| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Department of Animal Sciences, Purdue University, West Lafayette, IN 47907
Department of Animal Science, Iowa State University, Ames 50010
3 Corresponding author: sdonkin{at}purdue.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Key Words: gluconeogenesis • gene expression • glucagon
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Glucagon is a key hormone in glucose homeostasis. The release of glucagon is stimulated by hypoglycemic conditions and is inhibited by hyperglycemia and insulin (Cryer et al., 2003). Glucagon primarily acts on liver, brain, pancreas, kidney, intestine, and adipose tissue (Christophe, 1995; Burcelin et al., 1996). The primary action of glucagon on liver is to promote gluconeogenesis, promote glycogenolysis, and enhance glucose output. Exogenous glucagon serves to increase blood glucose concentrations in ruminants (de Boer et al., 1986; Hippen et al., 1999a). However, there is a lack of information regarding the effects of glucagon on expression of key gluconeogenic enzymes and hepatic glucose production in lactating dairy cows.
Exogenous glucagon in nonruminant liver increases phosphoenolpyruvate carboxykinase (PEPCK) mRNA concentrations (Iynedjian and Hanson, 1977; Beale et al., 1984; Christ et al., 1988) through activation of adenylate cyclase and targeted transcription of the PEPCK gene. Activity of pyruvate carboxylase (PC), a key enzyme for the metabolism of lactate to glucose, is increased when glucagon is administered to sheep (Brockman and Manns, 1974). A decrease in PEPCK mRNA and a corresponding increase in PC mRNA were noted when midlactation cows were continuously infused with glucagon over a 3.5-h interval (She et al., 1999). The decrease in PEPCK mRNA in that study was surprising, particularly when compared with results reported in nonruminant literature that indicated an acute action of glucagon to increase liver PEPCK mRNA (Beale et al., 1984; Christ et al., 1988). As discussed previously (She et al., 1999), the effects of acute infusion with glucagon may be the result of increased insulin concentration and the opposing actions of insulin with regard to PEPCK mRNA (Beale et al., 1984).
The objective of this study was to determine the effect of glucagon on expression of PC and PEPCK in lactating dairy cattle at the level of mRNA, enzyme activity, protein abundance, and gluconeogenic capacity. Based on the literature, we hypothesized that short-term treatment with glucagon would result in changes in the expression and activity of PC and PEPCK.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Cow Management and Sampling
All cows were fed a total mixed diet for 10% orts (Table 1
). Cows were fed daily at 0800 h, and feed was always available. To accommodate sample collection, cows were divided into 2 blocks consisting of 4 control and 4 glucagon-treated cows within each block. Each block received the same experimental conditions and sampling protocol but was separated in time by 2 d. The glucagon was a gift from Eli Lilly & Co. (Indianapolis, IN). Lyophilized bovine glucagon was dissolved before injections in 60 mL of 0.15 M NaCl (pH 10.25). Cows were injected in the neck with 3.75 mg of glucagon in 60 mL of 0.15 M NaCl (pH 10.25) at each treatment time. Cows were injected with a total of 4 doses of glucagon over a 21-h period for a total of 15 mg of glucagon given at –21, –14, –7, and 0 h relative to last injection. Control animals received subcutaneous injections of 60 mL of 0.15 M NaCl (pH 10.25) at each of the same time points. Site of injection was alternated from left to right, and animals were observed for any adverse reaction at injection sites.
|
Blood samples were obtained by coccygeal veinpuncture. On occasion when samples could not be collected from tail vein, samples were drawn from a jugular vein. All blood samples were collected into evacuated tubes (Becton Dickinson, Rutherford, NJ) immediately prior to glucagon or saline injections at –21, –14, –7, and 0 h relative to final injection. Additional samples were collected at 1.5, 3, 15, and 27 h relative to final injection. Tubes for NEFA and glucose assays contained sodium fluoride and potassium oxalate. Tubes for glucagon analysis contained Na2-EDTA. Tubes for plasma preparation were put on ice immediately after sampling and centrifuged at 4°C at 3,000 x g for 20 min within 1 h of collection. Samples were stored in borosilicate glass culture tubes (VWR Scientific Products, West Chester, PA) at –20°C until analysis. Prior to storage at –20°C, aprotonin (Boehringer-Mannheim, Indianapolis, IN) was added to samples intended for glucagon analysis as recommended by the product manufacturer (Diagnostic Products Corporation, Los Angeles, CA) to inhibit proteolytic degradation in the samples. Samples were analyzed for serum glucose (Glucose C2 Autokit, Wako Fine Chemicals Industries USA, Inc., Dallas, TX), NEFA (NEFA-C, Wako Fine Chemicals Industries USA, Inc.; Johnson and Peters, 1993), and BHBA (Gibbard and Watkins, 1968) in a 96-well microplate format and read on a Packard SpectraCount plate reader (Packard Instrument Co., Meriden, CT). Plasma glucagon and serum insulin were quantified by using radioimmunoassay kits and the human standards supplied (Diagnostic Products Corporation). The intraassay coefficient of variation for glucagon was 0.04%, and the interassay variation was 14%. For insulin, the intraassay coefficient of variation was 0.16%, and the interassay variation was 8.6%.
Liver biopsy samples (3 g total) were obtained by percutaneous needle biopsy (Greenfield et al., 2000) at 7 d prior to the start of injections and at 3 h after the last glucagon or saline injections. Liver samples were rinsed in saline, and 1 g of sample was placed in a 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 ß-mercaptoethanol], snap-frozen in liquid nitrogen, and placed in a –80°C freezer until analysis. A second aliquot (1 g) was rinsed in PBS, snap-frozen in liquid nitrogen, and placed in a –80°C freezer pending analysis by Western blotting. Samples for PC and PEPCK enzyme activity assay (0.4 g) were rinsed in saline, snap-frozen in liquid nitrogen, and stored at –80°C. An additional aliquot (0.6 g) for in vitro incubation was rinsed in saline and placed in a 50-mL conical tube containing 10 mL of Dulbecco’s Modified Eagles Media (DMEM) and 1% BSA, placed on ice, and transported to the laboratory.
cDNA Probe, RNA Extraction, and Northern Analysis
Plasmids bPC1000, containing a 1,051-bp fragment of bovine PC cDNA, and bPEPCK-700, containing a 1,150-bp fragment of bovine PEPCK-C cDNA, were cloned in our laboratory (Agca et al., 2002). The cDNA probe for PEPCK-M was also cloned in our laboratory and was generated by digestion of bPEPCK-M at an internal HindII site (Agca et al., 2002). Insert DNA was removed from plasmids by restriction enzyme digestion. Insert was separated by electrophoresis through a 1% agarose gel and purified by using the Wizard DNA Purification system (Promega, Madison, WI). Gamma-32P[dCTP] and Ready-to-go DNA labeling kit (Pharmacia, Piscataway, NY) were used to generate the radiolabelled cDNA probes. The specific activity of cDNA probes was approximately 109 cpm/µg of DNA.
Total RNA was extracted from liver biopsy samples as previously described (Chomczynski and Sacchi, 1987). A 20-µg aliquot was separated through a 1% agarose gel and transferred to a Genescreen membrane (NEN Life Science Products, Boston, MA). Transfer occurred for 20 h before RNA was cross-linked using UV light. Membrane was baked for 2 h at 80°C to remove any residual formaldehyde.
Membranes were prehybridized for 15 h at 42°C in a prehybridization solution consisting of 10% dextran sulfate, 5x SSPE (0.75 M NaCl, 0.05 M NaH2PO4·H2O, and 5 mM EDTA), 50% deionized formamide, 5x Denhardt’s (0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 0.1% BSA), 1% SDS, and 200 µg of denatured herring sperm DNA/mL). Hybridization with the 32P-labeled cDNA probes occurred for 18 h at 42°C. Membranes were then washed twice with 2x sodium chloride/sodium citrate (SSC; 0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) at room temperature for 5 min, twice with 1x SSC, 1% SDS at 65°C for 30 min, and twice in 0.1x SSC (0.015 M NaCl, 0.0015 M sodium citrate, pH 7.0) at room temperature for 30 min.
Kodak X-Omat AR film was used to visualize the mRNA transcripts. Expression was quantified by using Kodak Digital Science 1-D Image Analysis software (Eastman Kodak Co., Rochester, NY). Pooled samples of bovine liver mRNA were placed on the outside lanes of gel to account for variation in transfer of RNA, hybridization, and washing. Variation in loading between samples was adjusted using 18S mRNA abundance.
Protein Extraction and Immunoblots
Liver tissue (1 g) was thawed and minced on ice with scissors in PBS. To the minced tissue on ice, 5 mL of protein extraction buffer (20 mM Tris·HCl (pH 7.4), 10 mM NaCl, 10 mM KCl, 3 mM MgCl2, and 1 mg of Protease Inhibitor Cocktail/mL (Sigma, St. Louis, MO) was added and homogenized for 1 min using a Polytron homogenizer. Nonidet P-40 was added to 0.5%, and the homogenate was shaken in ice for 15 min and homogenized again using a Douce homogenizer. Homogenate was centrifuged at 10,000 x g for 15 min. The supernatant was removed, frozen in liquid nitrogen, and placed in –80°C freezer until quantification. Protein was quantified by using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL).
Liver protein samples were incubated at 60°C and separated (15 µg of protein per lane) by 7.5% SDS polyacrylamide gel electrophoresis (Laemmli, 1970). Purified bovine liver PC (Sigma-Aldrich) also was included in a separate lane in the gel. The separated proteins were electrotransferred to 0.45-µm pure nitrocellulose membrane, and protein bands were visualized by fast-green staining.
For analysis of biotin-dependent proteins, the membrane was blocked by incubation for 5 min in TBS/Tween buffer (30 mM Tris base, 300 mM NaCl, and 1 mL of Tween 20/L, pH 7.5) and for 1 h in blocking solution (30 mM Tris base, 300 mM NaCl, 1 mL of Tween 20/L, and 1.75% BSA, pH 7.5). The membrane was incubated for 1 h in blocking solution with 1.75% BSA containing an anti-biotin antibody (monoclonal anti-biotin clone, BN-34, horseradish peroxidase-conjugate; 1:6,000 dilution; Sigma-Aldrich). The membrane was washed 5 times for 5 min each in blocking solution and for 10 min in TBS/Tween. Membranes were blotted gently to remove excess moisture, and a chemiluminescent substrate (Pierce Supersignal chemiluminescent substrate, Rockford, IL) was applied for 5 min. The membranes were blotted gently, wrapped in plastic wrap, and exposed to x-ray film (Kodak X-Omat AR).
Determination of Molecular Size and Relative Protein Abundance
Digital images of the autoradiographic film from the immunoblots were scanned, and scanning densitometry was performed with the Kodak Digital Science 1-D Image Analysis Software (Eastman Kodak Co.) Relative molecular weights of biotin-dependent proteins were calculated by regressing the distance of migration of the protein through the gel, against the relative molecular weights of known markers, ranging from 9.1 to 184.5 kDa (Benchmark Prestained Protein Ladder, Invitrogen Corp., Carlsbad, CA).
Assay for PC and PEPCK Activity
Liver biopsy samples were thawed on ice, blotted dry, and weighed. Crude tissue extracts were prepared by homogenizing tissue in 3 volumes of ice-cold 0.1 M sucrose, 50 mM potassium phosphate (pH 7.4), and 0.25 mM EDTA (pH 7.4) with a Tissuemizer (Tekmar Inc., Cincinnati, OH). Samples were sonicated with a probe sonicator (Ultrasonics Inc., Plainview, NY) for 30 s to disrupt mitochondria and then centrifuged for 15 min at 600 x g to remove cell debris. Supernatant was removed and assayed for PC (Greenfield et al., 2000) and PEPCK (Agca et al., 2002) activities.
Preparation of Liver Slices and Measures of Gluconeogenesis
Liver biopsy samples were transported to the laboratory in ice-cold DMEM containing 1% BSA. Within 40 min of removal from the animal, the biopsy core samples were sliced using a razor blade into 3- to 4-mm uniform sections weighing 30 to 50 mg, and slices for each cow were incubated in DMEM containing 1% BSA and 2.5 mM propionate, 1.0 mM pyruvate, 1.0 mM lactate, and 2.0 mM glycerol 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 2 h. Incubations were terminated by the addition of 0.5 mL of 5 N H2SO4 to the media. The conversion of radiolabelled precursor to glucose was determined in the media as described previously (Donkin and Armentano, 1993). The rates of gluconeogenesis were calculated from the specific radioactivity of the precursor and nanomoles of product formed per hour. Rates of gluconeogenesis are expressed as nanomoles of precursor converted to glucose/milligram of liver per hour.
Statistical Analyses
The data were analyzed by using the PROC Mixed analysis of SAS (SAS Institute, 1988). For repeated measurements, the model included the fixed effects of block, treatment, and time relative to the last glucagon injection, random effects of cow within treatment by time, the interactions of fixed effects, and the residual error. The covariance structure was determined for anti-dependence, simple, unstructured, and autoregressive models. The model that yielded the minimal range of values for Akaike information criterion and Bayesian information criterion (BIC) for each variable was used for data analysis. First-order ante-dependence structure gave the lowest BIC coefficient for milk production, NEFA concentration, and BHBA concentration. Compound symmetry gave the lowest BIC coefficient for DMI, glucose, insulin, and glucagon concentrations. Liver samples obtained at 1 wk before glucagon injections served as a covariate in the analysis of PC, cytosolic PEPCK (PEPCK-C), and mitochondrial (PEPCK-M) mRNA abundance. If the effect of block was not significant, it was eliminated from the model. Means differed if P < 0.05 and tended to differ if 0.05 
P 0.15. The values reported are least squares means and standard errors unless noted otherwise. The relationships among mRNA expression, enzyme activity, protein abundance, and the in vitro incubation assay were examined. Pearson correlation coefficients were obtained using the PROC CORR procedure of SAS (SAS Institute, 1988).
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
; Table 2). An effect of day relative to final injection, however, was observed (P < 0.05). Dry matter intake was significantly decreased on the day of liver biopsy and during the 24-h glucagon injection period leading up to the biopsy for both glucagon- and saline-treated cows. The depression in DMI is a likely consequence of the increased frequency with which cows were handled because of repetitive subcutaneous injections over a 24-h period, as there was no differences in DMI for glucagon vs. saline cows. Previous studies have indicted a lack of effect of biopsy on DMI (Vazquez-Anon et al., 1994). Milk production did not differ between glucagon-and saline-treated animals (Figure 1; Table 2). This observation is in agreement with the literature, which describes a lack of relationship between glucagon injections and milk yield (Hippen et al., 1999b; Bobe et al., 2003a).
|
|
; Table 3). Glucagon concentrations for samples taken at 1.5 h after the final glucagon injection were 59% greater for cows receiving the glucagon injections compared with those for the saline-treated cows (149 vs. 89 pg/mL; P < 0.10). The value obtained for cows receiving saline injections is approximately equal to the level previously observed for mid-lactation dairy cattle (She et al., 1999). In lactating dairy cows, blood glucagon concentration is elevated by exogenous glucagon treatment (Hippen et al., 1999a, b; She et al., 1999; Bobe et al., 2003b). Plasma insulin concentrations were not altered by glucagon injections (Figure 2; Table 3). Several studies support an increase in insulin concentration when dairy cattle receive exogenous glucagon (Hippen et al., 1999a; She et al., 1999; Bobe et al., 2003a). The exception is a study reported by Hippen et al. (1999b), who observed that blood insulin concentrations were unchanged in response to glucagon infusions of 10.0 mg/d for 14 d.
|
|
). Glucagon-and saline-treated cows had elevated glucose concentrations at 1.5 h relative to final injection compared with blood glucose concentrations at 0 h (P < 0.05). During glucagon treatment, blood glucose concentrations typically increase, then rapidly decline to pretreatment values when glucagon treatment is terminated (Hippen et al., 1999a; She et al., 1999). Given the acute effects of glucagon treatment on glucose concentration (She et al., 1999; Bobe et al., 2003b), blood samples taken within 3 h of a glucagon injection are the best indication of the effects of glucagon on blood glucose. Three hours after the final glucagon and saline injections, both glucagon-and saline-treated cows exhibited elevated NEFA concentrations (P < 0.05; Figure 3). Exogenous glucagon does not affect NEFA concentrations when administered by short-term infusions (Hippen et al., 1999a; She et al., 1999). A decrease in NEFA concentrations was observed 4 h after subcutaneous injection, and an increase was observed at 8 h when midlactating cows were given 5 mg of glucagon (Bobe et al., 2003b). At 15 and 27 h after the final injection, BHBA concentrations were significantly elevated (Figure 3; P < 0.05), which may be associated with the elevated NEFA concentrations during the injection period. Overall, BHBA concentrations are not affected by exogenous glucagon treatment in dairy cattle (Hippen et al., 1999a; She et al., 1999).
|
). The present data indicate that PEPCK-C is not responsive to glucagon when given over a 24-h period despite a lack of change in insulin concentrations. Taken together, these data may indicate that PEPCK-C is not responsive in midlactation cows, but a change in insulin concentrations in response to glucagon injections may be dominant to any response to glucagon. Controlled studies have not been performed to determine the effects of insulin on PEPCK mRNA in cattle.
|
A single subcutaneous injection of either 2.5 or 5 mg of glucagon in midlactation cows resulted in blood glucagon concentrations that reached approximately 450 and 950 pg/mL, respectively, within 1 h (Bobe et al., 2003b). Continuous infusion of 10 mg of glucagon over 24 h via jugular catheter increased glucagon concentrations to 650 pg/mL (She et al., 1999). The maximal concentrations of glucagon observed in those experiments are approximately 4 to 7 times glucagon concentrations for a midlactation dairy cows. Glucagon concentrations returned to pretreatment levels within 2 h after the end of intravenous administration (Bobe et al., 2003b) and within 5 to 6 h after a single subcutaneous injection (She et al., 1999). The 15-mg dose of glucagon given as 3.75 mg every 7 h in the present experiment would be expected to elicit an increase in blood glucagons, which should dissipate by 5 to 6 h postinjection. This is reflected by the lack a striking difference in plasma glucagon between control and glucagons-treated cows. The shortest sampling time in the present experiment, 3 h after the last glucagon injection, resulted in a 1.7x greater concentration of glucagon compared with either control or baseline values (Figure 2), which similar is in magnitude to the increase in plasma glucagon observed 2 to 3 h after a single glucagon injection (Bobe et al., 2003b).
Glucagon treatment did not alter PEPCK-M mRNA expression when compared with the saline control (1.49 vs. 1.02; Table 4). Unlike the cytosolic isoform, PEPCK-M does not respond to hormones (Savon et al., 1993). During the transition to lactation in dairy cows, PEPCK-M transcript expression remains unchanged (Agca et al., 2002). However, it is important to quantify PEPCK-M gene expression, because this enzyme may be responsible for > 50% of the glucose synthesized in ruminant hepatocytes (Aiello and Armentano, 1987), especially in gluconeogenesis from lactate.
Glucagon injections did not alter PC mRNA expression (0.62 vs. 0.98; Table 4). Liver biopsy samples taken 1 wk before glucagon injections served as the covariate. There was no effect of covariate on PC mRNA expression. Pyruvate carboxylase is responsible for the conversion of pyruvate to oxaloacetate. Our data indicate a lack of effect of glucagon on PC mRNA expression. Previous data (She et al., 1999) indicated that PC mRNA expression increased 1.5-fold during a 3.5-h glucagon infusion and remained elevated up to 3.5 h postinfusion. The methods of glucagon administration differ between the present and previous study (She et al., 1999). In the latter, glucagon was infused intravenously over a 3.5-h period, but, in the present study, glucagon was injected subcutaneously as 4 injections over a 21-h period. In humans, continuous exposure to glucagon decreases its ability to stimulate hepatic glucose output (Rizza and Gerich, 1979). In vitro studies in rats indicate that the response in blood glucose to glucagon was equivalent for pulsatile administration of glucagon compared with continuous infusion of glucagon. The dose of glucagon required to elicit the observed response, however, was much greater when administered continuously (Komjati et al., 1986). To date, the exact mechanism by which glucagon influences PC gene expression is not well understood, but evidently, the timing and duration of exposure are critical. Data from the current study do not support an effect of glucagon exposure on PC expression, but continuous infusion data supports increased PC mRNA with glucagon (She et al., 1999). Additional work is necessary to determine the direct action of glucagon on PC expression in bovine liver.
Moreover, the failure of glucagon to increase PC activity (Table 4) corroborates the lack of glucagon effect on PC mRNA expression but is at odds with other data in ruminants, indicating an increase in PC activity with glucagon (Brockman and Manns, 1974). Glucagon treatment also did not influence PEPCK enzyme activity (Table 4). The PEPCK activity assay used here does not differentiate between the cytosolic and mitochondrial isoforms; however, a close relationship exists between total PEPCK activity and PEPCK-C mRNA (Hartwell et al., 1999). Despite a trend toward increased PEPCK-C mRNA expression, an increase in PEPCK activity was not observed. In hepatocytes, an increase in PEPCK-C mRNA precedes increased PEPCK activity by 2 to 3 h, and maximal PEPCK activity is observed with a 4-h exposure to glucagon (Christ et al., 1988). Similarly, PEPCK activity may be decreased rapidly because of a half-life of only 2.3 h, which is accelerated greatly by insulin (Christ et al., 1990). The timing of sample collection, relative to glucagon injection, might have masked our ability to detect changes in PEPCK-C mRNA or PEPCK activity. Previous research indicates a reduction in PEPCK mRNA with short-term glucagon infusion that reflects a response to increased circulating insulin concentrations (She et al., 1999). The lack of marked response to glucagon for PEPCK mRNA coupled with a lack of change in blood insulin concentrations suggests that PEPCK mRNA is either unresponsive to glucagon in midlactation cows or is maximally expressed under the experimental conditions utilized. Additional research that evaluates glucagon responsiveness across a spectrum of physiological states is necessary to determine the role of glucagon and other factors in regulating PEPCK in bovine.
Analysis of total PC protein abundance by immunoblotting for biotin-binding enzymes revealed a lack of effect of glucagon treatment on the relative amount of PC protein. This immunoblotting procedure detected a 146.2-kDa band and a 104.2-kDa band in the bovine liver that contains a biotin-binding region (Figure 4). Purified bovine PC standard is identified as a singly stained band at 137.5 kDa. There are 4 known biotin-dependent enzymes that are expressed in mammals: PC, acetyl-CoA carboxylase, propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase. The structure of PC consists of 4 identical subunits that are approximately 120 to 130 kDa each (Wallace and Easter-brook-Smith, 1985). The molecular weight of propionyl-CoA carboxylase is 75 kDa, and that of 3-methylcrotonyl-CoA carboxylase is 72 kDa (Salto et al., 1999). Acetyl-CoA carboxylase is the largest biotin-dependent carboxylase with a molecular weight of approximately 220 kDa (Haneji and Koide, 1989). By using these values and the molecular weight of bovine PC standard and given the resolving power of our PAGE analysis, we conclude that the band staining at 146.2 kDa is bovine PC. At present, it cannot be determined whether the 104.2-kDa band is propionyl-CoA carboxylase or 3-methylcrotonyl-CoA carboxylase. Previous work with rats has shown that 3-methylcrotonyl-CoA carboxylase is the most abundant biotin enzyme, and propionyl-CoA carboxylase is the least abundant (Salto et al., 1999). Regardless, the present analysis can be used to determine the relative abundance of PC in bovine liver samples, but additional optimization is necessary to determine the abundance of other biotin-binding proteins in bovine liver.
|
Glucagon did not affect the conversion of propionate or lactate to glucose (Table 4). Previous data indicate a direct effect of glucagon to increase gluconeogenesis from propionate in bovine hepatocytes (Donkin and Armentano, 1995). For glucagon-treated cows, the relationship between PC mRNA and PC protein abundance is diminished with a Pearson coefficient of 0.42 (P < 0.10; n = 8) in those animals, and there is a lack of correlation for PC mRNA and enzyme activity for those cows. In vitro experiments have demonstrated an effect of propionate to inhibit synthesis of glucose from lactate (Blair et al., 1973; Anderson and Bridges, 1984). Methylmalonyl-CoA, an intermediate in the metabolism of propionate to succinate, allosterically inhibits PC activity (Smith and Osborne-White, 1971). From these data, we propose that an increase in propionate use with glucagon treatment in midlactation cows leads to the inhibition of lactate utilization through an allosteric inhibition of PC activity.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
FOOTNOTES |
|---|
2 Current address: 313 Kildee Hall, Iowa State University, Ames 50010.
Received for publication March 24, 2005. Accepted for publication August 21, 2005.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  R. J. Collier, J. L. Collier, R. P. Rhoads, and L. H. Baumgard Invited Review: Genes Involved in the Bovine Heat Stress Response J Dairy Sci, February 1, 2008; 91(2): 445 - 454. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or saline injections (
) on DMI (upper panel) and milk production (lower panel). Glucagon or saline injections were administered during the period indicated, and liver biopsy samples were obtained on d –7 and within +3 h after the final injection. Data are least squares means and standard errors. There was an effect of time on both measures (P < 0.05), and no effect of glucagon (P > 0.05) or glucagon x time (P > 0.05).
