kg) for 2 weeks. At 2000 mg/kg, I3C induced Vg by over 100,000-fold compared to controls, which was comparable to 5 mg/kg 17 beta-estradiol (the dose resulting in maximum induction). I33' was five times as potent as I3C with equal efficacy. The potency of RXN was only 5% of I3C. Again, I33' and E(2) cotreatment resulted in additive Vg induction. I33' may have accounted for Vg increases observed in trout fed I3C as it is present in liver after oral dosing at concentrations (70 microM) expected to maximally induce Vg. In trout, results in vitro and in vivo document that I33' is estrogenic, consistent with our hypothesis that I3C promotes liver cancer in trout by estrogenic pathways., (Copyright 2001 Academic Press.)

Sturgeon are an ancient family (Acipenseridae) of fishes that lie close to the divergence of fish that eventually evolved into terrestrial animals and those that evolved into modern teleost species. Therefore, white sturgeon vitellogenin sequences fill a gap in the current understanding of the functional domains of this protein family. Vitellogenin cDNA was sequenced and used to investigate gene expression in white sturgeon, Acipenser transmontanus. Estrogen-induced vitellogenin mRNA was detected in the livers of both males and females and was also detected in undifferentiated gonads of estrogen-treated fish. Low levels of vitellogenin mRNA were also detected in the testis of both control and estrogen-treated males. The cDNA encoded a 186-kDa protein that was missing only six to seven of the amino-terminal amino acids. Comparisons to silver lamprey, Xenopus, and chicken vitellogenin sequences indicate that the overall structure of the yolk protein domains were highly conserved. There was considerable homology in three regions of the lipovitellin I domain. These conserved sequences are likely to be involved in vitellogenin receptor binding. The phosvitin domain of white sturgeon vitellogenin contained fewer and shorter serine repeats as predicted from yolk protein phosphate content of fish compared to Xenopus and chicken. However, the vitellogenin of white sturgeon had a lower serine content as compared with silver lamprey, indicating that an increased serine content is not strictly a function of evolutionary age.

The liver is an attractive target tissue for gene therapy. Current approaches for hepatic gene delivery include retroviral and adenoviral vectors, liposome/DNA, and peptide/DNA complexes. This study describes a technique for direct injection of DNA into liver that led to significant gene expression. Gene expression was characterized in both rats and cats following injection of plasmid DNA encoding several different proteins. Luciferase activity was measured after injection of plasmid DNA encoding the luciferase gene (pCMVL), beta-galactosidase (beta-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMV beta), and serum concentration of secreted human alpha-1-antitrypsin was measured following injection of plasmid DNA encoding this protein (pRC/CMV-sHAT). Several variables, including injection technique, DNA dose, and DNA diluent, were investigated. Direct injection of pCMVL resulted in maximal luciferase expression at 24-48 hr. beta-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human alpha-1-antitrypsin were detected in the serum of animals injected with pRC/CMV-sHAT. These findings demonstrate the general principle that direct injection of plasmid DNA into liver can lead to significant gene expression.

The critical physiological functions of the liver make hepatocytes important targets for therapeutic gene delivery. This study reports significant gene expression following direct injection of plasmid DNA into the livers of rats and cats. Transfection was characterized using luciferase and Lac Z expression from plasmids with the cytomegalovirus immediate early promoter/enhancer (CMV IE) or the Rous sarcoma virus long terminal repeat (RSV LTR). Dexamethasone treatment enhanced and prolonged transfected gene expression, possibly by activating gene expression. Southern analysis of total DNA extracted from liver at various times following injection detected persistent unintegrated plasmid DNA which maintained a prokaryotic methylation pattern. This study demonstrates the feasibility of direct DNA injection in the experimental analysis of hepatic gene expression in vivo.

Background: There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs. Methods: The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development. Results: Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality. Conclusions: This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation. [ABSTRACT FROM AUTHOR]

The objectives of this study were to determine the effects of dietary L-carnitine supplementation on liver lipid accumulation, hepatic nutrient metabolism, and lactation in multiparous cows during the periparturient period. Cows were assigned to treatments at d -25 relative to expected calving date and remained on the experiment until 56 d in milk. Treatments were 4 amounts of supplemental dietary carnitine: control (0 g/d of L-carnitine; n = 14); low carnitine (LC, 6 g/d; n = 11); medium carnitine (MC, 50 g/d; n = 12); and high carnitine (HC, 100 g/d; n = 12). Carnitine was supplied by mixing a feed-grade carnitine supplement with 113.5 g of ground corn and 113.5 g of dried molasses, which was then fed twice daily as a topdress to achieve desired daily carnitine intakes. Carnitine supplementation began on d -14 relative to expected calving and continued until 21 d in milk. Liver and muscle carnitine concentrations were markedly increased by MC and HC treatments. Milk carnitine concentrations were elevated by all amounts of carnitine supplementation, but were greater for MC and HC than for LC during wk 2 of lactation. Dry matter intake and milk yield were decreased by the HC treatment. The MC and HC treatments increased milk fat concentration, although milk fat yield was unaffected. All carnitine treatments decreased liver total lipid and triacylglycerol accumulation on d 10 after calving. In addition, carnitine-supplemented cows had higher liver glycogen during early lactation. In general, carnitine supplementation increased in vitro palmitate β-oxidation by liver slices, with MC and HC treatments affecting in vitro palmitate metabolism more potently than did LC. In vitro conversion of Ala to glucose by liver slices was increased by carnitine supplementation independent of dose. The concentration of nonesterified fatty acids in serum was not affected by carnitine. As a result of greater hepatic fatty acid β-oxidation, plasma β-hydroxybutyric acid was higher for the MC and HC treatments. Serum insulin was greater for all carnitine treatments, although plasma glucose was unaffected. Plasma urea N was lower and plasma total protein was higher for the MC and HC treatments. By decreasing liver lipid accumulation and stimulating hepatic glucose output, carnitine supplementation might improve glucose status and diminish the risk of developing metabolic disorders during early lactation. [ABSTRACT FROM AUTHOR]

Previously we determined that abomasal infusion of L-carnitine increased in vitro hepatic fatty acid oxidation, decreased liver lipid accumulation, and supported higher fat-corrected milk yield in feed-restricted lactating cows. The objectives of this study were to examine the effects of supplemental L-carnitine and amount of feed intake on free carnitine and carnitine ester concentrations in liver, muscle, milk, and plasma of lactating dairy cows. Eight lactating Holstein cows (132 + 36 d in milk) were used in a replicated 4 x 4 Latin square design with 14-d periods to test factorial combinations of water or L-carnitine infusion (20 g/d; d 5 to 14) and ad libitum or restricted (50% of previous 5-d intake; d 10 to 14) dry matter intake. Plasma was obtained 3 times daily on d 4, 8, and 12; milk samples were collected on d 8, 9, 13, and 14. Liver and muscle were biopsied on d 14 of each period. Free carnitine, short-chain acylcarnitine, and long-chain acylcarnitine concentrations were determined using a radioenzymatic assay coupled with ion exchange chromatography. Abomasal a-carnitine infusion increased total carnitine in plasma on d 8 and d 12. All liver carnitine fractions were increased by carnitine infusion. Feed restriction elevated concentrations of free carnitine, long-chain acylcarnitine, and total carnitine in liver tissue from carnitine-infused cows but not in those infused with water. In muscle, acid-soluble carnitine, long-chain acylcarnitine, and total carnitine concentrations were increased by carnitine infusion and feed restriction without significant interaction. Feed restriction increased free carnitine concentrations in muscle from water-infused cows but not in carnitine-infused cows. Carnitine infusion increased the concentration of each milk carnitine fraction as well as milk carnitine output on d 8 to 9. On d 13 to 14, all carnitine fractions except short-chain acylcarnitine were increased in milk from water-infused, feed-restricted cows, whereas all fractions were increased in carnitine-infused, feed-restricted cows. Carnitine infusion increased total carnitine in plasma, liver, muscle, and-milk during feed restriction, whereas feed restriction alone increased carnitine concentrations in muscle and milk but not in liver. Liver carnitine concentrations might limit hepatic fatty acid oxidation capacity in dairy cows during the periparturient period; therefore, supplemental L-carnitine might decrease liver lipid accumulation in periparturient COWS. [ABSTRACT FROM AUTHOR]

Our hypothesis was that peroxisomal proliferator-activated receptor (PPAR)-α agonists would increase hepatic β-oxidative capacity for fatty acids, which would decrease circulating NEFA and hepatic lipid accumulation in transition cows. The objectives of this study were 1) to determine if weaned Holstein calves are a suitable model for liver metabolism in periparturient cows, 2) to determine if PPAR-α agonists alter NEFA metabolism in bovine liver, and 3) to compare in vitro metabolism of palmitate and oleate by liver slices. Male Holstein calves (n=15) were assigned at 7 wk of age to 1 of 3 groups for a 5-d treatment period: untreated control, clofibrate (62.5 mg/kg BW), or fish oil (250 mg/kg BW). Calves were euthanized on d 6 and liver slices were incubated for 2, 4, and 8 h to determine conversion of [1-14C] palmitate and [1-14C] oleate to CO2, acid-soluble products (ASP), and esterified products (EP). Calves treated with clofibrate tended (P = 0.05) to consume less dry matter. Body weight, liver weight, and ratio of liver to body weight were not significantly different among treatments. In liver slices incubated for 8 h, conversion of palmitate to CO2 was greater (P < 0.05) for calves treated with clofibrate than for untreated or fish oil. Conversion of palmitate to ASP was numerically greater for calves treated with clofibrate or fish oil compared with controls. Palmitate conversion to EP, total palmitate metabolism, and metabolism of oleat did not differ among treatments Conversion of NEFA to CO2 was greater for palmitate than oleate for all treatments (P < 0.05), but rates of total metabolism were not different. Rates of palmitate oxidation by liver slices from calves in this study were nearly 10 times greater than rates previously observed for adult cows. Clofibrate increased the rate of oxidation of palmitate in liver slices from male Holstein calves. Applications of PPAR-α agonists may be of interest in enhancing the rate of hepatic fatty acid oxidation in transition dairy cows. [ABSTRACT FROM AUTHOR]

Our hypothesis was that supplemental L-carnitine would increase liver carnitine concentration and hepatic fatty acid oxidation. Previously, we have demonstrated that dietary L-carnitine effectively decreased liver lipid accumulation during the periparturient period. Multiparous Holstein cows (n = 56) were supplemented with four amounts of Carniking (50% L-carnitine; Lonza, Inc.), mixed with 227 g ground corn plus 227 g dried molasses, as a topdress from d 14 before expected calving until 21 days in milk. Treatments were: control (CON; 0 g L-carnitine), low (LC; 6 g L-carnitine), medium (MC; 50 g L-carnitine), and high (HC; 100 g L-carnitine). All cows were fed the same basal diet prepartum (21 d before expected calving) and same postpartum diet (calving until d 56). Liver and skeletal muscle were biopsied on d 21 before expected calving and at d 2, 10, and 28. Free, short-chain, and long-chain carnitine esters were quantified using a radioenzymatic procedure and ion exchange chromatography. Orthogonal contrasts were used to compare carnitine treatments vs. CON; LC vs. MC and HC; and MC vs. HC. Carnitine supplementation increased free carnitine and carnitine ester concentration in liver tissue (P = 0.01). The MC and HC treatments resulted in higher liver carnitine content than did the LC treatment (P < 0.01), although concentrations of free carnitine and carnitine esters did not differ between the MC and HC treatments at any point during the periparturient period (P > 0.15). Carnitine supplementation caused greater free carnitine and carnitine ester concentrations in liver on d 2 and 10 after calving compared with the CON treatment, but differences were diminished by d 28 after calving (treatment x time; P < 0.01). These results suggest that dietary L-carnitine increased liver carnitine concentration in periparturient cows, which likely contributed to the reduction in liver lipid accumulation around calving by stimulating fatty acid oxidation. [ABSTRACT FROM AUTHOR]