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Physiol. Genomics 27: 29-41, 2006. First published June 6, 2006; doi:10.1152/physiolgenomics.00036.2006
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Received 24 February 2006; accepted in final form 2 June 2006.
Physiological Genomics 27:29-41 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Plane of nutrition prepartum alters hepatic gene expression and function in dairy cows as assessed by longitudinal transcript and metabolic profiling

Juan J. Loor, Heather M. Dann, Nicole A. Janovick Guretzky, Robin E. Everts, Rosane Oliveira, Cheryl A. Green, Noah B. Litherland, Sandra L. Rodriguez-Zas, Harris A. Lewin and James K. Drackley

Department of Animal Sciences, University of Illinois, Urbana, Illinois


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Liver metabolism and health in dairy cows during the periparturient period are affected by plane of nutrition prepartum. Long-term adaptations in hepatic gene expression are important for complete understanding of liver function. We examined temporal gene expression profiles during the dry period and early lactation in liver of Holstein cows fed moderate dietary energy ad libitum or restricted during the entire dry period using a microarray consisting of 7,872 annotated cattle cDNA inserts and quantitative RT-PCR. We identified 85 genes with expression patterns that were affected by level of energy intake prepartum over time. Restricted energy intake prepartum resulted in more pronounced upregulation of genes with key functions in hepatic fatty acid oxidation (CPT1A, ADIPOR2), gluconeogenesis (PC), and cholesterol synthesis (SC4MOL). Ad libitum feeding upregulated a number of genes associated with liver triacylglycerol synthesis (DGAT1) and proinflammatory cytokines (TNFAIP3). Genomic responses to ad libitum feeding were accompanied by increased incorporation of palmitate to esterified products in vitro and increased liver triacylglycerol concentration in vivo. Overall, gene expression profiles due to plane of nutrition prepartum partly explained differences in rates of liver palmitate metabolism, blood serum metabolite concentrations, and liver tissue triacylglycerol concentration. Our data show that moderate overfeeding of energy in the dry period, in the absence of obesity, results in transcriptional changes predisposing cows to fatty liver and perhaps compromising overall liver health during the periparturient period. In this context, controlled energy intake may confer an advantage to the cow by triggering hepatic molecular adaptations well ahead of parturition.

lactation; bovine; energy balance; metabolism


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE MAJORITY OF HEALTH PROBLEMS affecting dairy cows occur during the "periparturient" period (i.e., the 6-wk period around parturition; Refs. 10, 14). Suboptimal nutritional management during the periparturient period can impact the incidence of clinical or subclinical disease and thus animal well-being. Many of the common periparturient health disorders are strongly linked to energy balance (36). Increasing evidence implicates subclinical ketosis and fatty liver as predisposing factors for energy-associated disease as well as other metabolic and infectious problems (14, 3). Hepatocyte differentiation and function of the adult liver as well as the performance of essential functions in the body are controlled through the coordinated expression of a large number of genes (6). Environmental (including nutrients), autocrine, endocrine, or paracrine signals all contribute to changes in hepatic gene expression (6).

Dairy cows, like most mammals, enter a period of negative energy balance soon after parturition when nutrient demands for lactation exceed nutrient intake (13). This results in marked elevation of circulating nonesterified fatty acids (NEFA) derived from adipose tissue triacylglycerol (TAG) catabolism, which are taken up by the liver and either esterified to TAG or oxidized to CO2 or ketone bodies (40). Type II ketosis, the most common form of ketosis on United States dairy farms today, results from abundant energy supply prepartum and typically affects cows in the first 2 wk of lactation (27). Ketosis is accompanied by marked accumulation of TAG in liver (27).

The present practice has been to increase energy density of prepartum diets and to maximize feed intake before parturition (22). Our recent data, however, suggest that cows that are moderately overfed (>140% of net energy requirements) during the dry period, even without becoming obese, may be placed at greater risk for periparturient health problems (7, 8). A consistent finding in our studies and others (22, 38) is that cows allowed ad libitum access to higher-energy diets (net energy >1.50 Mcal/kg dry matter) during the dry period have larger decreases in feed intake before parturition and lower feed intake postpartum. Therefore, lower-energy diets prepartum may be beneficial by maintaining more consistent feed intake around parturition (12).

Genomic technologies may help identify regulatory mechanisms in liver that are sensitive to nutrient balance during the dry period. We previously examined temporal hepatic gene expression in cows fed to current National Research Council (35) requirements during the dry period (33). Here we report temporal expression profiling of >6,300 unique genes in liver of dairy cows either moderately overfed or limit fed a moderate-energy diet throughout the dry period (8) using a cattle cDNA microarray (17). Our evidence indicates that moderate overfeeding during the dry period results in transcriptional changes predisposing cows to fatty liver and potentially compromising overall cow health.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and liver tissue collection.
All procedures were conducted under protocols approved by the University of Illinois Institutional Animal Care and Use Committee. Cows were selected randomly from two groups of 12 cows enrolled in an experiment (8) to assess the effects of ad libitum or restricted intake of moderate-energy diets during the entire dry period on prepartal metabolism and postpartal metabolism and performance. A diet based on corn silage and alfalfa silage and providing 1.59 Mcal net energy/kg during the far-off dry period (first 5 wk of an 8-wk dry period) or providing 1.61 Mcal net energy/kg during the close-up dry period (last 3 wk of the dry period) was fed either ad libitum or by restricted intake. For cows fed restricted energy, the amount offered was sufficient to supply only 80% of calculated net energy requirements (35). Eight multiparous Holstein cows were randomly selected from the ad libitum (n = 4) and restricted (n = 4) energy intake groups. Feed intake from dry-off through the day before parturition averaged 7.3 kg/day in restricted cows and 14.4 kg in cows fed ad libitum (Supplemental Fig. S1; online article contains supplemental data). Cows were housed in individual tie stalls throughout the experiment and were allowed to exercise daily in an outside lot from 0700 to 1000. All cows underwent normal parturition and were free from health disorders during the study. Liver was sampled via puncture biopsy on days –65, –30, –14, +1, +14, +28, and +49 relative to parturition from cows under local anesthesia at 0700, and RNA was extracted and stored as described previously (33).

Metabolic measurements.
A portion of liver tissue collected for RNA extraction was stored in liquid N2 for analysis of TAG (7, 8). Blood was sampled from the coccygeal vein/artery on days –63, –36, –22, –18, and –13; daily from day –10 through days 10, 14, 21, 28, and 42; and 56 days relative to parturition. Blood serum was assayed for concentrations of NEFA, total protein, urea nitrogen, ß-hydroxybutyrate (BHBA), glucose, and insulin as described previously (7, 8). Body weight for each cow was determined weekly from day –65 to day +56 relative to parturition (8). Energy balance was calculated individually for each cow (33). In vitro liver tissue palmitate metabolism procedures were according to Drackley et al. (11) as modified by Grum et al. (21). Briefly, a portion of biopsy tissue was placed in ice-cold phosphate (9.0 mM)-buffered 0.9% NaCl at pH 7.4, and liver slices were prepared using a Krumdieck tissue slicer (Alabama Research and Development, Munford, AL). Slices (~20 mg) were blotted and weighed into a 25-ml incubation flask. All incubations were performed in triplicate in 3 ml of Krebs-Henseleit buffer containing 25 mM Na-HEPES and L-carnitine (1 mM), pH 7.4. Substrate was sodium palmitate (2 mM) with 0.3 µCi of [1-14C]palmitic acid complexed to fatty acid-free bovine serum albumin in a 4:1 molar ratio. Production of acid-soluble products (predominately ketone bodies; ASP) and CO2 were measured simultaneously in the same flask. Esterification of palmitate in liver slices was measured in separate flasks after lipid extraction from tissue with chloroform-methanol (2:1 vol/vol) and calculated as total esterified products (EP; Ref. 21). Average rates of triplicate flasks were corrected for background radioactivity by subtracting values obtained from blank flasks.

Microarrays and quantitative real-time RT-PCR.
Details on the development, annotation (17), and use of this microarray for studies of bovine liver gene expression (33) have been reported. This platform is publicly accessible in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (GPL2108). Methods for aminoallyl labeling of cDNA, microarray hybridizations, and scanning were as reported by Loor et al. (33). Microarray data were deposited in the NCBI GEO database (accession no. GSE3331). For confirmation studies, quantitative real-time RT-PCR (qPCR) was conducted as described previously (33). Primers were designed to specifically amplify nine target cDNAs (Supplemental Methods and Supplemental Table S1) and an endogenous control gene, bovine ß-actin (ACTB) using Primer Express software v2.0 (Applied Biosystems, Foster City, CA).

Data analyses.
Details used for screening of microarray data and the criteria used to filter unreliable intensity values were as described previously (33). Data from a total of 106 microarrays were normalized for dye and array effects (i.e., Loess normalization and array centering) and used for statistical analysis. A mixed effects model with repeated measures was then fitted to the adjusted ratios (liver/reference standard) using Proc MIXED (SAS, SAS Institute, Cary, NC). The model consisted of day, treatment (i.e., ad libitum or restricted intake), and the interaction of day by treatment and dye as fixed effects and cow and array as random variables. Probability values for fixed effects were adjusted for the number of comparisons using Benjamini and Hochberg's false discovery rate (FDR) (39). Differences in relative expression were considered significant at an FDR-adjusted P ≤ 0.05, corresponding to raw P values ≤ 10–4. Data from qPCR were analyzed as described elsewhere (33), using the same statistical model described above. Differences were considered significant at P ≤ 0.05. Expression patterns for differentially expressed genes due to day x treatment and treatment alone were analyzed using k-means clustering (15) (GeneSpring 7.2, Silicon Genetics, Redwood City, CA). Normalized, log2-transformed ratios computed by GeneSpring were used for k-means clustering. Differences in concentrations of serum metabolites, body weight, energy intake, energy balance, palmitate metabolism, and liver TAG composition were assessed using the same statistical model as above. Differences were considered significant at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Metabolic measurements.
Calculated energy balance for the entire dry period was slightly negative and constant in cows fed restricted energy but positive for cows fed ad libitum (Supplemental Fig. S1). Both groups were in negative energy balance for the first 3 wk postpartum. Cows fed ad libitum had markedly greater concentrations of insulin in serum throughout the entire dry period (Fig. 1A). Serum NEFA and BHBA concentrations increased steadily in both groups as parturition approached (Fig. 1, B and C). Serum NEFA was numerically lower for cows fed ad libitum during the dry period but significantly higher during the first week postpartum. In contrast, serum BHBA was numerically lower for cows fed ad libitum during the dry period but significantly higher during the first week postpartum. Despite higher serum BHBA concentrations early postpartum, cows fed ad libitum were not ketotic. Temporal concentrations of serum total protein, urea nitrogen, and glucose are presented in Supplemental Fig. S2. Cows fed ad libitum had greater (P < 0.05) concentrations of total protein and tended (P = 0.10) to have greater serum glucose. There was a significant effect (P < 0.05) of time on the concentrations of all these serum metabolites. Liver TAG in both groups increased between days –14 and +14, but in cows fed ad libitum, concentrations on day +1 were 2-fold greater and those at day +14 were 2.5-fold greater than cows fed restricted energy (Fig. 1D). Total palmitate metabolism by liver slices in vitro was greater in cows fed ad libitum due to dietary effects in the dry period (days –30 and –14) (Fig. 2). Palmitate metabolized to EP in cows fed ad libitum was numerically greater in the dry period and significantly higher on day 1 postpartum.


Figure 1
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  		Fig. 1. Serum insulin (A), serum nonesterified fatty acid (NEFA; B), serum ß-hydroxybutyrate (BHBA; C), and liver tissue triacylglycerol (TAG) concentrations (D) during the dry period through early lactation. There were significant (*P ≤ 0.05) time x treatment effects for serum NEFA, insulin, and liver TAG. Serum BHBA tended (**P = 0.10) to be greater in cows fed energy ad libitum.

 

Figure 2
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  		Fig. 2. Palmitate metabolism in liver tissue. Data are expressed as nmol of palmitate oxidized to acid-soluble product (ASP) or CO2, or esterified product (EP). Time effect was significant (P ≤ 0.10) regardless of dietary treatment for all variables. Asterisks denote differences (P ≤ 0.05) due to dietary treatment (*), interaction of dietary treatment x time (**), or both (***) at the specified time point. Pooled SE were 21.8 (total metabolism), 18.4 (EP), 17.9 (ASP), and 1.4 (CO2).

 
Differential expression of genes in liver.
There were 6,781 cDNA elements representing 5,708 unique genes on the microarray that were expressed in liver (5,440 genes with unique UniGene identification and 268 nonredundant, unannotated sequences). Repeated-measures ANOVA at a cutoff FDR P value ≤ 0.05 resulted in a total of 85 differentially expressed genes due to day x diet interaction and 122 differentially expressed genes due to treatment alone (Tables 1 and 2). Nine clusters of genes accounted for the dominant expression patterns due to day x diet interaction (Fig. 3). Among these, clusters 5 (n = 11), 6 (n = 22), and 7 (n = 17) contained ~60% of the total number of differentially expressed genes. Clusters 2, 3, 4, 5, and 8 contain genes with expression patterns that were substantially higher in cows fed the restricted diet during the periparturient period. Some of these genes are associated with diverse aspects of metabolism, inflammatory responses, and insulin-like growth factor (IGF) action (Table 1). For example, PC, CPT1A, and ACADVL are key genes associated with gluconeogenesis and fatty acid ß-oxidation in liver. Similarly, HMGCR is involved in cholesterol biosynthesis (40), and SC4MOL is believed to function in cholesterol biosynthesis based on the similarity of its putative metal-binding motifs to the family of membrane desaturases-hydroxylases. Another distinct pattern of expression in cows fed restricted energy is depicted in cluster 7, which contained the second largest number of differentially expressed genes and clearly shows marked downregulation around parturition.


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  		Table 1. Complete list of differentially expressed genes due to interaction of time x treatment

 

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  		Table 2. Complete list of differentially expressed genes due to treatment effects

 

Figure 3
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  		Fig. 3. Mean expression patterns for 85 differentially expressed genes due to dietary treatment x time interaction, using k-means cluster analysis. See Table 1 for listing and description of genes within clusters.

 
Cows fed ad libitum had distinct expression patterns for a number of genes as evidenced in clusters 1, 6, 7, and 9 (Fig. 3). NFKB1, which functions as a transcription factor during cellular inflammatory responses (29), was among four genes with increased expression (cluster 1) between day –65 and day –14, followed by marked downregulation through day +14; the opposite pattern was observed in cows fed restricted energy. Cluster 6 exhibited the most distinctive pattern of expression in cows fed ad libitum. Namely, expressions of the 22 genes in this cluster were substantially higher during the periparturient period compared with cows fed the restricted diet.

Expression patterns for differentially expressed genes due to prepartum plane of nutrition alone, irrespective of time, were subdivided into four clusters (Fig. 4). Distinct patterns of expression during the periparturient period in cows fed restricted energy included pronounced upregulation of 30 genes (clusters 3 and 4) and marked downregulation of 70 genes (cluster 1). Cows fed ad libitum had more pronounced downregulation of 18 genes (cluster 2) around parturition. Among genes drastically downregulated by restricted feeding prepartum were some with essential functions in cell cycle regulation and apoptosis induced by cytokines such as IFN-{gamma} (S100A4, CEBPG) (1), TNF-{alpha}- and NF-{kappa}B-induced signaling (TNFAIP3) (29, 42), cellular volume and homeostasis (NFAT5) (28), activation of mitochondrial apoptotic cascades (E2IG5) (32), and regulation of macrophage/cell adhesion and migration (PLAUR) (23). Restricted energy feeding prepartum resulted in more pronounced upregulation of ADIPOR2 (30), CTF1, ACTN1, TDG, ROD1, and AKR1B1, among others (Fig. 4 and Table 2).


Figure 4
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  		Fig. 4. Mean expression patterns for 122 differentially expressed genes due to dietary treatment, using k-means cluster analysis. See Table 2 for listing and description of genes within clusters.

 
We observed differential expression due to prepartum dietary treatment or the interaction of treatment x time for a number of genes that are involved in regulation of transcription (NFAT5, RXRB, TAFL5, TCEA1, TCEB1, HDAC2), transport (AKR1B1, IVD, CCS, FOLR2), and regulation of cell growth (PLCE1, IGFBP4, TBRG4) (Tables 1 and 2). Interestingly, all the above genes associated with regulation of transcription were downregulated in the periparturient period in cows fed restricted energy (Figs. 3 and 4). Overall, the above gene expression and metabolic data clearly highlight long-term adaptive mechanisms in hepatic function in concordance with nutrient availability.

qPCR analysis.
Three of the ten genes assayed by qPCR (DGAT1, MTP, and GPAM) were not present on the array but are crucial components of TAG synthesis and lipoprotein transport pathways in rodents. The remaining seven genes (AGPAT1, SREBF1, ACADVL, ADIPOR2, SC4MOL, IFNGR2, and PC) were a subset of differentially expressed genes with essential functions in hepatic fatty acid oxidation, gluconeogenesis, signal transduction and lipid synthesis, cholesterol synthesis, and cytokine signaling. There were minor discrepancies in expression patterns for the six genes measured over all time points (Fig. 5 and Supplemental Fig. S3). For example, differences between the methods for PC, ADIPOR2, SC4MOL, ACADVL, and IFNGR2 were apparent on day –14 and for SREBF1 on day +1.


Figure 5
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  		Fig. 5. Comparison of expression patterns (fold change relative to day –30) observed with microarrays (left) vs. quantitative PCR (qPCR; right) for genes involved in hepatic fatty acid oxidation (ADIPOR2), gluconeogenesis (PC), cholesterol synthesis (SC4MOL), and glycerolipid synthesis (AGPAT1). See Supplemental Fig. S3 for remaining comparisons. Statistical analysis of qPCR data was performed on values normalized to ACTB. Results showed significant (P ≤ 0.01) time and time x treatment effects (*) for PC, ADIPOR2, SC4MOL, and AGPAT1 as assessed by qPCR.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The amount of energy intake prepartum is known to induce changes in blood and liver tissue metabolic indicators (7, 8, 12), and we show on a large scale that liver mRNA expression also is susceptible to prepartum plane of nutrition. Our findings that ad libitum feeding of a moderate-energy diet resulted in energy intakes greatly exceeding requirements confirm previous data from our group (7) and demonstrate that dairy cows in late pregnancy do not regulate energy intake to match their requirements. Overfeeding increased serum insulin, tended to increase glucose concentration, and decreased serum NEFA but did not change liver TAG concentration prepartum compared with cows fed restricted energy, similar to previous data (7). Serum NEFA increased linearly during the last 7 days of gestation, as expected (7, 33), but the extent of this increase and that of BHBA during the first week postpartum was greater for cows fed ad libitum prepartum (Fig. 1, B and C). The greater increase in serum NEFA likely explains greater serum BHBA early postpartum in cows fed ad libitum prepartum. Adipose lipid mobilization leads to increases in serum NEFA, uptake by liver, oxidation to ketone bodies and CO2, and increased incorporation into liver TAG (10, 40). Our measurements of palmitate metabolism in liver tissue indicate that greater liver TAG concentrations early postpartum in cows fed ad libitum energy were partly a result of both higher rates of esterification (Fig. 2) and greater NEFA availability (Fig. 1B) (11). Changes in liver gene expression help explain nutrition-induced differences in liver TAG concentration. Around parturition, cows fed ad libitum prepartum had increased expression of genes associated with TAG synthesis such as AGPAT1 (cluster 2; Figs. 3 and 5) and DGAT1 (Fig. 5). Expression of MTP (Fig. 5), which is critical for TAG assembly into VLDL for export (10), was increased by ad libitum feeding on day +1, providing evidence for a novel role in TAG formation in bovine liver as seen in rodents (37, 44). In contrast, expression of genes with key functions in various aspects of hepatic ß-oxidation, such as CPT1A, ACAA1, and ADIPOR2 (Fig. 4), was nearly twofold higher around parturition in cows fed the restricted diet. These cows also had significant (P < 0.05) positive correlations (data not shown) between mRNA expression for both CPT1A and ADIPOR2, with BHBA and NEFA concentrations in serum and palmitate metabolism to acid-soluble product. Correlations were weakly positive and nonsignificant in cows fed ad libitum energy. Despite the observed upregulation of fatty acid oxidation genes in cows fed restricted energy, the capacity of liver tissue to oxidize palmitate was not significantly different (Fig. 2), although means were somewhat larger for cows fed restricted energy at day 1 postpartum.

Some effects of moderate energy overfeeding prepartum on hepatic gene expression might have been mediated by the more than threefold increase in serum insulin in overfed cows (Fig. 1A). Stimulation of insulin signaling in rodent liver reduces gluconeogenesis primarily via downregulation of mRNA expression for PCK1 and G6PC (2) but also could adversely affect expression of PC. Liver PC expression in periparturient cows was fourfold higher on day 1 postpartum than on day –14 prepartum (19) and was apparently negatively correlated with insulin concentrations. In our study, PC mRNA was increased around parturition in both treatments (Fig. 5), but the increase was greater in cows fed the restricted diet. In these cows, higher PC mRNA was likely associated with the need to maintain blood glucose concentrations, whereas in the ad libitum group, a greater influx of propionate, greater hepatic glucose output, and the resulting hypersinsulinemia could have affected PC expression through a feedback mechanism (4, 45).

GPAM, SREBF1, MTP, and DGAT1 are important lipogenic proteins in rodent liver and help partition fatty acyl-CoAs toward TAG synthesis and VLDL secretion and away from oxidation (24, 34, 48, 49) (Fig. 6 and Supplemental Fig. S3). AGPAT1 is one of five isoforms that catalyzes the conversion of lysophosphatidic acid to phosphatidic acid, both of which are involved in signal transduction and lipid synthesis in various tissues (45). We attempted to simultaneously determine effects of different prepartum energy intakes on expression of these key lipogenic genes in bovine liver, with the aim of deciphering their involvement in TAG accumulation early postpartum. Expression patterns suggest that 1) concerted downregulation of GPAM and SREBF1 around parturition in dairy cows channels NEFA toward fatty acid oxidation (e.g., upregulation of ACADVL, CPT1A) and stimulates gluconeogenesis (e.g., PC upregulation), 2) the initial step of TAG synthesis catalyzed by GPAM is not limiting for liver lipid accumulation early postpartum, and 3) only AGPAT1, DGAT1, and MTP mRNA expressions appear susceptible to diet-induced hyperinsulinemia prepartum. Rodent studies have shown that insulin or diet-induced insulin resistance upregulates GPAM, SREBF1, MTP, and DGAT1 mRNA expression in various tissues (48, 49, 24, 34). Our data suggest that DGAT1, which catalyzes the last step in TAG synthesis (34), AGPAT1, and MTP are causally linked with liver TAG accumulation in response to prepartum energy intake level (Fig. 6). In cows fed ad libitum energy, the correlations between DGAT1 expression and palmitate metabolized to esterified product and to CO2 were 0.91 and –0.88 (P < 0.001). Similarly, AGPAT1 expression in these cows had positive correlations (P < 0.01) with serum NEFA (r = 0.67) and liver TAG (r = 0.85). These correlations provide additional evidence in support of a close involvement of both genes in liver TAG synthesis. Recently, a specific amino acid substitution in the bovine DGAT1 gene was shown to underlie a quantitative trait locus affecting milk fat concentration in cattle (20). Although the DGAT1 K232A mutation appears to be responsible for increased TAG synthesis in the mammary gland, DGAT1 expression in the liver could also have a similar effect. This raises the interesting possibility that both the phenotypic effects of DGAT1 alleles on milk fat composition and TAG concentration in liver may be regulated by diet. It may also explain why "low" alleles have persisted in the dairy cattle population, if liver lipid-related metabolic disorders associated with prepartum energy overfeeding are due to the more frequent "high" allele, which is more efficient in synthesizing triacylglycerol.


Figure 6
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  		Fig. 6. Expression patterns of GPAM, DGAT1, and MTP. Data are expressed as normalized values obtained by qPCR using SYBR Green. There were significant day and day x prepartum dietary treatment effects (P < 0.01) for the expression of these genes. Expression on day +1 for DGAT1 and MTP was significantly higher for cows fed ad libitum prepartum, and this difference persisted on day +28. GPAM expression was higher on day –14 for cows fed ad libitum. Pooled SE were 1.6 (GPAM), 1.05 (DGAT1), and 0.34 (MTP).

 
On the basis of the present findings, we propose that hyperinsulinemia might have primed hepatocytes to accumulate lipid postpartum when cows were fed the same diet and insulin concentrations were similar for both groups, i.e., hepatic capacity for fatty acid esterification increased and oxidation was hampered. Key evidence for this hypothesis, in addition to expression patterns of fatty acid oxidation, AGPAT1, and DGAT1 (see above), is the sustained lower expression of ADIPOR2 in cows fed ad libitum energy, particularly during days +1 through +14 (Fig. 5). ADIPOR2 is the liver-enriched isoform of the adiponectin receptor whose expression is necessary for adiponectin signaling of hepatic fatty acid ß-oxidation (30). Adipose tissue participates in the regulation of energy homeostasis through secretion of a number of biologically active adipokines (30). ADIPOR2 in liver serves as a receptor for adipose tissue-derived adiponectin and thus mediates the ability of this hormone to increase AMP-activated protein kinase activity, peroxisome proliferator-activated receptor (PPAR)-{alpha} expression and activity, and fatty acid oxidation (30, 51). Adiponectin mRNA expression and its concentration in serum are drastically reduced in a variety of states frequently associated with insulin resistance in rodents or humans, and this effect is mediated directly via upregulation of adipose TNF-{alpha} production (51). We propose that overfeeding energy prepartum through hyperinsulinemia promotes fat accumulation in visceral adipose depots that drain directly into the liver. Visceral fat accumulation is accompanied by increased visceral adipose TNF-{alpha} production and downregulation of adiponectin mRNA, which might decrease the adiponectin supply to the liver via the portal blood. The net result would be dampening of adiponectin effects in the liver, e.g., reduced ADIPOR2 mRNA and capacity for fatty acid oxidation. Our data would support a direct role of chronic hyperinsulinemia in downregulation of both ADIPOR2 and PPARA in the liver (30) (Fig. 7).


Figure 7
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  		Fig. 7. Schematic representation of interactions among gene expression patterns in liver and physiological events in visceral adipose, skeletal muscle, and liver tissue during the periparturient period in response to ad libitum or restricted feeding of moderate-energy diets. Data indicate that hyperinsulinemia induced by overfeeding a moderate-energy diet prepartum has a direct role in promoting 1) visceral adipose mass accumulation leading to upregulation of TNF-{alpha} and concomitant downregulation of adiponectin in adipose tissue, 2) selective upregulation of hepatic lipogenic genes, and 3) selective downregulation of hepatic fatty acid oxidation genes. Both lower serum adiponectin, due to excessive visceral adipose tissue accumulation, along with hyperinsulinemia could signal a reduction of or hamper ADIPOR2 expression in liver, whereas higher circulating serum TNF-{alpha} in portal blood is a positive signal for upregulation of hepatic proinflammatory cytokines (e.g., IFN-{gamma}). Upregulation of TNFAIP3 in overfed cows likely is a necessary response to dampen effects of TNF-{alpha}- and NF-{kappa}B-induced signaling (i.e., upregulated NFKB1). Overfed cows experiencing a heightened state of oxidative stress (partly through upregulated IFNGR2) might be at higher risk of excessive DNA damage, which in turn could be a feedback signal for upregulation of TNFAIP3. Upregulation of this protein mediates cytoplasmic signaling to activate NFKB in response to DNA damage. Excessive and prolonged activation of NFKB confers cells with anti-apoptotic activity, binds to DNA, and initiates gene expression (16, 25). Clearly, overfeeding prepartum might render the liver of cows more susceptible to oxidative stress and DNA damage through changes in gene expression. Another crucial effect of excessive visceral adipose tissue mass accumulation is higher serum NEFA early postpartum. Overfed cows appear to have lower capacity to completely oxidize NEFA in liver (e.g., higher serum BHBA), as shown by reduced expression of ADIPOR2, ACADVL, ACAA1, and CPT1A mRNA postpartum, but have higher capacity to store TAG, as shown by upregulation of AGPAT1, DGAT1, and MTP. Overfeeding prepartum had little effect on GPAM and SREBF1 pre- and postpartum, suggesting they do not play a crucial role in liver lipidosis early postpartum. However, these data reflect mRNA levels and do not discount effects of nutrition on protein expression. Restricted energy intake allows for upregulation of fatty acid oxidation, gluconeogenesis, cholesterogenesis, and normal nuclear hormone binding and prevents excessive oxidative stress and DNA damage while dampening proinflammatory responses in liver. It has been observed (43) that in nonruminants, there is a "normal" decrease in expression of liver nuclear hormone receptors (e.g., SREBF1, PPAR, RXRB), which likely contributes to the alterations in liver lipid metabolism early postpartum. Therefore, controlled energy intake prepartum would be advantageous to the cow as the liver "adjusts" its metabolic activity to the onset of parturition. It is possible, however, that caloric restriction might lead to excessive skeletal muscle tissue breakdown close to parturition and induce an inflammatory response in this tissue that could result in greater systemic TNF-{alpha}. Uncontrolled TAG accumulation in liver and oxidative stress increases the risk of periparturient health disorders by predisposing the cow to fatty liver and ketosis but also by upregulating TP53 expression, which could increase steatotic hepatocyte damage.

 
It remains to be determined whether plane of nutrition prepartum alters serum adiponectin and TNF-{alpha} levels in cows and the nature of their association with metabolic parameters. In studies with rats, an acute food deprivation (6 h) resulted in greater periepididymal adipose TNF-{alpha} production (47). Enhanced TNF-{alpha} production also is a marker in muscle-wasting conditions (e.g., disease, calorie restriction) and leads to muscle degradation through activation of NF-{kappa}B (25, 31). Thus concentrations of TNF-{alpha} in serum could be increased around parturition in cows fed restricted energy prepartum without the development of fatty liver early postpartum. In light of the above findings, determination of adipokine concentrations in portal blood due to prepartum plane of nutrition should shed light on the hypothesized cross talk between visceral adipose and the liver.

Our previous microarray experiment suggested a causal link between hepatic inflammation and liver TAG accumulation (33). The expression of TNFA mRNA is upregulated in the liver immediately after parturition under normal conditions (33). In mice, liver lipidosis due to the feeding of high-fat diets led to subacute hepatic inflammation through hepatic NF-{kappa}B activation (5). Those mice had not only liver TAG accumulation and elevated serum NEFA but also increased hepatic mRNA expression of proinflammatory cytokines (TNFA, IL6, IL1B). These previous results and our finding that TNFAIP3, whose expression is rapidly induced by TNF-{alpha} (29), was more than twofold lower around parturition than at dry-off in cows fed restricted energy (cluster 1, Fig. 4) are suggestive of nutrition playing a role in bovine hepatic inflammation. TNFAIP3 is a zinc finger protein and has been shown to inhibit NF-{kappa}B activation as well as TNF-{alpha}-mediated apoptosis (26). Knockout studies in mice suggest that this gene is critical for limiting inflammation by terminating TNF-{alpha}-induced NF-{kappa}B responses (29). Additional evidence for an enhanced inflammatory response in cows fed ad libitum energy is the upregulation of AGPAT1, which has been shown to play a role in amplifying proinflammatory cytokine-induced cellular signaling (47). Unlike previous suggestions (3), our present and previous (e.g., SAA1 expression; Ref. 33) data suggest that an inflammatory response precedes or predisposes cows to develop fatty liver early postpartum, particularly when allowed to overconsume moderately high-energy diets.

Analysis by qPCR showed that there was higher expression of IFNGR2 (Supplemental Fig. S3) in cows fed ad libitum from day –14 to day +28, suggesting that circulating IFN-{gamma} might exert effects on liver-signaling pathways (16). IFN-{gamma} transduces signals among cell types, leading to effects on cell proliferation and apoptosis, increased nitric oxide synthase production (i.e., oxidative stress), and regulation of various genes whose functions are yet to be identified (9). Low-level activation of NF-{kappa}B in livers of transgenic mice upregulated IFN-{gamma} as well as IFNGR2 mRNA (5). It is plausible that ad libitum energy intake affected liver TNF-{alpha} production either indirectly through adipose tissue (Fig. 7) or through increased ruminal endotoxin production, which is common in cattle fed high-grain diets (18). Higher endotoxin levels in the circulation resulted in an inflammatory response, as shown by increased blood concentrations of haptoglobin in cattle (18). Production of TNF-{alpha} by liver macrophages activates natural killer cells to produce IFN-{gamma}, which might help explain the upregulation of IFNGR2 in the present study (16). Expression patterns for a number of genes (Tables 1 and 2) support the assertion that inflammatory/immune responses are elicited by plane of nutrition prepartum.

We argue, as in other species (25), that energy restriction prepartum allows for upregulation of hepatic processes that modulate responses to a wide range of environmental and physiological stressors (e.g., parturition, copious milk production). These processes include, but are not limited to, reduced oxidative stress and DNA damage, enhanced tissue DNA repair, reduced TP53 expression (Fig. 3, cluster 7), and reduced apoptosis. Activation of TP53 expression plays an important role in the pathogenesis of fatty liver disease in other species (49). Expression of this gene was negatively correlated with serum NEFA (r = –0.88) and liver TAG (r = –0.70) in cows fed restricted diets but positively correlated (r = 0.55 and 0.60, respectively) in cows fed ad libitum. Inhibition of TP53 might prevent steatotic hepatocytes from being damaged and has been proposed as a therapy against fatty liver disease (49). The observed coordinated responses are of evolutionary significance in that, during periods of food scarcity (as seen early postpartum when the cow cannot consume enough food to meet requirements for milk production), these mechanisms help extend the total and productive life span of the animal. We have integrated transcript profiles and metabolic data into a model to explain complex physiological processes in liver and adipose tissue that are elicited by plane of nutrition during the periparturient period. Our model (Fig. 7) focuses on this relatively short period of the lactation cycle, because it is when the majority of health problems occur (10, 14) and because suboptimal nutritional management around this time has profound effects on clinical or subclinical disease (e.g., ketosis, fatty liver) (3) as well as subsequent productivity. Overall, we conclude that moderate overfeeding of energy prepartum results in transcriptional changes predisposing cows to fatty liver and potentially compromising liver health early postpartum.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Gene expression profiling was supported by award no. 2001-35206-10946 from the National Research Initiative Competitive Grants Program/Cooperative State Research, Education, and Extension Service/United States Department of Agriculture. Support for the animal work was provided by funds from the State of Illinois through the Illinois Council on Food and Agricultural Research.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the help from the staff of the University of Illinois Dairy Research and Teaching Unit for animal care.

Present address of H. M. Dann: William H. Miner Agricultural Research Institute, 586 Ridge Rd., PO Box 100, Chazy, NY 12921.


   FOOTNOTES
 
Address for reprint requests and other correspondence: J. J. Loor or J. K. Drackley, Dept. of Animal Sciences, Univ. of Illinois, 1207 West Gregory Drive, Animal Sciences Laboratory, Urbana, IL 61801 (e-mail: jloor{at}uiuc.edu or drackley{at}uiuc.edu).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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