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Nonesterified Fatty Acids and Glucose in Lactating Dairy Cows: Diurnal Variations and Changes in Responsiveness During Fasting to Epinephrine and Effects of Beta-Adrenergic Blockade
Nonesterified Fatty Acids and Glucose in Lactating Dairy Cows: Diurnal Variations and Changes in Responsiveness During Fasting to Epinephrine and Effects of Beta-Adrenergic Blockade D. M. FROHLI = and J.W. BLUM 2 Institute of Animal Science Swiss Federal Institute of Technology 8092 Z~Jrich,Switzerland ABSTRACT To examine whether the typical nightly rise of nonesterified fatty acids in high yielding dairy cows is due to enhanced sympathoadrenal activity, the /3-adrenergic blocker, propranolol, was infused from 1800 to 0805 h. Concentrations of nonesterified fatty acids continuously increased, whereas those of glucose and insulin decreased. Nonesterified fatty acid concentration decreased within minutes in response to concentrate feeding, starting at 0 7 0 0 h, in association with an increase of insulin and glucose. In a second experiment, adrenaline (.82 #mol/kg/min) was infused from 1800 to 1810 h, 0600 to 0610 h, and 0600 to 0610 h on the 1st, 2nd and 3rd d. After the second infusion, food was withdrawn for 23 h. Concentrations of adrenaline increased similary. Nonesterified fatty acids and glucose responses were higher during the second than the first infusion. During fasting, nonesterified fatty acid concentrations increased, whereas glucose and insulin concentrations decreased. During the third infusion nonesterified fatty acids responses were unchanged, whereas glucose responses were decreased. Thus, the nightly rise of nonesterified fatty acids was not the consequence of enhanced/3-adrenergic activity.
Responses of glucose and nonesterified f a t t y acids to adrenaline exhibited diurnal
differences. Responses of glucose to adrenaline were reduced within 1 d of fasting, whereas those of nonesterified fatty acids were not altered.
INTRODUCTION
Received August 3, 1987. Accepted December 21, 1987. 1Present address: F. Hoffmann-La Roche & Co. AG, Department of Vitamin and Nutrition Research, 4002 Basle, Switzerland. 2Present address, correspondence, and reprint requests: Department of Nutrition Pathology, Institute of Animal Breeding, University, Bremgartenstrasse 109a, 3012 Berne, Switzerland. 1988 J Dairy Sci 71 : 1170-1177
During early lactation, in dairy cows, fat oxidation is enhanced, whereas glucose oxidation is reduced, thus sparing glucose for lactose synthesis (3). Nevertheless, glucose concentrations are usually low as a consequence of the enhanced demand and insufficient gluconeogenesis (5, 10, 20). Concentrations of nonesterified fatty acids (NEFA) are elevated (9, 10). The magnitude of the rise depends on the degree of energy deficiency (9, 20). Concentrations of NEFA increase during the night in association with decreasing concentrations of immunoreactive insulin (IRI) as a consequence of decreased feed, i.e., energy intake (9). Glucose concentrations increase during the night, especially in the energy-deficient animals, likely as a result of low circulating insulin, which favors gluconeogenesis and glycolysis (9). Although somatotropin is the most potent hormone stimulating milk production (4), several other hormones regulate metabolism in a direction that favors milk secretion (12, 18). The possible role(s) of catecholamines (CA) for nutrient repartitioning in lactating cows has not been well-studied. This is somehow surprising, because adrenaline (epinephrine, E) and noradrenaline (norepinephrine, NE) cause NEFA and glucose to rise (8, 11). In addition, they cause insulin resistance (Blum, unpublished observations), as does somatotropin, at least during short-lasting administration (15). There is some evidence that CA can change metabolism in a direction typical for lactation. Thus, the number of/3-adrenergic receptors on
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CATECHOLAMINE EFFECTS IN COWS fat cells increase during lactation compared with numbers during the dry period (17). In addition, NEFA and glycerol responses are greater after parturition than before in cows as a consequence of reduced reesterification of NEFA and an increase in the active form of lipase in adipose tissue (23). Variable provision of food energy may be in part responsible for enhanced sensitivity to E, because responses of NEFA and glucose to E and NE are modified by fasting and low circulating IRI (1, 8, 13, 24, 26, 27). Also, E decreases acetate conversion to fatty acids (2). Furthermore, during the administration of somatotropin to lactating cows, effects of E on NEFA became enhanced (21). Endogenously produced somatotropin is usually elevated during lactation, especially when compared with levels during the dry period (14, 16, 20), which could contribute to enhanced fat mobilization. Experiments were conducted to study a possible contribution of endogenous CA on the nightly rise of NEFA and glucose, by inhibiting ~3-adrenergic effects with propranolol. Possible diurnal modifications and changes caused by fasting of NEFA, glucose, and lactate responses to the administration of E were also investigated. MATERIALS A N D METHODS Experimental Procedures
Effects of Propranolol on Plasma Nonesterified Fatty Acids, Glucose, and Immunoreactive Insulin. Six cows (two experiments with three animals) in their second to fourth lactations were studied 4 to 6 wk after parturition. Their milk production during 305 d of their preceding lactation was 5949 + 291 kg, and milk yield on the day before the start of the experiments was 32.8 -+ 2.0 kg/d. Animals were milked at 0515 and 1600 h. Prior to the experiment, animals received hay and corn silage ad libitum and concentrates at 0700 and 1700 h. During the experiment (starting at 1800 h), feed was completely removed from 1900 to 0700 h of the next morning, at which time the animals were fed. Catheters were implanted in both jugular veins on the morning before the start (1800 h) of the infusions of the ~-adrenergic blocking agent propranolol (19.3 nmol/kg/min for 845 rain, i.e., up to 0805 h of the next morning).
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Infusions of the ~-adrenergic agonist isoproterenol (286 pmol/kg/min from 0800 to 0805 h) were performed in addition to propranolol infusions through the same catheter. Blood samples were obtained from the contralateral vein at 1750, 1755, and 1759 (before the start of the propranolol infusions), 1900, 2000, 2100, 2200, 2300, 2400, 0100, 0200, 0300, 0400, 0500, 0515, 0530, 0545, 0600, 0615, 0630, 0645, 0700, 0730, 0755, 0758, 0800 (before the isoproterenol infusions), 0804, 0805, 0806, 0810, and 0820 h.
Effects of Epinephrine on Plasma Nonesterified Fatty Acids, Glucose, and Lactate in Normally Fed and Fasted Cows. Nine cows (three experiments with three animals) in their second to fourth lactations were studied 4 to 6 wk after parturition. Their milk production during 305 d of the preceding lactation was 7424 -+ 466 kg and milk yield on the day before the start of the experiments was 36.1 -+ 1.2 kg/d. The animals were milked twice daily (at 0515 and 1600 h). Prior to 0700 h of the 2nd d of the experiment animals received hay and corn silage ad libitum and concentrates twice daily (at 0700 and 1700 h). This corresponded to an intake of 135 -+ 3 MJ NE 1 and 2.84 +- 1.64 kg crude protein. From 0700 to 1900 h the animals received only hay ad libitum, which corresponded to an intake of 39.9 -+ 1.3 MJ NE I and .39 +- .13 kg crude protein. Then feed was completely removed until 0700 h of the next morning. Catheters were implanted in both jugular veins at 1800 h of the experiment. Epinephrine (.82 nmol/kg/min for 10 rain) was infused starting on the 1st d at 1800 h (infusion A), on the 2nd d at 0600 h (infusion B), and on the 3rd d again at 0600 h (infusion C). Blood samples were obtained from the contralateral veins at 10.5 and .5 rain before and 4, 6, 8, 10, 15, 20, 30, 45, and 60 min after the start of the 10-min infusions. Laboratory Procedures
Blood samples were immediately transferred to tubes containing heparin (50 U USP/ml blood) or in ice-cold perchloric acid (1 ml/1 ml blood) and centrifuged at 4°C within 2 h after collection. Plasma aliquots of heparinized blood samples were transferred to polystyrol cups and Journal of Dairy Science Vol. 71, No. 5, 1988
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FR(3HLI AND BLUM
frozen at - 2 0 ° C until analyzed for E, NE, IRI, triiodothyronine (T3), NEFA, and glucose. Deproteinized supernatants were filtered and frozen at - 2 0 ° C until analyzed for the determination of acetoacetate (AAC) and 1lactate. Concentrations of 1~ and NE were determined by a radioenzymatic method, IRI and T3 by radioimmunassay, and metabolites colorimetrically (9). All samples from one experiment were determined within the same assay. Control sera were measured to evaluate interassay variations. Drugs
DL-Propranolol was obtained from Imperial Chemical Industries, Macclesfield/England; epinephrine-bitartrate from Fluka AG, Buchs/ Switzerland; and isoproterenol-HC1 from Winthrop Products Co., Surbiton on Thames/ England. Epinephrine and isoproterenol were dissolved in .9% NaC1. Solutions of propranolol also contained 31.5 mg citric acid in 100 ml. During the experiments the solutions were kept on ice in light-protected bottles. Statistical Analysis
Means (+ SE) at various times during the infusions were compared to mean basal concentrations by paired t-test and total incremental or decremental changes after calculation of the area under the concentration curve (concentration × volume - 1 × m i n ) f o r each individual. R ESU LTS
Effects of Propranolol on Plasma Nonesterified Fatty Acids, Glucose, and Immunoreactive Insulin
Concentrations of NEFA, after a slight initial decrease (after I h; P<.05) continuously increased (higher than preinfusion concentrations at 2400 and later; P<.05), then rapidly decreased when animals were fed at 0700 h to values lower than those before the start of the infusions (P<.05), as shown in Figure 1. Concentrations of glucose started to decrease at 2200 h and were lower at 0100 h than preinfusion concentrations (P<.05) and rapidly increased towards preinfusion concentrations after the animals were again fed at 0700 h. Journal of Dairy Science Vol. 71, No. 5, 1988
Concentrations of IRI decreased during the first hours of the propranolol infusions (P<.05) and then remained low until 0700 h, after which time they rapidly increased to concentrations slightly higher than preinfusion concentrations. From the start of the experiment to 0700 h and from 0700 to 0800 h, IRI was negatively related to NEFA (r = --.88 and - . 9 7 , respectively). During infusions of isoproterenol (from 0800 to 0805 h) concentrations of IRI, NEFA, and glucose did not change. Effects of Epinephrine on Plasma Nonesterified Fatty Acids, Glucose, and Lactic Acid in Normally Fed and Fasted Cows
Concentrations of NEFA before the three infusions increased during the experiment (P<.01) and transiently increased in response to the E infusions (Figure 2). The total incremental change (mmol ° L - 1 ° 60 min) was significantly smaller in response to the first infusion (A) than in response to the following infusions, but changes in response to infusions B and C were similar. Concentrations of glucose were highest before the second infusion (B), lowest before the third infusion (C), and different before infusions A and B as well as B and C (P<.001). The total incremental changes (mmol • L - 1 60 min) were different from each other (P<.05) and greatest in response to infusion B and smallest in response to infusion C. Concentrations of lactate before the infusions were similar. The total incremental changes (mmol • L - 1 • 60 rain) were highest in response to infusion B and lowest in response to infusion C, but there were no significant differences between the three infusions. Concentrations of E (not shown) were higher before infusion C (.60 -+ .06 ~tmol/L) than before infusions A and B (.36 -+ .04 and .43 + .04 /amol/L, respectively) (P<.O1), reached maximal concentrations between 8 and 10 rain of the infusions (8.47 -+ .42, 8.72 -+ .48, and 8.76 + .51 /lmol/L above basal concentrations during infusions A, B, and C, respectively; P<.001), and then rapidly decreased. The total incremental changes were similar during the infusions (150 + 8, 152 -+ 9, and 155 + 9/amol • L - 1 • 60 min). Basal concentrations of NE were similar (2.1 + .3, 1.9 + .3, and 2.1 -+ .3 /amol/L before infusions A, B, and C, re-
C A T E C H O L A M I N E E F F E C T S IN COWS
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1788
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Figure 1. Changes o f nonesterfied fatty acids, glucose and insulin in blood of lactating cows during t h e night in the presence of propranolol, c o m b i n e d w i t h fasting, realimentation, and 5-min i s o p r o t e r e n o l i n f u s i o n s .
J o u r n a l of Dairy Science Vol. 71, No. 5, 1988
FROHLIAND
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to lO-min infusions of epinephrine, starting on the 1st d at 1800 h and on the 2rid and 3rd d at 0600 animals were fasted, starting after the second until the end of the third infusion.
J o u r n a l o f D a i r y S c i e n c e V o l . 7 1 , N o . 5, 1 9 8 8
h. T h e
CATECHOLAMINE EFFECTS IN COWS spectively). Norepinephrine concentrations did not change significantly during the E infusion (not shown). Concentrations before infusions A, B, and C of IRI (.67 + .16, .26 + .05, and .09 + .04 gg/L, respectively) and T3 (2.76 -+ .11, 2.25 + .15, and 1.94 + .11 nmol/L) continuously decreased and were different from each other (P<.05) (not shown). Concentrations of (AAC) before infusion A (108 + 28 ;zmol/L) were higher than before infusion B (61 + 31/amol/L) and highest before infusion C (188 + 33 /amol/L but basal AAC concentrations were different only between infusion B and C (P<.05) (not shown). Milk yield was 36.1 -+ 1.2 kg/d on the day before the start of the experiment, 36.0 + 1.0 kg on the 1st d, 36.8 +- 1.0 kg on the 2nd d, 31.7 -+ 1.4 kg on the 3rd d of the experiment, and 35.1 + 1.1 kg on the day after the experiment (not shown). DISCUSSION
Feed restriction during the night in experiments with propranolol simulated the normal situation, in which feed intake is markedly reduced during the night in dairy cows (9). In the present study cows were unaffected by the short time without feed, and metabolic and endocrine changes were relatively small. Feed removal starting at 0600 h for 23 h was characterized by lowered glucose, IRI, and T3 concentrations and elevated NEFA and ketone bodies, which are typical for cattle during energy restriction (10, 20). The increase of E concentrations during fasting was possibly due to hypoglycemia. Thus, insulin-induced reduction of glucose concentrations was associated with an immediate increase of E and NE concentrations in calves (7, 22), as found in other species. Stimulation of lipolysis in cattle by CA is mediated entirely by 13-adrenergic receptors on fat cells because the rise of NEFA concentrations in response to the administration of E, NE, and ~3-adrenergic agonists can be suppressed by ¢-adrenergic blockade with propranolol (8). The continuous increase of NEFA during the night in the presence of propranolol was comparable to results of a previous study performed in the absence of propranolol (9). Because propranolol could not prevent the increase of NEFA during the night indicates
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that enhanced lipolysis was not the consequence of an increased sympathoadrenal activity. Propranolol was infused in amounts sufficient to inhibit the typical rise of NEFA in response to the administration of the/3-adrenergic agonist isoproterenol seen otherwise in the absence of/3-adrenergic blockade (9). The'high negative relationship between concentrations of NEFA and IRI and in particular the rapid fall of NEFA concentrations in response to the increase of IRI concentrations during refeeding suggest that insulin is important for the regulation of NEFA concentrations. In this study, glucose concentrations decreased during the night in the presence of propranolol, suggesting that reduction of sympathoadrenal activity was responsible for this effect. However, amounts of plasma E and NE do not change significantly during the night in lactating dairy cows (9). The increase of glucose concentrations observed after the intake of concentrates in the morning was also surprising because glucose concentrations decreased in the absence of propranolol in previous studies (9). Thus, ~-adrenergic blockade obviously reversed the normal pattern of glucose during the night and in response to food intake. The greater responses of both NEFA and glucose in response to the administration of E in the morning vs. night suggested enhanced sensitivity or responsiveness of fat and liver cells to the catecholamine. Because lactate response was not changed, glycogenolysis in liver and muscle is probably modified differently by time of day. The marked diurnal variations of NEFA and glucose responses to E has to our knowledge not been previously reported. In our study, diurnal variations in sensitivity or responsiveness of target organs to E were likely modified by changes in blood concentrations of insulin, which were lower in the morning than night, thus favoring lipolysis and glycogenolysis in the morning. In lactating cows, IRI concentrations and sensitivity to insulin were lower in the morning than night and were associated with elevated glucose concentrations and reduced clearance rates of glucose in the morning (25). It is possible that other hormones, whose blood concentrations also exhibit diurnal variations as well as inherent diurnal patterns in the activity of
Journal of Dairy Science Vol. 71, No. 5, 1988
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FROHLI AND BLUM
enzymes involved in lipo- and glycogenolysis, also contributed to changes in sensitivity to E. Greater NEFA and glycerol responses and reduced glucose and lactate responses to E after 3 or 5 d of fasting has been reported previously in cattle (8, 13). In starvation ketotis, the increase of NEFA to the administration of E was enhanced (19). The present study shows that during fasting the time required for fat tissue to become more responsive and for liver and muscle to become less responsive to E is only about 1 d, much shorter than noted previously. The marked decrease of circulating insulin presumably favors enhanced E-stimulated lipolysis. Elevated blood somatotropin concentrations were associated with enhanced responsiveness of NEFA to E (21). The reduced increase of glucose in response to E after 24 h of feed withdrawl was presumably in part the consequence of decreased glycogen stores in the liver. The decrease of plasma T 3 concentrations during fasting probably modified the sensitivity of fat, liver, and muscle cells to E. Low thyroid hormone concentrations in hypothyroidism are usually associated with reduced lipolysis (6), which was not the case in our experiment. Because identical amounts of E were administered and since E concentrations in plasma increased in a comparable manner, amounts of E expected to interact with target organs were similar and do not explain diurnal variations or altered responses to E during fasting of glucose and NEFA. The interesting new information from this investigation is the marked diurnal variations of NEFA and glucose responses to E. Changes in metabolic responses to E during fasting could be observed within 24 h. Thus, high yielding dairy cows rapidly adapt in response to changes in feed intake. We speculate that metabolic alterations under basal conditions are regulated by CA largely at target organs and possibly only to a minor extent by changes in sympathoadrenal activity and circulating CA concentrations. This holds in part also for other hormones (12). Changes in partitioning of nutrients in favor of the mammary gland are considered to be largely the consequence of altered responsiveness of key tissues to homeostatic signals (3).
Journal of Dairy Science Vol. 71, No. 5, 1988
ACKNOWLEDGMENTS
This study has been supported by a grant of the Swiss Federal Institute of Technology (No. .330.057.15/9) and by F. Hoffmann-La Roche & Co. AG. The technical assistance of R. Admaty, M. Janeziz, and W. Moses is greatly acknowledged. We thank H. Schneeberger, Swiss Federal Research Station of Animal Production, Grangeneuve, 1724 Posieux, for providing us with the experimental facilities. REFERENCES
1 Arner, P., P. Engfeldt, and J. Nowak. 1981. In vivo observations on the lipolytic effect of noradrenaline during therapeutic fasting. J. Clin. Endocrinol. Metab. 53:1207. 2 Bauman, D. E. 1976. Intermediary metabolism of adipose tissue. Fed. Proc. 35:2308. 3 Bauman, D. E., and W. B. Currie. 1980. Partitioning of nutrients during pregnancy and lactation: a review of mechanisms involving homeostasis and homeorhesis. J. Dairy Sci. 63:1514. 4 Bauman, D. E., P. J. Eppard, M. J. De Geeter, and G. M. Lanza. 1985. Responses of high-producing dairy cows to longterm treatment with pituitary somatotropin and recombinant somatotropin. J. Dairy Sci. 68:1352. 5 Bergman, E. N. 1973. Glucose metabolism in ruminants as related to hypoglycemia and ketosis. Cornell Vet. 63:341. 6 Bilezikian, J. P., and J. N. Loeb. 1983. The influence of hyperthyroidism and hypothyroidism of a- and B-adrenergic receptor systems and adrenergic responsiveness. Endocrine Rev. 4:378. 7 Bloom, S. R., A. V. Edwards, R. N. Hardy, K. M. Malinkowsky, and M. Silver. Endocrine response to insulin hypoglycaemia in the young calf. J. Physiol. Lond. 244:783. 8 Blum, J. W., D. FriJhli, and P. Kunz. 1982. Effect of catecholamines on plasma free fatty acids in fed and fasted cattle. Endocrinology 110:452. 9 Blum, J. W., F. Jans, W. Moses, D. Ffdhli, M. Zemp, M. Wanner, I. C. Hart, R. Thun, and U. Keller. 1985. Twenty-four hour pattern of blood hormone and metabolite concentrations in highyielding dairy cows: effects of feeding low or high amounts of starch, or crystalline fat. Zentralbl. Veterinaermed. A 32:401. 10 Blum, J. W., P. Kunz, H. Leuenberger, K. Gautschi, and M. Keller. 1983. Thyroid hormones, blood plasma metabolires and haematological parameters in relationship to milk yield in dairy cows. Anita. Prod. 36:93. 11 Brockman, R. P., and B. Laarveld. 1986. Hormonal regulation of metabolism in ruminants: a review. Livest. Prod. Sci. 14:313. 12 Collier, R. J, J. p. Mc Namara, C. R. Wallace, and M. H. Dehoff. 1984. A review of endocrine regulation of metabolism during lactation. J. Anita.
CATECHOLAMINE EFFECTS IN COWS Sci. 59:498. 13 FriShli, D., and J. W. Blum. 1985. Epinephrine and norepinephrine during fasting: blood levels, metabolism and metabolic effects. Proc. 13th Int. Congr. Nutr., August 18--23, 1985 in Bringhton/ England (Abstr. 0034). 14 Hart, I. C., J. A. Bines, S. V. Morant, and J. L. Ridley. 1978. Endocrine control of energy metabolism in the cow: comparison of levels of hormones (prolactin, growth hormone, insulin and thyroxine) and metabolites in the plasma of highand low-yielding cattle at various stages of lactation. J. Endocrinol. 77:333. 15 Hart, I. C., P.M.E. Chadwick, T. C. Boone, K. E. Langley, C. Rudman, and L. M. Souza. 1984. A comparison of the growth-promoting, lipolytic, diabetogenic and immunological properties of pituitary and recombinant DNA-derived bovine growth hormone (somatotropine). Biochem. J. 224:93. 16 Herbein, J. H., R. J. Aiello, L. I. Ecklor, R. E. Pearson, and R. M. Akers. 1985. Glucagon, insulin, growth hormone, and glucose concentrations in blood plasma of lactating dairy cows. J. Dairy Sci. 68:320. 17 Jaster, E. H., and T. N. Wegner. 1981. Betaadrenergic receptor involvement in lipolysis of dairy cattle subcutaneous adipose tissue during dry and lactating state. J. Dairy Sci. 64:1655. 18 Karg, H., and H. Mayer. 1987. Manipulation der Laktation. Uebersichten Tierern'ahr. 15:29. 19 Kronfeld, D. S. 1965. Plasma non-esterified fatty acid concentrations in the dairy cow: responses to
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22
23
24
25
26
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nutritional and hormonal stimuli, and significance in ketosis. Vet. Rec. 77:30. Kunz, P., J. W. Blum, I. C. Hart, H. Bickel, and J. Landis. 198"5. Effects of different energy intakes before and after calving on food intake, performance and metabolic variables in dairy cows. Anim. Prod. 40:219. McCutcheon, S. N., and D. E. Bauman. 1986. Effect of chronic growth hormone treatment on responses to epinephrine and thyrotropin-releasing hormone in lactating cows. J. Dairy Sci. 69:44. Richer, E., M.-J. Davicco, M. Dalle, and J. P. Barlet. 1985. Rdponses endocrines ~ l'insuline et ~ge conceptionnel chez le veau. Reprod. Nutr. Ddv. 25:427. Sidhu, K. S., and R. S. Emery. 1972. Regulation of blood fatty acids and glycerol in lactating cows. J. Dairy Sci. 55:926. Sidhu, K. S., and R. S. Emery. 1973. Blood fatty acids and glycerol response to diet and norepinephrine. J. Dairy Sci. 56:258. Stangassinger, M., W. Thevis, and D. Giesecke. 1986. Ueber den Einfluss der Tageszeit auf die Insulinfunktion bei normalen und fetten Kiihen. J. Anim. Physiol. Anim. Nutr. 56:157. Zapf, J., M. Waldvogel, and E. R. Froesch. 1977. Increased sensitivity of rat adipose tissue to the lipolytic action of epinephrine during fasting and its reversal during refeeding. FEBS Lett. 76:135. Zumstein, P., J. Zapf, M. Waldvogel, and E. R. Froesch. 1980. Increased sensitivity to lipolytic hormones of adenylate cyclase in fat cells of diabetic rats. Eur. J. Biochem. 105:187.
Journal of Dairy Science Vol. 71, No. 5, 1988
Responses of glucose and nonesterified f a t t y acids to adrenaline exhibited diurnal
differences. Responses of glucose to adrenaline were reduced within 1 d of fasting, whereas those of nonesterified fatty acids were not altered.
INTRODUCTION
Received August 3, 1987. Accepted December 21, 1987. 1Present address: F. Hoffmann-La Roche & Co. AG, Department of Vitamin and Nutrition Research, 4002 Basle, Switzerland. 2Present address, correspondence, and reprint requests: Department of Nutrition Pathology, Institute of Animal Breeding, University, Bremgartenstrasse 109a, 3012 Berne, Switzerland. 1988 J Dairy Sci 71 : 1170-1177
During early lactation, in dairy cows, fat oxidation is enhanced, whereas glucose oxidation is reduced, thus sparing glucose for lactose synthesis (3). Nevertheless, glucose concentrations are usually low as a consequence of the enhanced demand and insufficient gluconeogenesis (5, 10, 20). Concentrations of nonesterified fatty acids (NEFA) are elevated (9, 10). The magnitude of the rise depends on the degree of energy deficiency (9, 20). Concentrations of NEFA increase during the night in association with decreasing concentrations of immunoreactive insulin (IRI) as a consequence of decreased feed, i.e., energy intake (9). Glucose concentrations increase during the night, especially in the energy-deficient animals, likely as a result of low circulating insulin, which favors gluconeogenesis and glycolysis (9). Although somatotropin is the most potent hormone stimulating milk production (4), several other hormones regulate metabolism in a direction that favors milk secretion (12, 18). The possible role(s) of catecholamines (CA) for nutrient repartitioning in lactating cows has not been well-studied. This is somehow surprising, because adrenaline (epinephrine, E) and noradrenaline (norepinephrine, NE) cause NEFA and glucose to rise (8, 11). In addition, they cause insulin resistance (Blum, unpublished observations), as does somatotropin, at least during short-lasting administration (15). There is some evidence that CA can change metabolism in a direction typical for lactation. Thus, the number of/3-adrenergic receptors on
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CATECHOLAMINE EFFECTS IN COWS fat cells increase during lactation compared with numbers during the dry period (17). In addition, NEFA and glycerol responses are greater after parturition than before in cows as a consequence of reduced reesterification of NEFA and an increase in the active form of lipase in adipose tissue (23). Variable provision of food energy may be in part responsible for enhanced sensitivity to E, because responses of NEFA and glucose to E and NE are modified by fasting and low circulating IRI (1, 8, 13, 24, 26, 27). Also, E decreases acetate conversion to fatty acids (2). Furthermore, during the administration of somatotropin to lactating cows, effects of E on NEFA became enhanced (21). Endogenously produced somatotropin is usually elevated during lactation, especially when compared with levels during the dry period (14, 16, 20), which could contribute to enhanced fat mobilization. Experiments were conducted to study a possible contribution of endogenous CA on the nightly rise of NEFA and glucose, by inhibiting ~3-adrenergic effects with propranolol. Possible diurnal modifications and changes caused by fasting of NEFA, glucose, and lactate responses to the administration of E were also investigated. MATERIALS A N D METHODS Experimental Procedures
Effects of Propranolol on Plasma Nonesterified Fatty Acids, Glucose, and Immunoreactive Insulin. Six cows (two experiments with three animals) in their second to fourth lactations were studied 4 to 6 wk after parturition. Their milk production during 305 d of their preceding lactation was 5949 + 291 kg, and milk yield on the day before the start of the experiments was 32.8 -+ 2.0 kg/d. Animals were milked at 0515 and 1600 h. Prior to the experiment, animals received hay and corn silage ad libitum and concentrates at 0700 and 1700 h. During the experiment (starting at 1800 h), feed was completely removed from 1900 to 0700 h of the next morning, at which time the animals were fed. Catheters were implanted in both jugular veins on the morning before the start (1800 h) of the infusions of the ~-adrenergic blocking agent propranolol (19.3 nmol/kg/min for 845 rain, i.e., up to 0805 h of the next morning).
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Infusions of the ~-adrenergic agonist isoproterenol (286 pmol/kg/min from 0800 to 0805 h) were performed in addition to propranolol infusions through the same catheter. Blood samples were obtained from the contralateral vein at 1750, 1755, and 1759 (before the start of the propranolol infusions), 1900, 2000, 2100, 2200, 2300, 2400, 0100, 0200, 0300, 0400, 0500, 0515, 0530, 0545, 0600, 0615, 0630, 0645, 0700, 0730, 0755, 0758, 0800 (before the isoproterenol infusions), 0804, 0805, 0806, 0810, and 0820 h.
Effects of Epinephrine on Plasma Nonesterified Fatty Acids, Glucose, and Lactate in Normally Fed and Fasted Cows. Nine cows (three experiments with three animals) in their second to fourth lactations were studied 4 to 6 wk after parturition. Their milk production during 305 d of the preceding lactation was 7424 -+ 466 kg and milk yield on the day before the start of the experiments was 36.1 -+ 1.2 kg/d. The animals were milked twice daily (at 0515 and 1600 h). Prior to 0700 h of the 2nd d of the experiment animals received hay and corn silage ad libitum and concentrates twice daily (at 0700 and 1700 h). This corresponded to an intake of 135 -+ 3 MJ NE 1 and 2.84 +- 1.64 kg crude protein. From 0700 to 1900 h the animals received only hay ad libitum, which corresponded to an intake of 39.9 -+ 1.3 MJ NE I and .39 +- .13 kg crude protein. Then feed was completely removed until 0700 h of the next morning. Catheters were implanted in both jugular veins at 1800 h of the experiment. Epinephrine (.82 nmol/kg/min for 10 rain) was infused starting on the 1st d at 1800 h (infusion A), on the 2nd d at 0600 h (infusion B), and on the 3rd d again at 0600 h (infusion C). Blood samples were obtained from the contralateral veins at 10.5 and .5 rain before and 4, 6, 8, 10, 15, 20, 30, 45, and 60 min after the start of the 10-min infusions. Laboratory Procedures
Blood samples were immediately transferred to tubes containing heparin (50 U USP/ml blood) or in ice-cold perchloric acid (1 ml/1 ml blood) and centrifuged at 4°C within 2 h after collection. Plasma aliquots of heparinized blood samples were transferred to polystyrol cups and Journal of Dairy Science Vol. 71, No. 5, 1988
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FR(3HLI AND BLUM
frozen at - 2 0 ° C until analyzed for E, NE, IRI, triiodothyronine (T3), NEFA, and glucose. Deproteinized supernatants were filtered and frozen at - 2 0 ° C until analyzed for the determination of acetoacetate (AAC) and 1lactate. Concentrations of 1~ and NE were determined by a radioenzymatic method, IRI and T3 by radioimmunassay, and metabolites colorimetrically (9). All samples from one experiment were determined within the same assay. Control sera were measured to evaluate interassay variations. Drugs
DL-Propranolol was obtained from Imperial Chemical Industries, Macclesfield/England; epinephrine-bitartrate from Fluka AG, Buchs/ Switzerland; and isoproterenol-HC1 from Winthrop Products Co., Surbiton on Thames/ England. Epinephrine and isoproterenol were dissolved in .9% NaC1. Solutions of propranolol also contained 31.5 mg citric acid in 100 ml. During the experiments the solutions were kept on ice in light-protected bottles. Statistical Analysis
Means (+ SE) at various times during the infusions were compared to mean basal concentrations by paired t-test and total incremental or decremental changes after calculation of the area under the concentration curve (concentration × volume - 1 × m i n ) f o r each individual. R ESU LTS
Effects of Propranolol on Plasma Nonesterified Fatty Acids, Glucose, and Immunoreactive Insulin
Concentrations of NEFA, after a slight initial decrease (after I h; P<.05) continuously increased (higher than preinfusion concentrations at 2400 and later; P<.05), then rapidly decreased when animals were fed at 0700 h to values lower than those before the start of the infusions (P<.05), as shown in Figure 1. Concentrations of glucose started to decrease at 2200 h and were lower at 0100 h than preinfusion concentrations (P<.05) and rapidly increased towards preinfusion concentrations after the animals were again fed at 0700 h. Journal of Dairy Science Vol. 71, No. 5, 1988
Concentrations of IRI decreased during the first hours of the propranolol infusions (P<.05) and then remained low until 0700 h, after which time they rapidly increased to concentrations slightly higher than preinfusion concentrations. From the start of the experiment to 0700 h and from 0700 to 0800 h, IRI was negatively related to NEFA (r = --.88 and - . 9 7 , respectively). During infusions of isoproterenol (from 0800 to 0805 h) concentrations of IRI, NEFA, and glucose did not change. Effects of Epinephrine on Plasma Nonesterified Fatty Acids, Glucose, and Lactic Acid in Normally Fed and Fasted Cows
Concentrations of NEFA before the three infusions increased during the experiment (P<.01) and transiently increased in response to the E infusions (Figure 2). The total incremental change (mmol ° L - 1 ° 60 min) was significantly smaller in response to the first infusion (A) than in response to the following infusions, but changes in response to infusions B and C were similar. Concentrations of glucose were highest before the second infusion (B), lowest before the third infusion (C), and different before infusions A and B as well as B and C (P<.001). The total incremental changes (mmol • L - 1 60 min) were different from each other (P<.05) and greatest in response to infusion B and smallest in response to infusion C. Concentrations of lactate before the infusions were similar. The total incremental changes (mmol • L - 1 • 60 rain) were highest in response to infusion B and lowest in response to infusion C, but there were no significant differences between the three infusions. Concentrations of E (not shown) were higher before infusion C (.60 -+ .06 ~tmol/L) than before infusions A and B (.36 -+ .04 and .43 + .04 /amol/L, respectively) (P<.O1), reached maximal concentrations between 8 and 10 rain of the infusions (8.47 -+ .42, 8.72 -+ .48, and 8.76 + .51 /lmol/L above basal concentrations during infusions A, B, and C, respectively; P<.001), and then rapidly decreased. The total incremental changes were similar during the infusions (150 + 8, 152 -+ 9, and 155 + 9/amol • L - 1 • 60 min). Basal concentrations of NE were similar (2.1 + .3, 1.9 + .3, and 2.1 -+ .3 /amol/L before infusions A, B, and C, re-
C A T E C H O L A M I N E E F F E C T S IN COWS
1173
NEFR mmol/[
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.95
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1788
1988
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2380
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Figure 1. Changes o f nonesterfied fatty acids, glucose and insulin in blood of lactating cows during t h e night in the presence of propranolol, c o m b i n e d w i t h fasting, realimentation, and 5-min i s o p r o t e r e n o l i n f u s i o n s .
J o u r n a l of Dairy Science Vol. 71, No. 5, 1988
FROHLIAND
1174
NEFR
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.
BLUM
25
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i
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.58
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II ] ] I[
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I
o
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1]~ : : : : : : : : 0600 8780 q
3r.d DRY in b l o o d
of lactating cows in response
to lO-min infusions of epinephrine, starting on the 1st d at 1800 h and on the 2rid and 3rd d at 0600 animals were fasted, starting after the second until the end of the third infusion.
J o u r n a l o f D a i r y S c i e n c e V o l . 7 1 , N o . 5, 1 9 8 8
h. T h e
CATECHOLAMINE EFFECTS IN COWS spectively). Norepinephrine concentrations did not change significantly during the E infusion (not shown). Concentrations before infusions A, B, and C of IRI (.67 + .16, .26 + .05, and .09 + .04 gg/L, respectively) and T3 (2.76 -+ .11, 2.25 + .15, and 1.94 + .11 nmol/L) continuously decreased and were different from each other (P<.05) (not shown). Concentrations of (AAC) before infusion A (108 + 28 ;zmol/L) were higher than before infusion B (61 + 31/amol/L) and highest before infusion C (188 + 33 /amol/L but basal AAC concentrations were different only between infusion B and C (P<.05) (not shown). Milk yield was 36.1 -+ 1.2 kg/d on the day before the start of the experiment, 36.0 + 1.0 kg on the 1st d, 36.8 +- 1.0 kg on the 2nd d, 31.7 -+ 1.4 kg on the 3rd d of the experiment, and 35.1 + 1.1 kg on the day after the experiment (not shown). DISCUSSION
Feed restriction during the night in experiments with propranolol simulated the normal situation, in which feed intake is markedly reduced during the night in dairy cows (9). In the present study cows were unaffected by the short time without feed, and metabolic and endocrine changes were relatively small. Feed removal starting at 0600 h for 23 h was characterized by lowered glucose, IRI, and T3 concentrations and elevated NEFA and ketone bodies, which are typical for cattle during energy restriction (10, 20). The increase of E concentrations during fasting was possibly due to hypoglycemia. Thus, insulin-induced reduction of glucose concentrations was associated with an immediate increase of E and NE concentrations in calves (7, 22), as found in other species. Stimulation of lipolysis in cattle by CA is mediated entirely by 13-adrenergic receptors on fat cells because the rise of NEFA concentrations in response to the administration of E, NE, and ~3-adrenergic agonists can be suppressed by ¢-adrenergic blockade with propranolol (8). The continuous increase of NEFA during the night in the presence of propranolol was comparable to results of a previous study performed in the absence of propranolol (9). Because propranolol could not prevent the increase of NEFA during the night indicates
1175
that enhanced lipolysis was not the consequence of an increased sympathoadrenal activity. Propranolol was infused in amounts sufficient to inhibit the typical rise of NEFA in response to the administration of the/3-adrenergic agonist isoproterenol seen otherwise in the absence of/3-adrenergic blockade (9). The'high negative relationship between concentrations of NEFA and IRI and in particular the rapid fall of NEFA concentrations in response to the increase of IRI concentrations during refeeding suggest that insulin is important for the regulation of NEFA concentrations. In this study, glucose concentrations decreased during the night in the presence of propranolol, suggesting that reduction of sympathoadrenal activity was responsible for this effect. However, amounts of plasma E and NE do not change significantly during the night in lactating dairy cows (9). The increase of glucose concentrations observed after the intake of concentrates in the morning was also surprising because glucose concentrations decreased in the absence of propranolol in previous studies (9). Thus, ~-adrenergic blockade obviously reversed the normal pattern of glucose during the night and in response to food intake. The greater responses of both NEFA and glucose in response to the administration of E in the morning vs. night suggested enhanced sensitivity or responsiveness of fat and liver cells to the catecholamine. Because lactate response was not changed, glycogenolysis in liver and muscle is probably modified differently by time of day. The marked diurnal variations of NEFA and glucose responses to E has to our knowledge not been previously reported. In our study, diurnal variations in sensitivity or responsiveness of target organs to E were likely modified by changes in blood concentrations of insulin, which were lower in the morning than night, thus favoring lipolysis and glycogenolysis in the morning. In lactating cows, IRI concentrations and sensitivity to insulin were lower in the morning than night and were associated with elevated glucose concentrations and reduced clearance rates of glucose in the morning (25). It is possible that other hormones, whose blood concentrations also exhibit diurnal variations as well as inherent diurnal patterns in the activity of
Journal of Dairy Science Vol. 71, No. 5, 1988
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FROHLI AND BLUM
enzymes involved in lipo- and glycogenolysis, also contributed to changes in sensitivity to E. Greater NEFA and glycerol responses and reduced glucose and lactate responses to E after 3 or 5 d of fasting has been reported previously in cattle (8, 13). In starvation ketotis, the increase of NEFA to the administration of E was enhanced (19). The present study shows that during fasting the time required for fat tissue to become more responsive and for liver and muscle to become less responsive to E is only about 1 d, much shorter than noted previously. The marked decrease of circulating insulin presumably favors enhanced E-stimulated lipolysis. Elevated blood somatotropin concentrations were associated with enhanced responsiveness of NEFA to E (21). The reduced increase of glucose in response to E after 24 h of feed withdrawl was presumably in part the consequence of decreased glycogen stores in the liver. The decrease of plasma T 3 concentrations during fasting probably modified the sensitivity of fat, liver, and muscle cells to E. Low thyroid hormone concentrations in hypothyroidism are usually associated with reduced lipolysis (6), which was not the case in our experiment. Because identical amounts of E were administered and since E concentrations in plasma increased in a comparable manner, amounts of E expected to interact with target organs were similar and do not explain diurnal variations or altered responses to E during fasting of glucose and NEFA. The interesting new information from this investigation is the marked diurnal variations of NEFA and glucose responses to E. Changes in metabolic responses to E during fasting could be observed within 24 h. Thus, high yielding dairy cows rapidly adapt in response to changes in feed intake. We speculate that metabolic alterations under basal conditions are regulated by CA largely at target organs and possibly only to a minor extent by changes in sympathoadrenal activity and circulating CA concentrations. This holds in part also for other hormones (12). Changes in partitioning of nutrients in favor of the mammary gland are considered to be largely the consequence of altered responsiveness of key tissues to homeostatic signals (3).
Journal of Dairy Science Vol. 71, No. 5, 1988
ACKNOWLEDGMENTS
This study has been supported by a grant of the Swiss Federal Institute of Technology (No. .330.057.15/9) and by F. Hoffmann-La Roche & Co. AG. The technical assistance of R. Admaty, M. Janeziz, and W. Moses is greatly acknowledged. We thank H. Schneeberger, Swiss Federal Research Station of Animal Production, Grangeneuve, 1724 Posieux, for providing us with the experimental facilities. REFERENCES
1 Arner, P., P. Engfeldt, and J. Nowak. 1981. In vivo observations on the lipolytic effect of noradrenaline during therapeutic fasting. J. Clin. Endocrinol. Metab. 53:1207. 2 Bauman, D. E. 1976. Intermediary metabolism of adipose tissue. Fed. Proc. 35:2308. 3 Bauman, D. E., and W. B. Currie. 1980. Partitioning of nutrients during pregnancy and lactation: a review of mechanisms involving homeostasis and homeorhesis. J. Dairy Sci. 63:1514. 4 Bauman, D. E., P. J. Eppard, M. J. De Geeter, and G. M. Lanza. 1985. Responses of high-producing dairy cows to longterm treatment with pituitary somatotropin and recombinant somatotropin. J. Dairy Sci. 68:1352. 5 Bergman, E. N. 1973. Glucose metabolism in ruminants as related to hypoglycemia and ketosis. Cornell Vet. 63:341. 6 Bilezikian, J. P., and J. N. Loeb. 1983. The influence of hyperthyroidism and hypothyroidism of a- and B-adrenergic receptor systems and adrenergic responsiveness. Endocrine Rev. 4:378. 7 Bloom, S. R., A. V. Edwards, R. N. Hardy, K. M. Malinkowsky, and M. Silver. Endocrine response to insulin hypoglycaemia in the young calf. J. Physiol. Lond. 244:783. 8 Blum, J. W., D. FriJhli, and P. Kunz. 1982. Effect of catecholamines on plasma free fatty acids in fed and fasted cattle. Endocrinology 110:452. 9 Blum, J. W., F. Jans, W. Moses, D. Ffdhli, M. Zemp, M. Wanner, I. C. Hart, R. Thun, and U. Keller. 1985. Twenty-four hour pattern of blood hormone and metabolite concentrations in highyielding dairy cows: effects of feeding low or high amounts of starch, or crystalline fat. Zentralbl. Veterinaermed. A 32:401. 10 Blum, J. W., P. Kunz, H. Leuenberger, K. Gautschi, and M. Keller. 1983. Thyroid hormones, blood plasma metabolires and haematological parameters in relationship to milk yield in dairy cows. Anita. Prod. 36:93. 11 Brockman, R. P., and B. Laarveld. 1986. Hormonal regulation of metabolism in ruminants: a review. Livest. Prod. Sci. 14:313. 12 Collier, R. J, J. p. Mc Namara, C. R. Wallace, and M. H. Dehoff. 1984. A review of endocrine regulation of metabolism during lactation. J. Anita.
CATECHOLAMINE EFFECTS IN COWS Sci. 59:498. 13 FriShli, D., and J. W. Blum. 1985. Epinephrine and norepinephrine during fasting: blood levels, metabolism and metabolic effects. Proc. 13th Int. Congr. Nutr., August 18--23, 1985 in Bringhton/ England (Abstr. 0034). 14 Hart, I. C., J. A. Bines, S. V. Morant, and J. L. Ridley. 1978. Endocrine control of energy metabolism in the cow: comparison of levels of hormones (prolactin, growth hormone, insulin and thyroxine) and metabolites in the plasma of highand low-yielding cattle at various stages of lactation. J. Endocrinol. 77:333. 15 Hart, I. C., P.M.E. Chadwick, T. C. Boone, K. E. Langley, C. Rudman, and L. M. Souza. 1984. A comparison of the growth-promoting, lipolytic, diabetogenic and immunological properties of pituitary and recombinant DNA-derived bovine growth hormone (somatotropine). Biochem. J. 224:93. 16 Herbein, J. H., R. J. Aiello, L. I. Ecklor, R. E. Pearson, and R. M. Akers. 1985. Glucagon, insulin, growth hormone, and glucose concentrations in blood plasma of lactating dairy cows. J. Dairy Sci. 68:320. 17 Jaster, E. H., and T. N. Wegner. 1981. Betaadrenergic receptor involvement in lipolysis of dairy cattle subcutaneous adipose tissue during dry and lactating state. J. Dairy Sci. 64:1655. 18 Karg, H., and H. Mayer. 1987. Manipulation der Laktation. Uebersichten Tierern'ahr. 15:29. 19 Kronfeld, D. S. 1965. Plasma non-esterified fatty acid concentrations in the dairy cow: responses to
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22
23
24
25
26
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nutritional and hormonal stimuli, and significance in ketosis. Vet. Rec. 77:30. Kunz, P., J. W. Blum, I. C. Hart, H. Bickel, and J. Landis. 198"5. Effects of different energy intakes before and after calving on food intake, performance and metabolic variables in dairy cows. Anim. Prod. 40:219. McCutcheon, S. N., and D. E. Bauman. 1986. Effect of chronic growth hormone treatment on responses to epinephrine and thyrotropin-releasing hormone in lactating cows. J. Dairy Sci. 69:44. Richer, E., M.-J. Davicco, M. Dalle, and J. P. Barlet. 1985. Rdponses endocrines ~ l'insuline et ~ge conceptionnel chez le veau. Reprod. Nutr. Ddv. 25:427. Sidhu, K. S., and R. S. Emery. 1972. Regulation of blood fatty acids and glycerol in lactating cows. J. Dairy Sci. 55:926. Sidhu, K. S., and R. S. Emery. 1973. Blood fatty acids and glycerol response to diet and norepinephrine. J. Dairy Sci. 56:258. Stangassinger, M., W. Thevis, and D. Giesecke. 1986. Ueber den Einfluss der Tageszeit auf die Insulinfunktion bei normalen und fetten Kiihen. J. Anim. Physiol. Anim. Nutr. 56:157. Zapf, J., M. Waldvogel, and E. R. Froesch. 1977. Increased sensitivity of rat adipose tissue to the lipolytic action of epinephrine during fasting and its reversal during refeeding. FEBS Lett. 76:135. Zumstein, P., J. Zapf, M. Waldvogel, and E. R. Froesch. 1980. Increased sensitivity to lipolytic hormones of adenylate cyclase in fat cells of diabetic rats. Eur. J. Biochem. 105:187.
Journal of Dairy Science Vol. 71, No. 5, 1988