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Division of Nutrition and Physiology, Institute of Animal Genetics, Nutrition and Housing, Faculty of Veterinary Medicine, University of Berne, CH-3012 Berne, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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0.05) plasma concentrations of glucose, insulin, and glucagon more in colostrum-fed than in formula-fed calves but increased (P 0.05) urea concentrations and decreased (P 0.05) concentrations of NEFA, ACTH, and cortisol independent of colostrum vs. formula feeding. Colostrum feeding increased (P < 0.05) plasma glucose, but decreased (P < 0.05) plasma urea concentrations. Glucagon-to-insulin ratios in DEXA-treated and colostrum-fed calves were decreased (P < 0.05). Dexamethasone treatment decreased hepatic mRNA levels and activities of PC (P < 0.001 and P < 0.10) and activities of PEPCK (P < 0.001) but increased (P < 0.001) the glycogen content. Colostrum feeding increased (P < 0.05) mitochondrial PEPCK mRNA levels and PEPCK activities in calves not treated with DEXA but decreased (P < 0.1) amounts of PC mRNA. In conclusion, increased plasma glucose concentrations after DEXA treatment were not associated with a stimulation of hepatic gluconeogenic enzyme activities; however, colostrum feeding probably raised plasma glucose concentrations because of increased hepatic gluconeogenic activities.
Key Words: Calves • Colostrum • Glucocorticoids • Gluconeogenesis • Phosphoenolpyruvate Carboxykinase • Pyruvate Carboxylase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Hammon and Blum, 1998; Rauprich et al., 2000). Thus, glycogenolysis and especially gluconeogenesis are important for neonatal glucose homeostasis (Girard et al., 1992; Liggins, 1994). Cortisol enhances glucose production and glycogen storage around birth in humans and in precocious species like ruminants (Liggins, 1994; Fowden, 1997). Plasma cortisol concentrations markedly increase in the calf fetus and remain elevated during the neonatal period (Liggins, 1994; Hammon and Blum, 1998); however, it is not known whether cortisol stimulates gluconeogenesis in neonatal calves. In addition, colostrum feeding may affect the activity of enzymes involved in gluconeogenesis.
Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and pyruvate carboxylase (PC; EC 6.4.1.1) are rate-limiting enzymes for gluconeogenesis in liver (Rognstad, 1979; Girard et al., 1992; Donkin, 1999) and are regulated by glucocorticoids (Pilkis and Granner, 1992; Hanson and Reshef, 1997; Jitrapakdee and Wallace; 1999). Both enzymes are active in the bovine fetus (Prior and Scott, 1977). High hepatic gluconeogenic rates, such as during the postpartum period of lactating cows, are associated with increased expression of PEPCK and PC (Greenfield et al., 2000; Agca et al., 2002).
The objective of this study was to determine the effects of dexamethasone (DEXA) on plasma metabolites and hepatic gene expression in newborn calves fed either colostrum or milk-based formulas (Rauprich et al., 2000). Diets were designed to supply identical levels of macronutrients but differed with respect to hormones and growth factors present in colostrum. We tested the hypothesis that DEXA treatment and different feeding variably affect glucoregulatory hormones, PEPCK and PC gene expression and/or enzyme activities, and hepatic glycogen content.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Twenty-eight male calves (11 Holstein Friesian, 12 Simmental x Red Holstein, one Red Holstein x Limousin, and four Brown Swiss) were studied. They were born at the Federal Research Station for Animal Production (Posieux, Switzerland) or at neighboring farms. Calves were born without extrinsic help and were separated immediately after birth from their dams and held on straw in boxes for 5 d.
The colostrum fed to calves was from cows of the Federal Research Station for Animal Production (Posieux, Switzerland). Cows were milked twice daily, and the colostrum of Milkings 1, 3, and 5 was stored separately in plastic bottles at -20°C. Three separate pools, one each from Milkings 1, 3, and 5, were prepared at the beginning of the study. Each of these pools was stored in multiple aliquots in plastic bottles at -20°C. Before feeding, colostrum was warmed to 40°C and then fed immediately. In addition, three formulas were created that contained nutrients (protein, fat, lactose) in amounts comparable to those of the colostrum of Milkings 1, 3, and 5 after parturition but that contained almost no biologically active substances, such as immunoglobulins, hormones, and growth factors, and were fed on d 1, 2, and 3 after birth, respectively. Formulas were produced by UFA AG (Sursee, Switzerland) and consisted of calcium caseinate (Emmi Milch AG, Lucerne, Switzerland), lactalbumin 90 (Emmi Milch AG), milk fat (55% of DM; Institute Agricole de l’Etat de Fribourg, Grangeneuve, Switzerland), and a vitamin and mineral premix (Provimi, Cossonay-Gare, Switzerland). The three formulas were dissolved by adding water and stored in plastic bottles at -20°C until used. Before feeding, the bottles were warmed to 40°C and then fed immediately. The milk replacer (UFA 200 Natura, without antibiotics; UFA AG) was prepared as a 100 g/L solution. Compositions of colostrum and formula and milk replacer are given in Table 1.
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). Each group consisted of seven calves. Calves were primarily grouped with the goal of obtaining similar BW and breed distributions in all four groups. Calves of CD- and CD+ received pooled colostrum obtained from Milkings 1, 3, and 5 (d 1, 2, and 3 after parturition, respectively) on the first 3 d of life. Calves of FD- and FD+ were fed three milk-based formulas on d 1, 2, and 3 that contained amounts of nutrients similar to those of the colostrum of Milkings 1, 3, and 5, respectively. Calves of all groups received a milk replacer on d 4. Amounts of colostrum and formula fed were 6% of BW on d 1, 8% of BW on d 2, and 10% of BW from d 3 on. Calves were fed by bottle twice daily. To ensure that all calves received the same amounts of formula or colostrum, calves with reduced appetite were tube-fed their planned amounts of formula or colostrum. Calves received their first meal at 3.1 ± 0.04 h (FD-), 2.2 ± 0.02 h (FD+), 2.4 ± 0.03 h (CD-), and 3.4 ± 0.04 h (CD+) after birth, respectively. The following feedings were at 8, 24, and 32 h after the first feeding. From d 3 on, calves were fed daily at 0800 and 1600. Additionally, calves of FD+ and CD+ were injected i.m. with DEXA (30 µg/kg BW x d; DEXA, Lohmann Animal Health, Cuxhaven, Germany) from d 1 to 4. We chose the dose of DEXA within the range of doses given to neonatal calves (Coxam et al., 1990).
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Blood Analyses
Blood samples were taken from the jugular vein into evacuated tubes on d 1, 2, and 5 and with a catheter on d 4. Tubes containing dipotassium-EDTA (1.8 g/L blood) were used for the determination of pre- and postprandial values of glucose, lactate, NEFA, urea, insulin, glucagon, and ACTH at 0, 1, 2, 4, and 8 h after the first, third, and seventh feeding and on d 5, respectively. These tubes were also used to collect blood for the measurement of plasma concentrations of cortisol in pre- and postprandial samples on d 1 and 2; in 25 samples on d 4, taken before (0 h) and every 20 min after morning feeding for 8 h for the evaluation of secretory patterns; and on d 5. Tubes were put on crushed ice until centrifuged at 1,000 x g for 20 min. Supernatants were aliquoted and stored at -20°C.
Plasma concentrations of glucose and urea were measured using kits from Hoffmann-La Roche, Basle, Switzerland (catalog No. 07 3671 6 and 07 3685 6, respectively), L-lactate measured with a kit from Bio Mérieux, Marcy l’Etoile, France (catalog No. 61192), and NEFA measured with a kit from Wako Chemicals, Neuss, Germany (catalog No. 994-75409). For the analysis of these compounds, we used the automatic analyzer Cobas Mira Plus (Roche). Concentrations of insulin, glucagon, and cortisol were measured by RIA as described by Hammon and Blum (1998). Plasma concentrations of ACTH were analyzed using a kit from Nichols Institute Diagnostics (San Juan Capistrano, CA).
Analyses in Formula, Colostrum, and Milk Replacer
Samples of Formula 1, 2, and 3, and samples of the individual pools of colostrum of Milkings 1, 3, and 5 were lyophilized to determine DM, CP (by Kjeldahl method), crude fat (by Berntrop method), and ash (after combustion at 550°C) using standard procedures at the Swiss Federal Research Station for Animal Production (Posieux, Switzerland). Contents of nitrogen-free extract (NFE) and GE (based on energy equivalents of 36.6, 17.0, and 24.2 MJ/kg fat, NFE, and crude protein, respectively) were calculated. Information on contents of the milk replacer was given by the producer. Concentrations of insulin and IGF-I in formula and colostrum were analyzed as described (Hammon and Blum, 1998; Sauter et al., 2003a).
Analyses in Liver
Calves were slaughtered on d 5 of life and liver samples were transferred either in liquid nitrogen for measurement of enzyme activities and glycogen or in TRIzol reagent (Gibco BRL, Basle Switzerland) and then frozen in liquid nitrogen for mRNA measurements. Liver samples were stored at -80°C until analyzed for mRNA of PEPCK and PC and for measurement of activities of PEPCK and PC and glycogen concentrations.
Quantification of PEPCK and PC mRNA by Real-Time PCR.
For mRNA measurements, total RNA was extracted using TRIzol reagent (Gibco BRL) and resuspended in RNase-free water, which was treated with diethyl pyrocarbonate (DEPC, Sigma-Aldrich, Deisendorf, Germany). The integrity and purity of RNA were tested by measurement of optical density (ratios at 260 and 280 nm being greater than 1.9) and by electrophoresis using ethidium bromide staining. Total RNA was reverse-transcribed into cDNA using hexamer primers (Pharmacia Biotech, Buckinghamshire, U.K.) as described (Pfaffl et al., 2002).
Real-time reverse transcriptase (RT)-PCR was performed by LightCycler (Roche Molecular Biochemicals, Rotkreuz, Switzerland) using SYBR Green I as fluorescence dye (Pfaffl et al., 2002). Primers were created for cytosolic PEPCK (PEPCK-C), mitochondrial PEPCK (PEPCK-M), and PC. The design of the primers is shown in Table 3. Primers were designed to flank a region that contains at least one intron to ensure that no contaminating genomic DNA was amplified that could lead to false signals. Primers for PEPCK-C and PC were based on bovine sequences (Agca et al., 2000, 2002). The forward primer for PEPCK-M was designed based on the human PEPCK-M sequence (Modaressi et al., 1998) because the known bovine sequence (Agca et al., 2002) used for the design of the PEPCK-M reverse primer does not contain introns.
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). Products were verified by sequencing. The PCR products were purified with the High Pure PCR purification kit (Roche Molecular Diagnostics) and directly sequenced with the Rhodamine dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) using the appropriate PCR primers. After purification of sequencing products by ethanol precipitation, they were run on an ABI 3100 Genetic Analyzer (Applied Biosystems). Sequences were edited and proofread in both directions using Sequencher (GeneCodes, Ann Arbor, MI). The PCR products for PC and PEPCK-C showed 100% and 99% homology with the bovine sequences used for primer design. The PCR product of PEPCK-M showed 91% homology with human PEPCK-M, but only 72% homology with bovine PEPCK-C.
Quantification of mRNA was performed by relative expression using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene transcript (Pfaffl, 2001). For each calf, the crossing point (CP) values, which represent the intersection of a fixed threshold above background fluorescence and the amplification curve (Rasmussen, 2001), were determined for each enzyme and for GAPDH. Values of CP for enzymes and for GAPDH were corrected for different runs by an internal standard (
CP). For each sample, CPGAPDH was used for normalization of the amount of every target gene (CPtarget) of corresponding sample, which resulted in CP (= CPtarget - CPGAPDH). Relative expression levels, R, within each enzyme were given by the arithmetic formula R = 2-CP (Pfaffl, 2001). The logarithm (log 2) is based on an optimum efficiency, E, of PCR, which is E = 2, when the PCR product is replicated every cycle. Based on CP evaluation, the expression of GAPDH in our study was not affected by different feeding or DEXA treatment. Intraassay and interassay CV for PEPCK-C were 0.5% and 0.4% and for PEPCK-M were 0.7% and 0.9%. Intraassay and interassay CV for PC were 0.4% and 0.5%.
Enzyme Activities.
Measurements of PEPCK and PC activities in bovine liver followed Greenfield et al. (2000) and Agca et al. (2002). Liver tissue was homogenized in buffer (1:3, wt/vol) containing cold 0.1 M sucrose, 50 mM potassium phosphate (pH 7.4), and 0.25 mM EDTA (pH 7.4). Samples were sonicated for 10 s to disrupt mitochondria. The homogenates were kept on ice at all times. Activities of PEPCK were determined in cell homogenates by measuring the carboxylation of phosphoenolpyruvate to oxaloacetate using NaH14CO3 (Ballard and Hanson, 1967) with the addition of NADH and malate dehydrogenase to ensure the conversion of the reaction product, oxaloacetate, to malate (Atkin et al., 1979). Activities of PC were assayed in the crude homogenate by NaH14CO3 incorporation into oxaloacetate and citrate in the presence of pyruvate (Atkin et al., 1979).
Glycogen and Protein Determination.
Hepatic glycogen concentrations were converted to glucose by amyloglucosidase (EC 3.2.1.33), as described by Roehrig and Allred (1974). Glucose concentrations were measured as described above. The protein concentration in liver was determined using a kit (BCA Protein Assay Reagent; Pierce, Rockford, IL).
Statistical Procedures
Values for blood traits and mRNA concentrations were expressed as means ± SEM. For plasma concentrations, areas under the concentration curves were computed for each day as measures of mean concentrations between 0 and 8 h after the morning meal. Because data showed normal distribution, data were evaluated using the RANDOM and REPEATED methods of the MIXED procedure (SAS Inst. Inc., Cary, NC). Separate models were applied for the calculation of preprandial and mean plasma concentrations during the whole experimental period of 5 d and for the calculation of postprandial effects on d 1, 2, and 4, respectively. The DEXA treatment, diet, and time were used as fixed effects, and the individual calves were used as random effects. For the evaluation of differences in DEXA responses with regard to different diet and for evaluation of differences in time pattern within DEXA or feeding groups, interactions (DEXA x diet; DEXA x time; diet x time) were included in the model. Treatment, diet, and time differences were localized by Bonferroni t-test (P < 0.10 for a trend and P < 0.05 for significant difference). Episodic secretion of cortisol on d 4 (mean concentrations, basal concentrations, peak amplitudes, and peak frequencies) were analyzed according to Merriam and Wachter (1982). Group differences were analyzed by the GLM procedure, and differences were localized by Bonferroni t-test (P < 0.10 for a trend and P < 0.05 for significant difference).
Amounts of mRNA, enzyme activities, and glycogen content in liver were evaluated using the GLM with DEXA treatment and diet as main effects. The DEXA x diet interaction was included into the model. The DEXA treatment and diet differences were localized by Bonferroni t-test (P < 0.05).
The PROC CORR procedure of SAS was used to calculate correlations between abundance of mRNA, enzyme activities, glycogen content, and plasma concentrations of metabolites on d 5.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Blood Analyses
Mean plasma glucose concentrations (Figure 1) increased (P < 0.001) in all four groups from d 1 to 2. Postprandial concentrations increased in all four groups on d 2 (P < 0.001) and d 4 (P < 0.05). Concentrations were higher (P < 0.01) in DEXA-treated calves than in untreated calves on all days and were higher (P < 0.05) in colostrum-fed than in formula-fed calves on d 4. There were significant DEXA x diet interactions on d 4 and 5, and glucose concentrations increased more (P < 0.05) after DEXA treatment in colostrum-fed than in formula-fed calves. Plasma lactate concentrations (Table 4) decreased (P < 0.001) with time, but neither feeding nor DEXA treatment had an effect. Plasma urea concentrations (Table 4) changed (P < 0.001) with time and were affected (P < 0.001) by DEXA and diet. Urea concentrations were higher (P < 0.01) on d 4 and 5 in DEXA-treated calves than in untreated calves and were higher (P < 0.05) on d 4 in formula-fed than in colostrum-fed calves. Plasma concentrations of NEFA (Table 4) decreased (P < 0.001) with time. Plasma concentrations of NEFA were influenced by DEXA treatment and were lower (P < 0.001) in DEXA treated than in untreated calves on d 4 and 5.
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) changed (P < 0.10 or less) after feeding on d 1, 2, and 4. On d 1, preprandial insulin concentrations were lower (P < 0.05) in DEXA-treated calves than in untreated calves, and the postprandial insulin response tended to be higher (P < 0.10) in colostrum-fed than in formula-fed calves. On d 2, the postprandial insulin response was greater (P < 0.05) in DEXA-treated than in untreated calves. On d 4 and 5, insulin concentrations were higher (P < 0.001) in DEXA-treated than in untreated calves. There were significant DEXA x diet interactions on d 4 and 5, and insulin concentrations increased more (P < 0.05) after DEXA treatment in colostrum-fed than in formula-fed calves. Mean plasma glucagon concentrations (Figure 3) decreased in untreated groups (P < 0.05) from d 2 to d 4. Glucagon concentrations changed (P < 0.05) with time after feeding on d 1 and 4. Glucagon concentrations were higher (P < 0.05) in DEXA-treated than in untreated calves on d 4 and 5. There was a significant DEXA x diet interaction on d 5, and glucagon concentrations after DEXA treatment increased more (P < 0.05) in colostrum-fed than in formula-fed calves. The glucagon-to-insulin ratios of preprandial plasma concentrations on a molar basis were lower (P < 0.01) in DEXA-treated than in untreated calves and were lower (P < 0.05) in CD- than in FD- (mean ratios of preprandial plasma concentrations from d 2 to d 5 were 0.59 ± 0.03, 0.29 ± 0.03, 0.43 ± 0.03, and 0.26 ± 0.02 for FD-, FD+, CD-, and CD+, respectively).
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) tended to decrease (P < 0.10) from d 1 to d 4. The DEXA treatment reduced (P < 0.05) preprandial and mean ACTH concentrations on d 2 and 4 (mean ACTH concentrations on d 2 were 41 ± 2 ng/L for FD- and CD- and 31 ± 3 ng/L for FD+ and CD+ and on d 4 were 28 ± 2 ng/L for FD- and CD- and 23 ± 2 ng/L for FD+ and CD+). Cortisol concentrations (Figure 4) decreased (P < 0.001) in all four groups from d 1 to d 4. Plasma cortisol concentrations decreased (P < 0.05) after feeding on d 1 in all groups and on d 2 and 4 in untreated groups. Four hours after the first DEXA treatment on d 1, cortisol concentrations were lower (P < 0.05) in DEXA-treated calves than in untreated calves and then remained lower (P < 0.001) during the investigated period. Analyses of the secretory pattern of cortisol on d 4 revealed no secretory peaks of cortisol in DEXA-treated groups. Mean and basal cortisol concentrations as well as amplitudes were lower (P < 0.001) in DEXA-treated than in untreated calves. There was a significant DEXA x diet interaction (P < 0.05) on d 5, and cortisol concentrations were higher in FD- than in CD.
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) were higher (P < 0.05) in colostrum-fed than in formula-fed calves, but PEPCK-C mRNA levels showed no group differences. Amounts of PC mRNA were lower (P < 0.001) in DEXA-treated than in untreated calves and tended to be lower (P < 0.10) in colostrum-fed than in formula-fed calves. Activities of PEPCK (Table 5) on d 5 were decreased (P < 0.001) in DEXA-treated calves and were higher (P < 0.05) in CD- than in CD+. Activities of PEPCK were higher (P < 0.05) in CD- than in FD-. Activities of PC tended to be lower (P < 0.10) in DEXA-treated than in untreated calves. Protein concentration did not differ between groups (mean: 0.14 ± 0.02 g/g tissue).
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Correlations
There were positive correlations between mRNA levels and activities of PC (r = 0.35; P < 0.10), between mRNA levels of PEPCK-C and PC (r = 0.42; P < 0.05), and between activities of PEPCK and PC (r = 0.66; P < 0.001), but there were no significant correlations between PEPCK-C or PEPCK-M mRNA levels and PEPCK activities. Plasma glucose concentrations correlated negatively with PC mRNA levels (r = -0.47; P < 0.05), with activities of PEPCK (r = -0.51; P < 0.01) and PC (r = -0.5; P < 0.01), but correlated positively with hepatic glycogen content (r = 0.68; P < 0.001), and with plasma concentrations of insulin (r = 0.5; P < 0.01) and glucagon (r = 0.4; P < 0.05). Plasma insulin concentrations correlated negatively with PC mRNA levels (r = -0.47; P < 0.05) and with PEPCK activity (r = -0.46; P < 0.05), but correlated positively with hepatic glycogen content (r = 0.71; P < 0.001), and with plasma concentrations of glucagon (r = 0.69; P < 0.001). Plasma glucagon concentrations correlated negatively with PEPCK activities (r = -0.41; P < 0.05), but correlated positively with hepatic glycogen content (r = 0.4; P < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Agca et al., 2002). However, transition cows differ in their physiological and developmental state from neonatal calves. Measurements of PEPCK and PC mRNA in transition cows by real-time PCR resulted in the same pattern of mRNA levels before and after calving as previously reported (Greenfield et al., 2000; Agca et al., 2002; M. Reist, H. M. Hammon, and J. W. Blum, unpublished observations). Furthermore, ratios of PEPCK-C to PEPCK-M mRNA levels in our study were in the same range as seen in cows (Agca et al., 2002). Therefore, we believe that our method of relative quantification of PC and PEPCK mRNA levels by real-time PCR is valid and provides in other situations the expected data. Differences in PC mRNA regulation between neonatal and adult cattle may also result from different splicing of the 5' untranslated region of the PC mRNA that was recently shown in cattle (Agca et al., 2000). Furthermore, PC activities, but not mRNA levels, depend on the availability of biotin, which may differ in neonates and adults (Pearce and Balnave, 1978; Rodriguez-Melendez et al., 1999).
The hyperglycemic effect of glucocorticoids is well established in cattle (McDowell, 1983; Brockman and Laarveld, 1986; Maciel et al., 2001), including veal calves (Sternbauer et al., 1998). The increase of plasma glucose concentrations by glucocorticoids is thought to be at least in part due to enhanced hepatic gluconeogenesis (McDowell, 1983; Kraus-Friedmann, 1984; Brockman and Laarveld, 1986). In addition, PEPCK and PC are known as rate-limiting enzymes for gluconeogenesis (Rognstad, 1979; Girard et al., 1992; Donkin, 1999) and their activities are stimulated by glucocorticoids (Kraus-Friedmann, 1984; Pilkis and Granner, 1992; Jitrapakdee and Wallace, 1999). Therefore, it was very surprising that expression and activities of PEPCK and PC were either unaffected or were even reduced after DEXA administration in the present study. An additional study, in which calves from d 3 of life up to d 42 were treated with DEXA, also showed inhibitory effects of DEXA treatment on hepatic PC and PEPCK mRNA levels and/or activities, which supports the present findings (Hammon et al., 2003). Provided that PEPCK and PC activities correspond with hepatic gluconeogenic activities, the elevated plasma glucose concentrations in DEXA-treated calves probably did not result from increased hepatic gluconeogenesis in our calves. However, we have not measured glucose production in liver of these calves. Elevated plasma glucose concentrations might be a result of reduced glucose utilization, as shown in humans and veal calves (Shamoon et al., 1980; Sternbauer et al., 1998). Glucocorticoids provoke decreased insulin sensitivity in muscle tissue, which leads to inhibition of glucose uptake by muscle and to peripheral insulin resistance (McDowell, 1983; Brockman and Laarveld, 1986). This concept is supported by the present study because glucose concentrations were increased despite elevated insulin concentrations in DEXA-treated calves. Furthermore, we have found increased plasma urea concentrations, which indicates accelerated protein breakdown (Dardevet et al., 1999). On the contrary, decreased fat mobilization in DEXA-treated calves, indicated by lower NEFA plasma concentrations, was probably caused by elevated insulin concentrations (McDowell, 1983). Reduced plasma GH concentrations in DEXA-treated calves (Sauter et al., 2003a) may have contributed to the decreased fat mobilization.
Although we presume insulin resistance in muscle tissue, this seems not to be the case in liver. Insulin resistance in liver is characterized by an elevated glucagon-to-insulin ratio, increased mRNA expression and activities of PEPCK and PC, and stimulation of gluconeogenesis (Hanson and Reshef, 1997; Jitrapakdee and Wallace, 1999; Dirlewanger et al., 2000). This was not the case in our study. Because plasma insulin and glucose concentrations partly showed negative associations with gluconeogenic enzyme expression and/or activities in the present study, insulin and glucose might have inhibited hepatic PEPCK and PC activities (Hanson and Reshef, 1997; Cournarie et al., 1999; Jitrapakedee and Wallace, 1999) and have decreased hepatic glucose production (McDowell, 1983; Brockman and Laarveld, 1986; Hostettler-Allen et al., 1993). Glucose inhibits hepatic glucose production independent of endocrine regulation (Moore et al., 1998; Vella et al., 2003). However, evidence that glucose and/or insulin had directly inhibited PEPCK and PC activities and PC expression could not be determined in the present study and it is not known whether glucose itself inhibits hepatic PEPCK or PC synthesis in ruminants. Insulin has a dominant inhibitory effect on DEXA-stimulated expression of gluconeogenetic enzymes, as shown for PEPCK gene expression (Sasaki et al., 1984). In addition, inhibition of PEPCK activity after glucocorticoid treatment due to increased insulin secretion was described in rats (Exton, 1979).
The DEXA treatment increased glucagon concentrations on d 4 and 5 of life. Marco et al. (1973) and Wise et al. (1973) found increased basal glucagon levels in humans after DEXA treatment. They speculated that the increase of amino acids, such as alanine, that resulted from protein catabolism after glucocorticoid treatment might have contributed to enhanced glucagon secretion (Marco et al., 1973; Wise et al., 1973). Administration of DEXA resulted in decreased ACTH and cortisol concentrations in the present study, indicating a suppression of the hypothalamic-pituitary-adrenal axis, as expected (Kooistra et al., 1997; Ng et al., 1997).
In calves treated with DEXA, glucose storage in liver was increased as indicated by elevated glycogen content. An increase of hepatic glycogen content after glucocorticoid administration was also seen in lactating cows (McDowell, 1983). The increased glycogen synthesis might be a result of higher plasma glucose and/or insulin concentrations after DEXA treatment (McDowell, 1983; Moore et al., 1998), a suggestion that is supported by close correlations between hepatic glycogen concentrations and plasma concentrations of glucose and insulin in the present study. If insulin-stimulated hepatic glycogen synthesis was enhanced, this indicates that insulin maintained its hepatic effects despite DEXA treatment. In support of that, cortisol induces hepatic glycogen deposition in the fetus close to term (Kraus-Friedmann, 1984; Fowden, 1997).
Low plasma glucose concentrations at birth, followed by rising concentrations during the ensuing days, are typical for neonates, including calves (Girard et al., 1992; Hammon and Blum, 1998; Rauprich et al., 2000). Colostrum feeding resulted in higher glucose concentrations on d 4 than did formula feeding, an effect that is in agreement with previous studies (Hammon and Blum, 1998; Rauprich et al., 2000). Because nutrient intake was the same in both groups, this may have mainly been the consequence of higher concentrations of hormones and growth factors in colostrum than in formula. Colostrum feeding increased the capacity to absorb glucose and other sugars from the intestine, as demonstrated by enhanced xylose absorption in colostrum-fed vs. formula-fed calves (Rauprich et al., 2000; Sauter et al., 2003b). Additionally, colostrum intake may have stimulated gluconeogenesis, as seen in neonatal pigs (Lepine et al., 1991). This suggestion is supported by higher PEPCK activities in CD- than in FD-. The more marked expression of PEPCK-M in colostrum-fed than in formula-fed calves was surprising, because PEPCK-M is supposed not to be regulated by metabolic changes in mammals (Girard et al., 1992; Hanson and Reshef, 1997), including cows (Agca et al., 2002). However, hepatic PEPCK-M activities increased during neonatal development in guinea pigs (Wicheanvonagoon and Arinze, 1984). Therefore, elevated PEPCK-M expression might be a consequence of improved neonatal development by colostrum feeding (Blum and Hammon, 2000). Contrary to these findings, hepatic expression of PC was depressed in colostrum-fed calves, but PC activities were numerically higher after colostrum feeding in the absence of DEXA treatment.
Lactate, propionate, glycerol as well as amino acids (first of all, alanine) are important precursors of hepatic gluconeogenesis in neonates (Girard et al., 1992), including calves (Donkin and Armentano, 1994). Plasma lactate concentrations declined during the experimental period, but were not affected by feeding. Plasma urea concentrations were higher in formula-fed than in colostrum-fed calves. The greater rate of amino acid desamination should have increased the supply of substrates for gluconeogenesis. However, gluconeogenic enzyme activities were lower or unchanged in formula-fed when compared to colostrum-fed calves, although the glucagon-to-insulin ratio was higher in FD- than in CD-. Therefore, the glucoregulatory system in the liver of formula-fed calves was probably less mature than in colostrum-fed calves. As the protein intake was comparable in formula- and colostrum-fed calves, but protein absorption (mainly, immunoglobulins) was greater in colostrum-fed than in formula-fed calves (Norrman et al., 2003), lower urea concentrations in colostrum-fed calves likely expressed higher protein synthesis and/or reduced protein degradation when compared with formula-fed calves. Nonnutrient components of colostrum may have caused stimulation of protein synthesis, as shown in neonatal pigs (Burrin et al., 1995). Factors like insulin or IGF-I, which are present in high amounts in bovine colostrum, might have affected neonatal intestinal protein metabolism because receptors for IGF and insulin are present in the intestinal mucosa of neonatal calves (Hammon and Blum, 2002; Georgiev et al., 2003).
In contrast to previous studies (Hammon and Blum, 1998; Rauprich et al., 2000), plasma concentrations of insulin, glucagon, and cortisol were not influenced by colostrum feeding, but colostrum feeding modulated DEXA effects on plasma concentrations of insulin, glucagon, and cortisol. Furthermore, the glucagons-to-insulin ratios were greater in FD- than in CD-. The greater glucagons-to-insulin ratio in formula-fed calves was not associated with enhanced activities of gluconeogenic enzymes. Because the glucagon-to-insulin ratio is critical for glucose homeostasis (McDowell, 1983), the higher glucagon-to-insulin ratios in FD-, combined with lower glucose plasma concentrations, indicate a decreased glucagon efficiency on hepatic glucose metabolism in formula-fed than colostrum-fed calves.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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2 This study was supported by the Swiss National Science Foundation (grant No. 32-59311.99).
3 Part of a thesis of S. N. Sauter for doctor of veterinary medicine, accepted by the faculty of veterinary medicine, Univ. of Berne, in 2002.
5 Present address: Federal Veterinary Office, 3097 Liebefeld-Berne, Switzerland.
4 Correspondence: Experimental Station of the Division of Animal Nutrition and Physiology, University of Berne, Route de la Tioleyre 4, CH-1725 Posieux, Switzerland (phone: +41-26-4077294; fax: +41-26-4077297; E-mail: harald.hammon{at}itz.unibe.ch).
Received for publication May 26, 2003. Accepted for publication August 22, 2003.
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-MSH and the differential effects of dexamethasone and haloperidol on the secretion of -MSH and ACTH in dogs. J. Endocrinol. 152:113–121.[Abstract]
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and CD-: 
) and with dexamethasone (DEXA) injections (FD+: • and CD+: 
). On d 4, all calves were fed with milk replacer. Values are means ± SEM, n = 7. D = DEXA effect (P < 0.05); F = feeding effect (P < 0.05); T = time effect (P < 0.05); D x F = DEXA x feeding interaction (P < 0.05); D x T = DEXA x time interaction (P < 0.05); F x T = feeding x time interaction (P < 0.05). Arrows mark time of feeding.
