QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Online Supporting Material Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bradford, B. J. Articles by Allen, M. S. Search for Related Content PubMed PubMed Citation Articles by Bradford, B. J. Articles by Allen, M. S.

---

Nutrient Metabolism

Phlorizin Administration Increases Hepatic Gluconeogenic Enzyme mRNA Abundance but Not Feed Intake in Late-Lactation Dairy Cows^1,2,3

Department of Animal Science, Michigan State University, East Lansing, MI 48824

⁴To whom correspondence should be addressed. E-mail: allenm{at}msu.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Gluconeogenic capacity may be an important factor regulating dry matter intake (DMI) in lactating dairy cows. To determine whether increased glucose demand affects feed intake and hepatic gene expression, lactating Holstein cows were treated with phlorizin or vehicle (propylene glycol) for 7 d. Multiparous cows (n = 12; 269 ± 65 d in milk, mean ± SD) were randomly assigned to treatment sequence in a crossover design and were adapted to a common diet for 7 d before the beginning of the experiment. Phlorizin injected s.c. at 4 g/d caused glucose excretion in urine at the rate of 474 g/d. Although phlorizin decreased lactose synthesis and milk production (both P < 0.01), DMI and 3.5% fat-corrected milk production were not altered by treatment. A net deficit of 383 g glucose/d in milk and urine for phlorizin (relative to control) was likely replaced partially through increased gluconeogenesis. The molar insulin:glucagon ratio was decreased 17% by phlorizin (P < 0.001) and hepatic phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and pyruvate carboxylase mRNA abundance increased (all P < 0.05). Late-lactation dairy cows adapted quickly to an increase in peripheral glucose demand; adaptation mechanisms likely included enhanced gluconeogenic capacity, whereas DMI was not altered.

KEY WORDS: • dairy cows • phlorizin • glucose demand • gluconeogenesis • pyruvate carboxylase

Dry matter intake (DMI)⁵ is regulated by a wide range of signals related to the intake of specific nutrients, gut distention, adiposity, cephalic stimulation, and other factors (1). Forbes (2) suggested that these signals are integrated, driving feeding behaviors that minimize discomfort to the animal. Although this signal integration likely occurs in the hypothalamus, there is a large body of evidence indicating that some signals that regulate DMI originate in the liver and are delivered via the hepatic vagus (3,4). These signals are related to the energy status of the liver; increased hepatic ATP concentrations coincide with the ends of meals in rats (5).

Propionate is an important fuel for ruminants because it is the primary substrate for gluconeogenesis in lactating cows fed highly fermentable diets. Its importance in intake regulation was demonstrated by Oba and Allen (6), who showed that intraruminal infusions of propionate linearly decreased metabolizable energy intake (including both feed and infusions) compared with isomolar infusions of acetate. The liver was likely responsible for these effects because hepatic vagotomy eliminated hypophagic responses to portal infusions of propionate (7). These treatments have physiological importance for the regulation of meal size because propionate flux to the liver increases rapidly during meals (8).

If meal size and intermeal interval are dependent on the energy status of the liver, the intake effects of propionate should depend on the extent to which it stimulates hepatic oxidation. Therefore, increasing the flux of propionate to glucose might decrease its hypophagic effects while simultaneously providing more glucose for the mammary gland and other peripheral tissues. This mechanism, mediated by changes in insulin and glucagon secretion, may provide the link between increases in peripheral energy demand and increased DMI. Although this relation has been established in a variety of circumstances (9–11), well-controlled mechanistic studies in ruminants have not been conducted.

To test the effects of increased glucose demand on metabolic regulation and DMI, we used phlorizin to cause irreversible glucose loss in urine (12). We hypothesized that increased peripheral glucose demand would cause upregulation of gluconeogenic pathways, and that increased gluconeogenic capacity would increase DMI by increasing meal size. Therefore, the objective of this study was to measure responses in hepatic mRNA abundance, milk yield, DMI, feeding behavior, and plasma hormones and metabolites during phlorizin treatment. Although several studies have measured the effect of phlorizin on lactating cows (13,14), only one has considered its effect on DMI, and that study used only 4 cows with treatment periods of 2 d (14). To our knowledge, there have been no reports evaluating the effects of phlorizin on expression of metabolic genes in bovine liver.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Experimental procedures were approved by the All-University Committee on Animal Use and Care at Michigan State University.

    Design and treatments. Multiparous Holstein cows (n = 12; 269 ± 65 d in milk; 30.7 ± 5.0 kg/d milk yield; 2.6 ± 0.7 lactations; mean ± SD) were selected from the Michigan State University Dairy Cattle Teaching and Research Center and were randomly assigned to treatment sequence in a crossover design. Phlorizin, an inhibitor of renal glucose reabsorption (12), was administered via s.c. injection at the rate of 4 g/d, with propylene glycol as vehicle and control. Treatment periods were 7 d, and injections were given every 6 h during these periods. The cows were adapted to a single diet for a 7-d period before the first treatment period, and a 7-d rest period was included between the 2 treatment periods. The experimental diet (419 g dry matter/kg diet) contained dry, finely ground corn grain, corn silage, alfalfa silage, a premix of protein supplements, and a premix of minerals and vitamins (Table 1). The diet was formulated for 280 g/kg neutral detergent fiber and 160 g/kg crude protein.

Urine was collected for a 24-h period starting on d 4 (1100 h) of each experimental period. Urinary catheters (Bardex Lubricath Foley 24FR, Bard Medical) were inserted and urine was collected into a container with 0.23 mol HCl to prevent glycolysis. Volumes were measured at 12-h intervals and samples were taken and frozen until analysis. According to the data of Amaral-Phillips et al. (14), phlorizin injected every 6 h causes glucose excretion at a constant rate, indicating that a single 24-h collection provided a valid estimate of daily glucose excretion. During the same 24-h period, blood samples were collected hourly from indwelling jugular catheters. Collected blood was immediately emptied into 2 tubes, one containing potassium EDTA and the other containing potassium oxalate with sodium fluoride as a glycolytic inhibitor (Vacutainer, Becton Dickinson). Both were centrifuged at 2000 x g for 15 min immediately after sample collection, and plasma was harvested and frozen at –20°C until analysis. One plasma sample from each tube containing K₃EDTA was preserved with benzamidine (0.05 mol/L final concentration), a proteolytic inhibitor that prevents glucagon degradation (16,17).

Liver biopsies were collected from phlorizin-treated cows immediately before the first injection on d 1 of each treatment period (control sample) and shortly after the final injection at the end of d 7 (treated sample). After local anesthetization with 2% lidocaine hydrochloride, biopsy instruments (14-gauge Vet-Core biopsy needles, Global Veterinary Products) were inserted between the 11th and 12th ribs on a line between the olecranon and the tuber coxae on the right side; 10 samples of 20 mg were collected and immediately (<5 s) frozen in liquid nitrogen; the 200 mg sample was stored at –80°C until further processing.

    Sample analysis. Diet ingredients, orts, and fecal samples were dried in a 55°C forced-air oven for 72 h and analyzed for dry matter concentration. All samples were ground with a Wiley mill (1-mm screen; Authur H. Thomas). Samples were analyzed for ash, neutral detergent fiber, indigestible neutral detergent fiber, crude protein, and starch. Ash concentration was determined after 5 h of oxidation at 500°C in a muffle furnace. Neutral detergent fiber was analyzed according to Van Soest et al. [(18), method A]. Indigestible neutral detergent fiber was measured as neutral detergent fiber residue after 240 h of in vitro fermentation (19). Ruminal fluid for the in vitro incubations was collected from a nonpregnant dry cow fed alfalfa hay only. Crude protein was analyzed according to Hach et al. (20). Starch was measured by an enzymatic method (21) after samples were gelatinized with sodium hydroxide; glucose concentration was measured using the glucose oxidase method. Concentrations of all nutrients were expressed as percentages of dry matter determined from drying at 105°C in a forced-air oven. Indigestible neutral detergent fiber was used as an internal marker to calculate total-tract digestibility of other nutrients.

Milk samples were analyzed for fat, true protein, and lactose with infrared spectroscopy by the Michigan Dairy Herd Improvement Association. Urinary nitrogen was quantified in duplicate by Dumas combustion using a commercial analyzer (LECO FP-2000). Urine and plasma samples were analyzed in duplicate for glucose content by the glucose oxidase method. Plasma samples were analyzed in duplicate using commercial kits to determine concentrations of FFA [NEFA C-kit; Wako Chemicals, as modified (22)], ß-hydroxybutyrate (BHBA; procedure #2440, Stanbio Laboratory), insulin (Coat-A-Count, Diagnostic Products), and glucagon (Glucagon kit #GL-32K, Linco Research). Plasma L-lactate content was quantified with a clinical analyzer (YSI 1500, YSI Life Sciences).

Total RNA was isolated from liver tissue using a commercial kit (RiboPure, Ambion) and samples were treated with DNase to remove any DNA contamination. The quality of all RNA isolates was verified by analysis with an Agilent 2100 Bioanalyzer (Agilent Technologies). mRNA abundance for phosphoenolpyruvate carboxykinase (cytosolic form: PCK1, EC 4.1.1.32), glucose-6-phosphatase (EC 3.1.3.9, catalytic subunit: G6PC, regulatory subunit: SLC37A4), pyruvate carboxylase (PC, EC 6.4.1.1), and pyruvate dehydrogenase kinase 4 (PDK4) was analyzed by quantitative real-time RT-PCR (qRT-PCR) using a commercial kit (Superscript III Platinum Two-Step qRT-PCR Kit, Invitrogen). Reverse transcription was conducted using oligo-dT primers with 1 µg total RNA added as a template. After RNase H treatment, the cDNA product was quantified by spectrophotometer. Real-time PCR was carried out in duplicate using 1 µg cDNA and was monitored using the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Labeled LUX primers (FAM label) and complementary unlabeled primers (Invitrogen) were designed with online LUX Designer software (23) and included at 200 nmol/L in the PCR mix (Supplemental Table 1). Melting point analysis confirmed that only the transcripts of interest were amplified during PCR. Copy numbers for each gene were measured with 2 separate qRT-PCR analyses using standard curves to allow for absolute quantification (24). Clones used for standards were as follows: PCK1, bPEPCK-C2000 [(25), a gift from Dr. Shawn Donkin, Purdue University, West Lafayette, IN]; G6PC, GenBank accession #CB465348 (a gift from Dr. Tim Smith, USDA Meat Animal Research Center, Clay Center, NE); SLC37A4, accession #BF075927; PC, accession #BF604240; PDK4, accession #BE589194 (all from the Center for Animal Functional Genomics, Michigan State University, East Lansing, MI). Clones were cultured, plasmids were harvested (Wizard Plus Minipreps, Promega), and copy numbers were quantified by spectrophotometer for serial dilution. mRNA abundance was normalized using the geometric mean of copy numbers of cyclophilin, ß-actin, and phosphoglycerate kinase 1 in each sample (26). Control genes were selected from genes used for normalization in the literature, based on consistent expression patterns in a preliminary study (unpublished data). Quantification of copy numbers for control genes was carried out in the manner described above, with the following clones used for standards: cyclophilin, accession #BG690429; ß-actin, accession #BG689033; phosphoglycerate kinase 1, accession #BG688981 (all from the Center for Animal Functional Genomics, Michigan State University, East Lansing, MI).

    Statistical analysis. Data were analyzed by the fit model procedure of JMP (version 5.0, SAS Institute) using the REML method according to the following model:

where Y_ijk is a dependent variable, µ is the overall mean, P_i is the fixed effect of period (i = 1 to 2), T_j is the fixed effect of treatment (j = 1 to 2), C_k is the random effect of cow (k = 1 to 12), PT_ij is the interaction of period and treatment, and e_ijk is the residual error. Plasma analyses were conducted using the above model, but also included fixed effects of sample time. Sample time was a significant factor for all plasma variables except glucagon. mRNA abundance was analyzed with a model that included fixed effects of treatment and qRT-PCR run and random effect of cow. Values for PCK1, G6PC, PC, and PDK4 mRNA abundance were log-transformed for analysis, and values reported here are back-transformed. Although statistical analysis of mRNA abundance was carried out using values for copy numbers/µg cDNA, abundance is reported relative to control values for ease of interpretation. For all main effects, significance was declared at P < 0.05, and tendencies were declared at P < 0.10. There was a tendency for an interaction between period and treatment for plasma glucagon concentration (P = 0.14), but not for other variables of interest (all P > 0.15).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Glucose loss, milk production, and intake. Phlorizin treatment caused urinary excretion of 474 g glucose/d (Table 2), equivalent to 40% of daily lactose secretion in this group of cows. Milk yield was depressed by treatment (P = 0.001) primarily because of an 8% decrease in milk lactose production with phlorizin (P < 0.01). Milk fat and 3.5% fat-corrected milk yield were not altered by treatment, although milk protein production tended to decrease with phlorizin (P = 0.08). Although the response in milk lactose yield was significant, the decrease of 90 g/d accounted for a relatively small proportion of the 474 g/d of glucose lost in urine from phlorizin treatment. Contrary to our hypothesis, DMI was not altered by treatment (Table 2), nor was digestible DMI. Phlorizin did not alter feeding behavior as quantified by meal size and the number of meals/d (data not shown).

View larger version (40K): [in this window] [in a new window]   FIGURE 1 Effects of phlorizin administration in lactating Holstein cows on hepatic mRNA abundance relative to control for genes involved in gluconeogenesis. Values are means ± SEM, n = 12. *Different from control, P < 0.05; ***different from control, P < 0.001.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Regulation of transcription and/or mRNA stability is an important component of PC regulation, because Greenfield et al. (30) demonstrated that mRNA abundance was highly related to PC activity in bovine liver biopsies. The expression of PC is regulated by both insulin and glucagon (31), and short-term infusions of glucagon increased PC transcript abundance (32) and enzyme activity (33) in ruminants. Therefore, in this study, increased glucose demand probably increased transcription of PC by causing a decrease in plasma glucose concentration and a corresponding decrease in the insulin:glucagon ratio (Table 3). The significant increase in PC mRNA abundance in response to increased glucose demand by phlorizin also agrees with recent reports in transition dairy cows with increased glucose demand (30,34) and suggests that the liver responds to increased glucose demand in part by limiting oxidation of intracellular pyruvate.

Pyruvate carboxylase is generally recognized as an important enzyme for the production of glucose from lactate and glucogenic amino acids (30,35). However, its importance in regulating the flux of propionate to glucose is not generally discussed, with notable exceptions (36,37). For net oxidation of propionate to occur, it must be converted to pyruvate (through oxaloacetate and phosphoenolpyruvate), oxidized by pyruvate dehydrogenase, and directed toward the tricarboxylic acid cycle as acetyl-CoA (see Supplemental Fig. 1). In one of the few studies in which this pathway was studied in lactating cows, Black et al. (38) calculated that 13% of propionate was converted to acetyl-CoA. Although this seems to be a relatively insignificant proportion of the propionate entering the liver, it represents the only net loss of glucogenic carbon from propionate in the bovine liver; all other propionate that is not utilized in gluconeogenesis is converted to lactate or glucogenic amino acids. Enhancing PC activity, therefore, should not only increase gluconeogenesis from lactate and amino acids, but also increase the proportion of propionate that can be utilized for glucose production. This may be especially true because of the transient nature of propionate absorption (8); during meals, propionate is rapidly taken up by the liver, and propionate influx may exceed the liver’s capacity for gluconeogenesis. If this build-up causes an increase in pyruvate concentration in hepatocytes, the relative activities of PC and pyruvate dehydrogenase are important in determining the proportion of glucogenic substrates that are preserved.

Past research using phlorizin indirectly supports these assertions. Veenhuizen et al. (29) used phlorizin to increase irreversible loss of glucose by 36% and measured the end-products of propionate metabolism. Phlorizin significantly increased the proportion of propionate that was utilized for gluconeogenesis, both with and without supplemental dietary propionate. Similarly, the relative proportion of propionate used for glucose production vs. CO₂ production was increased in hepatocytes harvested from phlorizin-treated wethers (39). Although no enzyme activity or gene expression data are available from these studies, it is possible that enhanced PC activity was responsible for increasing the efficiency of gluconeogenesis from propionate.

Although phlorizin treatment increased the demand for glucose specifically, adaptations to the treatment included altered metabolism of fatty acids as well as glucose and its precursors. Phlorizin increased plasma FFA concentration, which likely increased hepatic FFA uptake because the liver takes up FFA in proportion to its concentration in plasma (40). The increased uptake of FFA likely resulted in greater hepatic fatty acid oxidation because plasma BHBA concentration was increased by phlorizin. Although lipolysis contributes only small amounts of carbon for gluconeogenesis (glycerol), hepatic fatty acid oxidation plays an important role in supporting gluconeogenesis (41). Acetyl-CoA and NADH produced by oxidation of fatty acids provide energy and reducing equivalents to drive gluconeogenesis and allosterically inhibit pyruvate dehydrogenase activity, which increases the proportion of pyruvate retained for glucose production. Therefore, pyruvate dehydrogenase activity was likely decreased by phlorizin, despite the fact that mRNA abundance of the regulatory protein PDK4 (42) was not altered by treatment. Work by Chow and Jesse (43) indicated that gluconeogenesis from propionate was decreased when fatty acid oxidation, and therefore acetyl-CoA and NADH accumulation, was limited by tetradecylglycidic acid. The authors were surprised by this result because acetyl-CoA does not activate any of the enzymes in the direct gluconeogenic pathway from propionate through phosphoenolpyruvate. However, carbon cycling among oxaloacetate, phosphoenolpyruvate, and pyruvate (see Supplemental Fig. 1) could explain this surprising result because of the allosteric effects of acetyl-CoA and NADH on PC and pyruvate dehydrogenase (41).

Plasma lactate concentrations were increased by phlorizin in this study; however, this does not indicate whether glucose production from lactate was altered in phlorizin-treated cows. Baird et al. (44) found that lactate turnover through the Cori cycle was decreased in lactating cows that were food deprived for 4 d, even though plasma lactate concentrations nearly doubled. Although net hepatic uptake of lactate increases with increasing demand for glucose production in ruminants (45), the majority of this lactate is apparently not used for glucose production because it accounted for only 7% of total glucose production in early-lactation cows (46). Fatty acid oxidation inhibits the conversion of lactate to pyruvate by decreasing the NAD:NADH ratio, and nonessential amino acids are probably more important sources of gluconeogenic substrate in cases of increased glucose demand (39). In this study, the tendency for an increase in urinary N excretion during phlorizin treatment suggests that increased PC activity may have increased glucose production from nonessential amino acids.

Despite the indirect evidence that gluconeogenic capacity was increased (and the possibility that propionate oxidation was decreased) by phlorizin treatment, voluntary DMI did not increase. Although this result is contrary to our hypothesis, the treatment effect on hepatic fatty acid oxidation makes it impossible to reject the hypothesis. A decrease in propionate oxidation was expected to increase DMI only if it delayed prandial increases in hepatic energy status. In this study, however, enhanced hepatic fatty acid oxidation could have replaced an energy deficit created by a decrease in propionate oxidation.

The decision to use late-lactation cows in this study was based on the assumption that DMI is limited primarily by metabolic factors rather than distention of the reticulorumen when cows are producing at less than the peak of lactation (47). In cows whose intake is limited primarily by rumen distention, the energy status of the liver likely has less effect on DMI. However, considering the results of the present study, other animal or experimental models may be more appropriate for testing the effects of increased glucose demand on DMI. Early-lactation cows generally mobilize large amounts of fat from adipose tissue, making it possible that a decrease in the insulin:glucagon ratio would have little additional effect on lipolysis. Maintaining a constant rate of lipolysis across treatments would likely prevent hepatic fatty acid oxidation from confounding the effects of glucose demand on DMI. An alternative model would be to use late-lactation cows and to administer tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase, during both phlorizin and control injections. The drawback to this model is that tetradecylglycidic acid would limit fatty acid oxidation in muscle and adipose tissue in addition to the liver. Nevertheless, the strength of the evidence relating meal patterns to the energy status of the liver in rodents (48) encourages continued investigation of this potential mechanism of DMI regulation in cattle.

	ACKNOWLEDGMENTS

 
The authors thank R. E. Kreft, T. H. Herdt, R. A. Longuski, D. G. Main, Y. Ying, C. S. Mooney, J. A. Voelker, R. J. Tempelman, and S. P. Suchyta for technical assistance; S.S. Donkin, S. Koser, T.P.L. Smith, and K. S. Tennill for help in acquiring cDNA clones; and West Central Soy for donation of SoyPlus protein supplement.

	FOOTNOTES

 
¹ Presented in part at the 10th International Symposium on Ruminant Physiology, Aug. 30–Sept. 4, 2004, Copenhagen, Denmark [Bradford, B. J. & Allen, M. S. (2004) Increasing glucose demand increases hepatic pyruvate carboxylase mRNA concentration but not feed intake in late-lactation dairy cows. J. Anim. Feed Sci. 13 (suppl. 1): 377].

² Supported in part by National Research Initiative Competitive Grant no. 2004–35206-14167 from the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service, and by a National Science Foundation Graduate Research Fellowship.

³ Supplemental Table 1 and Supplemental Figure 1 are available as Online Supporting Material with the online posting of this paper at www.nutrition.org.

⁵ Abbreviations used: BHBA, ß-hydroxybutyrate; DMI, dry matter intake; G6PC, glucose-6-phosphatase catalytic subunit; PC, pyruvate carboxylase; PCK1, cytosolic phosphoenolpyruvate carboxykinase; PDK4, pyruvate dehydrogenase kinase 4; qRT-PCR, quantitative RT-PCR; SLC37A4, glucose-6-phosphatase translocase.

Manuscript received 4 March 2005. Initial review completed 28 April 2005. Revision accepted 27 May 2005.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Allen, M. S., Bradford, B. J. & Harvatine, K. J. (2005) The cow as a model to study food intake regulation. Annu. Rev. Nutr. 25:523-547.[Medline]

2. Forbes, J. M. (1996) Integration of regulatory signals controlling forage intake in ruminants. J. Anim. Sci. 74:3029-3035.[Abstract]

3. Langhans, W. & Scharrer, E. (1987) Evidence for a vagally mediated satiety signal derived from hepatic fatty acid oxidation. J. Auton. Nerv. Syst. 18:13-18.[Medline]

4. Scharrer, E. (1999) Control of food intake by fatty acid oxidation and ketogenesis. Nutrition 15:704-714.[Medline]

5. Koch, J. E., Ji, H., Osbakken, M. D. & Friedman, M. I. (1998) Temporal relationships between eating behavior and liver adenine nucleotides in rats treated with 2,5-AM. Am. J. Physiol. 274:R610-R617.

6. Oba, M. & Allen, M. S. (2003) Intraruminal infusion of propionate alters feeding behavior and decreases energy intake of lactating dairy cows. J. Nutr. 133:1094-1099.[Abstract/Free Full Text]

7. Anil, M. H. & Forbes, J. M. (1988) The roles of hepatic nerves in the reduction of food intake as a consequence of intraportal sodium propionate administration in sheep. Q. J. Exp. Physiol. 73:539-546.[Abstract/Free Full Text]

8. Benson, J. A., Reynolds, C. K., Aikman, P. C., Lupoli, B. & Beever, D. E. (2002) Effects of abomasal vegetable oil infusion on splanchnic nutrient metabolism in lactating dairy cows. J. Dairy Sci. 85:1804-1814.[Abstract/Free Full Text]

9. Blundell, J. E., Stubbs, R. J., Hughes, D. A., Whybrow, S. & King, N. A. (2003) Cross talk between physical activity and appetite control: does physical activity stimulate appetite?. Proc. Nutr. Soc. 62:651-661.[Medline]

10. Dohoo, I. R., Leslie, K., DesCoteaux, L., Fredeen, A., Dowling, P., Preston, A. & Shewfelt, W. (2003) A meta-analysis review of the effects of recombinant bovine somatotropin. 1. Methodology and effects on production. Can. J. Vet. Res. 67:241-251.[Medline]

11. Johnson, M. S., Thomson, S. C. & Speakman, J. R. (2001) Limits to sustained energy intake. I. Lactation in the laboratory mouse Mus musculus. J. Exp. Biol. 204:1925-1935.[Abstract/Free Full Text]

12. Young, J. W., Schmidt, S. P., Akowuah, E. S., Hess, G. S. & McGilliard, A. D. (1974) Effects of phlorizin on glucose kinetics in the bovine. J. Dairy Sci. 57:689-694.

13. Rutter, L. M. & Manns, J. G. (1988) Follicular phase gonadotropin secretion in cyclic postpartum beef cows with phlorizin-induced hypoglycemia. J. Anim. Sci. 66:1194-1200.

14. Amaral-Phillips, D. M., McGilliard, A. D., Lindberg, G. L., Veenhuizen, J. J. & Young, J. W. (1993) Effects of decreased availability of glucose for dairy cows. J. Dairy Sci. 76:752-761.[Abstract/Free Full Text]

15. Dado, R. G. & Allen, M. S. (1993) Continuous computer acquisition of feed and water intake, chewing reticular motility, and ruminal pH of cattle. J. Dairy Sci. 76:1589-1600.[Abstract]

16. Ensinck, J. W., Shepard, C., Dudl, R. J. & Williams, R. H. (1972) Use of benzamidine as a proteolytic inhibitor in the radioimmunoassay of glucagon in plasma. J. Clin. Endocrinol. Metab. 35:463-467.[Abstract/Free Full Text]

17. Matsunaga, N., Goka, T., Nam, K. T., Oda, S., Ohneda, A. & Sasaki, Y. (1997) Inhibition of GH releasing factor (GRF)-induced GH secretion by intraruminal infusion of volatile fatty acids (VFA) in sheep. Endocr. J. 44:133-140.[Medline]

18. Van Soest, P. J., Robertson, J. B. & Lewis, B. A. (1991) Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583-3597.[Abstract]

19. Goering, H. K. & Van Soest, P. J. (1970) Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Forage Fiber Analysis (Apparatus, Reagents, Procedures, and Some Applications) Agric. Handbook No. 379. ARS/USDA Washington, DC.

20. Hach, C. C., Bowden, B. K., Lopelove, A. B. & Brayton, S. V. (1987) More powerful peroxide Kjeldahl digestion method. J. Assoc. Off. Anal. Chem. 70:783-787.

21. Karkalas, J. (1985) An improved enzymatic method for the determination of native and modified starch. J. Sci. Food Agric. 36:1019-1027.

22. McCutcheon, S. N. & Bauman, D. E. (1986) Effect of chronic growth hormone treatment on responses to epinephrine and thyrotropin-releasing hormone in lactating cows. J. Dairy Sci. 69:44-51.

23. LUX Fluorogenic Primers. www.invitrogen.com/lux. [Last accessed Jan. 6, 2005].

24. Whelan, J. A., Russell, N. B. & Whelan, M. A. (2003) A method for the absolute quantification of cDNA using real-time PCR. J. Immunol. Methods 278:261-269.[Medline]

25. Agca, C., Greenfield, R. B., Hartwell, J. R. & Donkin, S. S. (2002) Cloning and characterization of bovine cytosolic and mitochondrial PEPCK during transition to lactation. Physiol. Genomics 11:53-63.[Abstract/Free Full Text]

26. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. & Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3:RESEARCH0034.[Medline]

27. Pilkis, S. J. & Granner, D. K. (1992) Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu. Rev. Physiol. 54:885-909.[Medline]

28. Nordlie, R. C., Foster, J. D. & Lange, A. J. (1999) Regulation of glucose production by the liver. Annu. Rev. Nutr. 19:379-406.[Medline]

29. Veenhuizen, J. J., Russell, R. W. & Young, J. W. (1988) Kinetics of metabolism of glucose, propionate and CO₂ in steers as affected by injecting phlorizin and feeding propionate. J. Nutr. 118:1366-1375.

30. Greenfield, R. B., Cecava, M. J. & Donkin, S. S. (2000) Changes in mRNA expression for gluconeogenic enzymes in liver of dairy cattle during the transition to lactation. J. Dairy Sci. 83:1228-1236.[Abstract]

31. Jitrapakdee, S. & Wallace, J. C. (1999) Structure, function and regulation of pyruvate carboxylase. Biochem. J. 340(Pt. 1):1-16.

32. She, P., Lindberg, G. L., Hippen, A. R., Beitz, D. C. & Young, J. W. (1999) Regulation of messenger ribonucleic acid expression for gluconeogenic enzymes during glucagon infusions into lactating cows. J. Dairy Sci. 82:1153-1163.[Abstract]

33. Brockman, R. P. & Manns, J. G. (1974) Effects of glucagon on activities of hepatic enzymes in sheep. Cornell Vet 64:217-224.[Medline]

34. Hartwell, J. R., Cecava, M. J. & Donkin, S. S. (2001) Rumen undegradable protein, rumen-protected choline and mRNA expression for enzymes in gluconeogenesis and ureagenesis in periparturient dairy cows. J. Dairy Sci. 84:490-497.[Abstract]

35. Looney, M. C., Baldwin, R. L. & Calvert, C. C. (1987) Gluconeogenesis in isolated lamb hepatocytes. J. Anim. Sci. 64:283-294.

36. Armentano, L. E. (1992) Ruminant hepatic metabolism of volatile fatty acids, lactate and pyruvate. J. Nutr. 122:838-842.

37. Steinhour, W. D. & Bauman, D. E. (1988) Propionate metabolism: a new interpretation. Dobson, A. Dobson, M. J. eds. Aspects of Digestive Physiology in Ruminants :238-256 Comstock Ithaca, NY.

38. Black, A. L., Luick, J., Moller, F. & Anand, R. S. (1966) Pyruvate and propionate metabolism in lactating cows. Effect of butyrate on pyruvate metabolism. J. Biol. Chem. 241:5233-5237.[Abstract/Free Full Text]

39. Overton, T. R., Drackley, J. K., Ottemann-Abbamonte, C. J., Beaulieu, A. D., Emmert, L. S. & Clark, J. H. (1999) Substrate utilization for hepatic gluconeogenesis is altered by increased glucose demand in ruminants. J. Anim. Sci. 77:1940-1951.[Abstract/Free Full Text]

40. Emery, R. S. & Herdt, T. H. (1991) Lipid nutrition. Vet. Clin. North Am. Food Anim. Pract. 7:341-352.[Medline]

41. Gonzalez-Manchon, C., Martin-Requero, A., Ayuso, M. S. & Parrilla, R. (1992) Role of endogenous fatty acids in the control of hepatic gluconeogenesis. Arch. Biochem. Biophys. 292:95-101.[Medline]

42. Huang, B., Wu, P., Bowker-Kinley, M. M. & Harris, R. A. (2002) Regulation of pyruvate dehydrogenase kinase expression by peroxisome proliferator-activated receptor-alpha ligands, glucocorticoids, and insulin. Diabetes 51:276-283.[Abstract/Free Full Text]

43. Chow, J. C. & Jesse, B. W. (1992) Interactions between gluconeogenesis and fatty acid oxidation in isolated sheep hepatocytes. J. Dairy Sci. 75:2142-2148.[Abstract]

44. Baird, G. D., van der Walt, J. G. & Bergman, E. N. (1983) Whole-body metabolism of glucose and lactate in productive sheep and cows. Br. J. Nutr. 50:249-265.[Medline]

45. Baird, G. D., Lomax, M. A., Symonds, H. W. & Shaw, S. R. (1980) Net hepatic and splanchnic metabolism of lactate, pyruvate and propionate in dairy cows in vivo in relation to lactation and nutrient supply. Biochem. J. 186:47-57.[Medline]

46. van der Walt, J. G., Baird, G. D. & Bergman, E. N. (1983) Tissue glucose and lactate metabolism and interconversions in pregnant and lactating sheep. Br. J. Nutr. 50:267-280.[Medline]

47. Allen, M. S. (2000) Effects of diet on short-term regulation of feed intake by lactating dairy cattle. J. Dairy Sci. 83:1598-1624.[Abstract]

48. Horn, C. C., Ji, H. & Friedman, M. I. (2004) Etomoxir, a fatty acid oxidation inhibitor, increases food intake and reduces hepatic energy status in rats. Physiol. Behav. 81:157-162.[Medline]

This article has been cited by other articles:

				B. J. Bradford, L. K. Mamedova, J. E. Minton, J. S. Drouillard, and B. J. Johnson Daily Injection of Tumor Necrosis Factor-{alpha} Increases Hepatic Triglycerides and Alters Transcript Abundance of Metabolic Genes in Lactating Dairy Cattle J. Nutr., August 1, 2009; 139(8): 1451 - 1456. [Abstract] [Full Text] [PDF]

				C. S. Wilcox, M. M. Schutz, S. S. Donkin, D. C. Lay Jr., and S. D. Eicher Short Communication: Effect of Temporary Glycosuria on Molasses Consumption in Holstein Calves J Dairy Sci, September 1, 2008; 91(9): 3607 - 3610. [Abstract] [Full Text] [PDF]

				R. F. Cooke, J. D. Arthington, D. B. Araujo, G. C. Lamb, and A. D. Ealy Effects of supplementation frequency on performance, reproductive, and metabolic responses of Brahman-crossbred females J Anim Sci, September 1, 2008; 86(9): 2296 - 2309. [Abstract] [Full Text] [PDF]

				B. J. Bradford and M. S. Allen Depression in Feed Intake by a Highly Fermentable Diet Is Related to Plasma Insulin Concentration and Insulin Response to Glucose Infusion J Dairy Sci, August 1, 2007; 90(8): 3838 - 3845. [Abstract] [Full Text] [PDF]

				F.-Q. Zhao and A. F. Keating Expression and Regulation of Glucose Transporters in the Bovine Mammary Gland J Dairy Sci, June 1, 2007; 90(13_suppl): E76 - E86. [Abstract] [Full Text] [PDF]

				B. J. Bradford and M. S. Allen Phlorizin Induces Lipolysis and Alters Meal Patterns in Both Early-and Late-Lactation Dairy Cows J Dairy Sci, April 1, 2007; 90(4): 1810 - 1815. [Abstract] [Full Text] [PDF]

				B. J. Bradford and M. S. Allen Phlorizin Administration Does Not Attenuate Hypophagia Induced by Intraruminal Propionate Infusion in Lactating Dairy Cattle J. Nutr., February 1, 2007; 137(2): 326 - 330. [Abstract] [Full Text] [PDF]

				B. J. Bradford, A. D. Gour, A. S. Nash, and M. S. Allen Propionate Challenge Tests Have Limited Value for Investigating Bovine Metabolism J. Nutr., July 1, 2006; 136(7): 1915 - 1920. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Online Supporting Material Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bradford, B. J. Articles by Allen, M. S. Search for Related Content PubMed PubMed Citation Articles by Bradford, B. J. Articles by Allen, M. S.