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Department of Animal Sciences, Purdue University, West Lafayette, IN 47907
Corresponding author: S. S. Donkin; e-mail: sdonkin{at}purdue.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: energy • glucose • liver • gene
Abbreviation key: CPS-I = carbamoyl phosphate synthetase, FR = feed restricted, OTC = ornithine transcarbamylase, PC = pyruvate carboxylase, PEPCK = phosphoenolpyruvate carboxykinase, PEPCK-C = cytosolic phophoenolpyruvate carboxykinase, PEPCK-M = mitochondrial phosphoenolpyruvate carboxykinase, PUN = plasma urea nitrogen
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Starvation in rats induces both liver and kidney PC activity (Salto et al., 1996). Induction of PC mRNA and activity during the transition to lactation may be part of the physiological adaptations associated with the onset of parturition (Greenfield et al., 2000a), a consequence of the decline in voluntary feed intake that occurs in late gestation, or a combination of both factors. Imposed feed restriction has been used to study the metabolic consequences of nutrient insufficiency on metabolism and milk production (Drackley et al., 1991; Bertics and Grummer, 1999) and, therefore, may provide insight on nutrient-gene interactions in dairy cows.
Hepatic N disposal and urea synthesis are usually sufficient to accommodate acute increases in amino acid catabolism (Morris, 2002). However, long-term adaptations in the capacity of the urea cycle are observed in response to dietary calorie restriction (Tillman et al., 1996), dietary protein intake level, or catabolism of endogenous protein. Changes in rate of gene transcription represent a major portion of the long-term response in expression of urea cycle enzymes (Morris, 2002) and require several days to be fully manifested (Schmike, 1962). Changes in liver N metabolism, in response to changes in feed intake, occur within 5 d in cattle (Reynolds, 1992). To our knowledge, the molecular basis of these adaptations has not been investigated in cattle.
Despite intense investigation on control genes for key metabolic reactions in liver of nonruminants, there is a paucity of data describing nutritional and hormonal regulation of gene expression in liver of ruminants. The objective of the present study was to determine the effect of imposed feed restriction on expression of PEPCK, PC, carbamoyl phosphate synthetase I (CPS-I), ornitine transcarbamylase (OTC), and argininosuccinate synthase mRNA in liver of lactating dairy cattle. A second objective was to determine the biological consequences of changes in expression of gluconeogenic enzymes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Animals, Management, and Sampling: Experiment 1
Sixteen multiparous Holstein cows selected from the Purdue University Dairy Research and Education Center herd were stratified by previous production and days in milk and were assigned to one of 2 treatment groups. Cows averaged 34.2 ± 0.8 kg milk/d and were 156 ± 6 DIM at the beginning of the trial. Cows were housed in individual tie stalls, had free access to water, were milked twice daily at 0800 and 2000 h, and were fed once daily a diet formulated to meet or exceed requirements for milk production (NRC, 2001) (Table 1
). Feed intake was measured daily by difference of feed offered and refused. The Purdue Animal Care and Use Committee approved animal handling and sample collection procedures.
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). Individual milk yields were recorded electronically at each milking (HerdMaster Galaxy Management System, Alfa-Laval Agri Inc., Kansas City, MO). Milk samples were obtained for 2 consecutive milkings during each phase of the experiment and analyzed for fat, protein, and SCC by near infrared reflectance and for MUN by the Bentley Chemspec method at the DHIA Laboratory (East Lansing, MI).
Liver biopsy samples were obtained by blind percutaneous needle biopsy (Greenfield et al., 2000a) within 2 to 6 h of feeding; on d 5, 10, and 20 of the trial, which corresponded to the last day of the ad libitum feeding phase; the last day of the feed restriction phase; and after 10 d of realimentation. Liver samples were rinsed in saline, transferred into a 50-mL conical tube containing 10 mL of guanidinium thiocyanate solution [4 M guanidinium thiocyanate, 25 mM sodium citrate (pH = 7.4), 0.5% sarcosyl, and 0.1 M beta-mercaptoethanol], frozen in liquid nitrogen, and stored at –80°C pending RNA extraction.
Blood samples and BW were obtained on the days when liver biopsies were performed. Two blood samples were collected immediately prior to biopsy from a medial coccygeal blood vessel into vacutainers (Becton Dickinson, Franklin Lakes, NJ). One sample collection tube contained potassium oxalate and sodium fluoride, and the other contained heparin. Plasma was separated by centrifugation at 550 x g for 15 min and was frozen at –20°C pending analysis.
Animals, Management, and Sampling: Experiment 2
Five cows were selected from the Purdue University Dairy Research and Education Center herd and used to determine the relationship between gluconeogenesis and expression of PC and PEPCK mRNA in liver. Cows averaged 158 ± 6.5 DIM, were producing 40.75 ± 1.6 kg/d of milk/d, and were housed, fed, and managed as described previously for cows in Experiment 1.
Cows were offered feed for ad libitum consumption on d 1 through 6 of the experiment. Feed intake was measured daily by difference of feed offered and feed refused. Approximately 4 h after feeding, on d 6 of the experiment, liver samples were obtained by blind percutaneous needle biopsy (Greenfield et al., 2000b). Samples were rinsed in ice-cold saline, and an aliquot of approximately 250 mg was prepared for mRNA analysis as described previously. An additional 1 g of liver was placed in ice-cold Dulbecco’s Modified Eagles medium containing 1% BSA, transported on ice to the laboratory, subdivided into 30- to 50-mg explants, and used to measure the rate of gluconeogenesis from radiolabelled propionate and lactate. On d 7 through 11 of the experiment, all cows were given 50% of their previous ad libitum intake. Liver biopsy samples were obtained again on d 11 of the experiment for RNA analysis and measures of gluconeogenesis from propionate and lactate. The Purdue Animal Care and Use Committee approved animal handling and sample collection procedures.
cDNA Probes
Plasmids bPC1000, bPEPCKC3', and bPEPCKM3' were cloned in our laboratory (Agca et al., 2000, 2002) and contained insert DNA for bovine PC, PEPCK-C, and mitochondrial PEPCK (PEPCK-M), respectively. Plasmid pAS4, containing a 1.5-kb fragment of the human argininosuccinate synthetase cDNA; plasmid pOTC, containing a 1.2-kb fragment of the human OTC cDNA; and plasmid and plasmid pCPS-I, containing a 5.65-kb fragment of rat CPS-I, were purchased from American Type Culture Collection (Rockville, MD). The plasmid pDF8 containing a 1.06-kb fragment corresponding to the central region of the rat 18S rRNA gene was provided by Richard Torzynski (Cytoclonal Pharmaceutics Inc., Dallas, TX).
The cDNA inserts were excised from plasmids by restriction enzyme digestion followed by separation in low-melting temperature agarose gel and purified using the Wizard DNA purification system (Promega, Madison, WI). Radiolabelled cDNA probes were generated using 32P[dCTP] and the Ready-to-go DNA labeling kit (Pharmacia, Piscataway, NY) by oligonucleotide priming. The specific activity of cDNA probes was approximately 109 cpm/µg of DNA.
Northern Blotting
Total RNA was extracted from liver biopsy samples (Chomczynski and Sacchi, 1987), and a 20-µg aliquot was separated by electrophoresis through a 1% agarose gel (Tsang et al., 1993) and transferred to Genescreen membrane (NEN Life Science Products, Boston, MA) by capillary action. The RNA was crosslinked using UV light, and the membrane was baked at 80°C for 2 h to remove any residual formaldehyde as per manufacturer’s instructions. Membranes were prehybridized for 12 h in 50% deionized formamide, 5 x SSPE, 5x Denhardt’s, 10% dextran sulfate, and 200 µg/mL denatured herring sperm DNA at 42°C for 6 to 18 h. Hybridization was performed in the same solution with the addition of 32 P-labeled cDNA (2 x 106 cpm/mL) for 16 h at 42°C. Following hybridization, membranes were washed twice in 2x SSC (0.3 M NaCl, 0.03 M sodium citrate; pH = 7.0) for 5 min at room temperature, twice in 2x SSC, 1% SDS sodium dodecyl sulfate at 65°C for 30 min, and twice in 0.1x SSC for 30 min at room temperature.
Expression of mRNA was visualized by exposing membranes to Kodak X-Omat AR film and quantified using Kodak Digital Science 1-D Image Analysis software (Eastman Kodak Co., Rochester, NY). Multiple sets of combs within a gel were necessary to accommodate all samples for the experiment. To account for possible differences between sets of samples, a pooled sample (20 µg) of bovine liver RNA was added to outside lanes within each comb and was used to adjust for variation in transfer of RNA and hybridization conditions. Variations in sample loading were adjusted using18S rRNA within each sample.
Plasma Analysis
Glucose concentrations were determined by a glucose oxidase method (Sigma kit #510-A; Sigma Diagnostics, St. Louis, MO). Plasma NEFA was measured using the NEFA C kit (Wako Chemical Co., Dallas, TX). Plasma urea nitrogen (PUN) concentrations were determined by a colorimetric method (Sigma #535; Sigma Diagnostics). Plasma glucagon and insulin were determined using radioimmunoassay kits and the human standards supplied (Diagnostic Products Corporation, Los Angeles, CA). Variation within assay for insulin averaged 5.7%, and variation between assays was 5.1%. Variation within assay for glucagon averaged 4.9%, and variation between assays was 2.4%.
Preparation of Liver Slices and Measures of Gluconeogenesis
Liver biopsy samples from cows in Experiment 2 were obtained using a 9-mm diameter biopsy needle (Greenfield et al., 2000b), transported on ice to the laboratory in Dulbecco’s Modified Eagles medium containing 1% BSA. Within 40 min of removal from the animal, the biopsy core was sliced into 3- to 4-mm uniform sections weighing 30 to 50 mg; slices for each cow were incubated in Dulbecco’s Modified Eagles medium containing 1% BSA and 2.5 mM propionate, 1.0 mM pyruvate, and 1.0 mM lactate as outlined previously for bovine hepatocytes (Donkin and Armentano, 1993). The metabolism of [2-14C]propionate and [U-14C]lactate to glucose was determined over a 2-h interval as previously described (Donkin and Armentano, 1993). Measurements were performed in triplicate for 1-h and 2-h incubations. Incubations were terminated by the addition of 0.5 mL of 5N H2SO4 to the media. Explant cultures were removed from the flasks, homogenized in 1 mL of DNA buffer, and assayed for DNA content as described previously (Donkin and Armentano, 1993). The conversion of radiolabelled precursor to glucose was determined in the media as described previously (Donkin and Armentano, 1993). Data for 1- and 2-h incubations were used to test for linearity of rates of gluconeogenesis, which are expressed as nanomoles of precursor converted to glucose per microgram of DNA per hour.
Statistics
Data for experiment 1 were analyzed using PROC MIXED of SAS (1999). For repeated measurements, the model included the fixed effects of treatment and time, random effects of cow within treatment by time, the interactions of fixed effects, and the residual error. Time represented for d 5, 10, and 20. The covariance structure was determined for anti-dependence, simple, unstructured, and autoregressive models. The model that yielded the minimum range of values for Akaike information criterion and Bayesian information criterion for each variable was used for data analysis. Comparisons between means for control and FR groups were determined using single degree of freedom contrasts. For experiment 2, data were analyzed using PROC MIXED (1999). The model accounted for the effects of treatment (feed restriction or ad libitum feeding) and cow within treatment. Data for mRNA and gluconeogenic activity were combined for both sampling times (ad libitum and restricted feeding), and Pearson correlation coefficients were obtained using the PROC CORR of SAS (1999). Data are presented as least squares means and standard errors.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). Dry matter intake for the FR cows averaged 43% less than control cows during the period of imposed feed restriction. Average feed intake and milk production for the control cows were not different for d 1 through 5 compared with d 6 through 10 of the experiment.
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; Figure 1). The negative energy balance for the restricted cows represents a potential loss of 1.94 kg/d, whereas the ad libitum fed cows had a potential gain of 2.42 kg/d given a starting BCS of 3.5 (NRC, 2001). The imposed reduction in feed intake in the absence of an offsetting reduction in milk production provided a model to investigate changes in hepatic gene expression when energy intake was limiting.
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) likely because of compensatory decreases in milk production.
Glucose concentrations (mg/dL) were not different for the 2 groups of cows during d 6 through 10 d of the trial (Table 3). Blood glucose levels in FR cows with feed withdrawal for 24 to 48 h were previously observed to decrease (Baird et al., 1972; Reid et al., 1977; Athanasiou and Phillips, 1978), but in other instances a 6-d feed withdrawal period had relatively little effect on blood glucose (Lomax and Baird, 1983). The lack of change in blood glucose in the present trial suggests an effect of imposed feed restriction that permitted compensatory changes in glucose synthesis, glucose utilization, or both and maintenance of normal glycemia.
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). Nonesterified fatty acid levels were elevated for FR cows during d 6 through 10 of trial; values for control cows remained unchanged compared with samples taken on d 5. Plasma NEFA declined below pre-restriction values during d 11 through 20 of the trial. Both groups were at 40% of values obtained during d1 through 5. The differences between NEFA during these phases of the experiment are not readily apparent. The change in NEFA during partial feed restriction is less than values reported in the literature for feed restriction (Drackley et al., 1991), and it is also considerably less than changes in NEFA measured at calving (Greenfield et al., 2000b). Feed withdrawal increased NEFA by 25% within 24 h in dairy cattle, and concentrations declined during realimentation but failed to match levels prior to feed restriction after 48 h (Athanasiou and Phillips, 1978). An imposed reduction in feed intake of 80% caused a 2-fold increase in blood NEFA when compared with control cows (Drackley et al., 1991). In addition, net splanchnic uptakes of FFA were increased 400% during a 6-d feed withdrawal period for dairy cows (Lomax and Baird et al., 1983). The rise in NEFA is due to the lipid mobilization triggered by an imposed energy deficit. Decreased NEFA serum levels during d 11 through 20 of the present study suggest restoration to energy equilibrium.
Plasma urea nitrogen was similar for the 2 groups of cows during d 1 through 5 of trial. However, PUN levels tended to be lower for the FR cows compared with control cows during d 6 through 10 of the trial. Activity of the urea cycle, in nonruminants, increases during starvation and in response to high-protein diets (Morris, 1992). Levels of MUN decreased (P < 0.05) for FR cows during d 6 through 10 when compared with the control group (Table 2). Plasma urea nitrogen is directly proportional to MUN levels, and both are responsive to several factors in lactating cows (Roseler et al., 1993; Jonker et al., 1998). Changes in PUN and MUN during the trial suggest differences in protein supply during d 6 through 10. Excessive ruminal protein degradation results in increased urea in blood, milk, and urine. The decrease in PUN and MUN with feed restriction in the present study suggests a reduction in ammonia load from the rumen with reduced N intake as noted previously (Jonker et al., 1998) or more efficient use of absorbed protein.
The concentrations of insulin and glucagon were not altered by feed restriction (Table 3). Similarly, the molar ratios of these hormones were not changed in response to feed restriction. A similar lack of response to feed restriction has been previously observed for peripheral insulin and glucagon (Drackley et al., 1991). Plasma insulin and glucagon concentrations were not altered in lactating cows given 50% of ad libitum feed intake (De Boer et al., 1986). In rats, peripheral glucagon concentrations are relatively constant during feed deprivation, despite elevated hepatic portal blood concentrations of glucagon (Balks and Jungermann, 1984). In beef steers, peripheral insulin and glucagon are elevated with increased feed intake because of increased splanchnic flux of these hormones, which exceeds their hepatic removal (Lapierre et al., 2000). The severity of feed restriction in the present study might not have been adequate to alter insulin and glucagon release. Alternatively, changes in splanchnic flux of insulin and glucagon, in response to reduced feed intake, might have been offset by compensatory changes in hepatic extraction of these hormones.
Expression of PC mRNA (Figure 2) was similar between the 2 groups during the first 5 d of trial (P > 0.05). Conversely, during d 6 through 10, abundance of PC mRNA increased (P < 0.05) for FR cows compared with the previous 5-d period. Expression of PC mRNA for control cows was not different between d 1 through 5 and d 6 through 10 of the experiment. Elevated expression of PC and other gluconeogenic enzymes during feed withdrawal and starvation have been reported in the rat (Jitrapakdee and Wallace, 1999). There is an obligatory requirement for glucose as an oxidizable substrate for brain, erythrocytes, kidney medulla, and mammary tissue (Mayes, 1996). Therefore, feed restriction results in increased gluconeogenesis from lactate, amino acids, and glycerol to meet glucose needs (Baird et al., 1980). An increase in PC activity in feed deprived sheep supports increased use of lactate and alanine during feed withdrawal (Filsell et al., 1969). A strong correlation between PC mRNA and PC activity in bovine liver (Greenfield et al., 2000a) supports an increase in the synthesis of oxaloacetate from pyruvate during feed restriction because of increased PC activity.
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) indicate an increase in PC mRNA abundance (P = 0.06) during the feed restriction period. The changes in PC mRNA are mirrored by an increase (P < 0.05) in lactate conversion to glucose in the same biopsy samples. Furthermore, the Pearson correlation coefficient for the PC mRNA and lactate conversion to glucose was 0.64 (P < 0.05; n = 10). Taken together, these data indicate that changes in PC expression are indicative of a change in gluconeogenic capacity from lactate.
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). Expression of PEPCK, a key gluconeogenic enzyme in nonruminants, is upregulated in response to feed deprivation, glucagon, and glucocorticoids and is reduced when feeding a carbohydrate-rich diet or by elevated insulin (Pilkis and Granner, 1992). In nonruminants, the activity of PEPCK is primarily regulated at the level of transcription of the gene (O’Brien and Granner, 1991). The activity of PEPCK did not change during starvation in dairy cows but was induced in lactating compared with non-lactating cows (Ballard et al., 1969) and as cows approach peak milk production (Greenfield et al., 2000a). The lack of simultaneous changes of mRNA expression for PC and PEPCK during feed restriction suggests a change in capacity for pyruvate and lactate metabolism that may not be matched by increased capacity for glucose synthesis from precursors that contribute to the oxaloacetate pool in liver. An increase in gluconeogenesis from lactate coupled with lack of change in gluconeogenesis from propionate during feed restriction indicates specific changes in gluconeogenesis prior to the cytosolic phosphoenolpyruvate step of gluconeogenesis. Compartmentalization of oxaloacetate metabolism cannot be determined from these experiments; however, to maintain a net balance of NADH in glucose synthesis propionate and lactate, carbon would preferentially utilize PEPCK-C and PEPCK-M, respectively. Mitochondrial PEPCK is usually considered as unregulated and constitutive. These data suggest that the activity of PEPCK-M in dairy cattle is adequate to accommodate increased gluconeogenesis from lactate and points to PC as a controlling step for gluconeogenesis from lactate.
Expression of CPS-I mRNA abundance (Figure 3) during d 6 through 10 did not change (P > 0.05) for FR cows compared with controls. Expression of mRNA for argininosuccinate synthetase and OTC was not different between d 1 through 5 and d 6 through 10 for control and FR cows. Enzymes of the urea cycle catalyze the conversion of ammonia and carbon dioxide into urea, which serves to detoxify ammonia, the primary nitrogenous waste product derived from protein catabolism (Christowitz et al., 1981). It is important to note that induction of urea enzymes is slow, requiring 5 to 7 d to achieve a new steady state, and, in the case of arginase, the half-life of the enzyme is 5 d (Schimke, 1962, 1964). Previous work with rats suggests a slight increase in urea cycle enzyme activity after a 48-h feed withdrawal (Snodgrass et al., 1978). Urea cycle enzyme activity is elevated in response to starvation and high-protein diets (Morris, 1992). The current data from dairy cows would indicate that feed restriction reduces urea formation without a change in expression of urea cycle enzymes. Although feed restriction did not alter urea cycle enzyme mRNA compared with samples taken prior to feed restriction, there was a tendency (P = 0.08) for expression of CPS-I mRNA to increase in FR cows during the realimentation period. Elevated levels of CPS-I mRNA during the realimentation phase might be due to a delayed effect in response to feed restriction.
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FOOTNOTES |
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Current address: Abbott Laboratories, Abbott Park, IL.
Received for publication June 8, 2004. Accepted for publication April 5, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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