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Glucagon-lnduced Desensitization of Broiler Adipocyte Lipolysis1
Glucagon-lnduced Desensitization of Broiler Adipocyte Lipolysis1 T. P. OSCAR 2 Division of Animal and Veterinary Sciences, West Virginia University, Morgantoxvn, West Virginia 26506-6108
1992 Poultry Science 71:1015-1021
INTRODUCTION
glucagonemia induced by exogenous GLU administration is associated with a reducThe phenomenon by which the biologi- tion of target tissue responsiveness to cal action of a hormone on a cell decreases GLU (DeRubertis and Craven, 1976; Sogradually over time is known as desensiti- man and Felig, 1978). zation (Sibley and Lefkowitz, 1985). A It is well established that GLU acutely number of in vitro studies indicate that stimulates lipolysis from avian adipocytes glucagon (GLU) induces desensitization of (Leclercq, 1984). However, it has not been its metabolic effects in rat hepatocytes determined whether GLU induces desen(Plas and Nunez, 1975; Gurr and Ruh, sitization of its lipolytic effect in avian 1980; Iyengar et a\., 1980; Santos and adipocytes. Induction of desensitization by Blasquez, 1982; Heyworth and Houslay, GLU in hepatocytes results in down1983; Noda et ah, 1984; Murphy et ah, regulation of GLU receptors (Santos and 1987), canine kidney cell line MDCK (Rich Blasquez, 1982; Noda et ah, 1984), whereas et ah, 1984), and rat Sertoli cells (Attrama- in the canine kidney cell line MDCK dal et ah, 1988). Likewise, in vivo hyper- desensitization is associated with increased activity of the inhibitory guanine nucleotide binding protein (Gi) (Rich et ah, 1984). Thus, GLU-induced desensitization Received for publication October 24, 1991. of its metabolic effects in vitro may Accepted for publication January 15, 1992. 1 Scientific Article Number 2288, West Virginia Uni- provide a useful model system for investiversity Agricultural and Forestry Experiment Station. gating the role of cellular proteins in the Present address: USDA, Agricultural Research regulation of GLU biological action. Service, Poultry Research Laboratory, Georgetown, Therefore, the present study was underDE 19947. 1015
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ABSTRACT Continuous exposure of cells to a hormonal stimulus results in attenuation of the hormone's effects on the cell; a process known as desensitization. The present study was undertaken to determine whether glucagon (GLU) induces desensitization of its lipolytic effect in adipocytes isolated from the abdominal fat of market-age broilers. Preincubation of adipocytes with 10 to 100 n g / m L of porcine GLU (pGLU) or chicken GLU (cGLU) for 24 h reduced (P < .05) GLU-stimulated lipolysis. However, pGLU decreased (P < .05) lipolysis to a greater extent than cGLU. Maximal lipolysis was reduced 70% by pGLU and 55% by cGLU. Chicken GLU also exhibited lower biological potency for acutely stimulating lipolysis from control and cGLU-treated adipocytes. Glycerol release from control adipocytes incubated for 1 h with .3 n g / m L of cGLU or pGLU was 26 and 42 n m o l / h per 3% cells, respectively. The GLU-induced decrease in lipolysis occurred rapidly and was partially reversible. The results of the present study indicated that GLU induced desensitization of its lipolytic effect in broiler adipocytes. {Key words: broiler adipocytes, chicken glucagon, porcine glucagon, lipolysis, desensitization)
1016
OSCAR
Isolation and Culture of Adipocytes
Lipolysis Assay
Adipocytes were isolated under sterile conditions and maintained in primary culture using the method of Oscar (1991). In brief, broilers w e r e euthanatized by asphyxiation with carbon dioxide. Adipose tissue (2 g per digest) was minced in plastic containers and then digested with sterile digestion medium (SDM) for 1 h at 37 C in an orbital shaking (150 opm) water bath. The SDM contained Dulbecco's MEM supplemented with 10 m M glucose, 25 mM
To remove GLU before measurement of lipolysis, adipocytes were washed with 15, 10, and 6 mL of washing medium (WM). The WM was identical in composition to AM except that it contained 1% BSA rather than 3% BSA. In addition, a 20-min incubation at 37 C was included after the first two w a s h steps to facilitate removal of adipocyte-associated GLU. After washing, adipocytes were resuspended in AM. The packed cell volume of the suspension -was determined by centrifugation in microhematocrit tubes (Oscar, 1991). Adipocytes (total volume of 9 uL or a final concentration of 3% adipocyte volume per assay volume) were assayed for lipolysis by incubation for 1 h at 37 C in .3 mL of
MATERIALS AND METHODS Materials Porcine GLU (pGLU) was a gift from Eli Lilly.3 Chicken GLU (cGLU) was prepared by Litron Laboratories 4 and was a gift from John P. McMurtry. 5 Tissue culture ingredients were obtained as previously described (Oscar, 1991). Female broilers (Vantress x Cobb), 49 to 63 days old, were used as the source of abdominal adipose tissue.
Glucagon Solutions
^ l i Lilly and Co., Indianapolis, IN 46200. Litron Laboratories, Rochester, NY 14620. ^JSDA, Agricultural Research Service, Beltsville, MD 20705. 4
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Chicken GLU and pGLU were dissolved in .001 NHC1, pH 2.5 and the concentration of GLU (.2 to .3 mg/mL) in these solutions was determined spectrophotometrically (Pollock and Kimmel, 1975). Stock solutions of GLU (.05 mg/mL) were prepared by diluting the acidified solutions of pGLU and cGLU with assay medium (AM). Assay medium was composed of phenol red and pyruvate-free Dulbecco's modified Eagle's medium (MEM) supplemented with 10 mM glucose, 25 mM 4-(2-hydroxyethyl)-lpiperazine-ethanesulfonic acid (HEPES), and 3% BSA with a pH of 7.6 at 22 C. Aliquots (.2 mL) of the GLU stock solutions were stored at -20 C.
HEPES, 3% BSA, 250 units/mL collagenase, and .125% trypsin; p H 7.6 at 22 C. To obtain enough adipocytes for a replicate of an experiment, one digest was conducted for each treatment. After 1 h of digestion, 26 mL of sterile incubation medium (SIM) was added to each digest container. The SIM was composed of Dulbecco's MEM supplemented with 10 mM glucose, 25 mM HEPES, antibiotics (penicillin, streptomycin, and neomycin), and 1% BSA; p H 7.6 at 22 C. To remove undigested tissue, the diluted digestion mixture was filtered through polypropylene mesh and collected into separate 50-mL polypropylene centrifuge tubes. Once the adipocytes had floated to form a layer, the infranatant that contained digestion enzymes and stromal-vascular cells was removed and the adipocytes were resuspended in 30 mL of SIM. This washing procedure was repeated three more times except that the adipocytes were resuspended in 20,10, and 3 mL of SIM. After the final wash step, the adipocyte suspensions were combined to produce a single preparation of adipocytes. Adipocytes were cultured at 37 C in 50-mL polypropylene centrifuge tubes that contained 30 mL of SIM under an initial atmosphere of air. After 24 h in culture, pGLU or cGLU (0 to 100 ng/mL) were added and adipocytes were incubated for .5 to 24 h before removal of GLU by washing and assessment of lipolysis.
taken to determine whether GLU induces desensitization of lipolysis by broiler adipocytes maintained in primary culture.
DESENSITIZATION OF ADIPOCYTE LIPOLYSIS
(Figure 1). Likewise, preincubation with 10 n g / m L pGLU did not alter BL or SL but reduced ML 10%. Submaximal lipolysis was 55% lower and ML was 40% lower from adipocytes pretreated with 100 n g / mL pGLU, whereas BL was similar between control adipocytes and those incubated with 100 n g / m L pGLU. In a subsequent experiment (results not shown), similar reductions of SL and ML were noted when adipocytes were preincubated with 25 or 50 n g / m L pGLU for 24 h. To determine whether preincubation with the homologous hormone would also reduce lipolysis, adipocytes were incubated for 24 h with 0, 1, 10, or 100 n g / mL cGLU before removal of cGLU by washing and assessment of lipolysis (Figure 2). Similar to results with 1 n g / m L pGLU, preincubation of adipocytes with 1 n g / m L cGLU did not alter lipolysis. In
Statistical Analysis Treatment effects on characteristics of lipolysis were examined by ANOVA for a randomized complete block design using the General Linear Models (GLM) procedure of base SAS® software (SAS Institute, 1985). Each experiment consisted of three to five replicates with each replicate involving the preparation of a single batch of adipocytes from one donor bird. When a significant (P < .05) treatment effect was observed, least square means were compared using the PDIFF option (uses repeated t tests) of the GLM procedure of SAS® (SAS Institute, 1985).
RESULTS Preincubation of adipocytes with 1 n g / mL pGLU for 24 h did not alter lipolysis
1 10 pGLU (ng/mL)
100
FIGURE 1. Effect of porcine glucagon (pGLU) on lipolysis by broiler adipocytes in primary culture. Adipocytes were incubated with 0,1, 10, or 100 ng/ mL pGLU for 24 h before removal of pGLU by washing and assessment of basal lipolysis (•), submaximal lipolysis (•), and maximal lipolysis (•) with pGLU. Each point is the average of four replicates. Points within a line with no common letters differ (P < .05).
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AM that contained 0 to 10 n g / m L pGLU or cGLU (Oscar, 1991). Glycerol released into the medium was used as the index of lipolysis and was measured using the glycerol kinase method of Eggstein and Kuhlmann (1974). Four characteristics of lipolysis were examined in the present study. 1) Basal lipolysis (BL) was the amount of glycerol release that occurred in the absence of hormonal stimulation. 2) Maximal lipolysis (ML) was the amount of lipolysis in the presence of 10 n g / m L GLU, a dose of GLU that stimulates lipolysis maximally in primary cultured broiler adipocytes (Oscar, 1991). 3) Submaximal lipolysis (SL) was the quantity of glycerol release in the presence of a less than maximal dose of GLU (1 n g / mL). 4) Physiological lipolysis (PL) was the amount of lipolysis in the presence of .3 n g / mL GLU, which is a dose that is within the normal range of plasma GLU concentrations in broilers (Cogburn et ah, 1989). The DNA content of adipocytes used to assess lipolysis was determined using the fluorometric method of Labarca and Paigen (1980) with calf thymus DNA as the standard. Preincubation of adipocytes with GLU did not alter the amount of DNA per 9 uL or 3% adipocytes (results not shown). Therefore, lipolysis results were expressed as glycerol release in nanomoles per hour per 3% adipocytes.
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FIGURE 2. Effect of chicken glucagon (cGLU) on lipolysis by broiler adipocytes in primary culture. Adipocytes were incubated with 0,1, 10, or 100 ng/ mL cGLU for 24 h before removal of cGLU by washing and assessment of basal lipolysis (•), submaximal lipolysis (•), and maximal lipolysis (A) with porcine GLU (pGLU). Each point is the average of five replicates. Points within a line with no common letters differ (P < .05).
addition, BL was not affected by pretreatment with cGLU. However, SL was decreased 15% and ML was nonsignificantly (P < .10) decreased 10% by preincubation with 10 ng/mL cGLU. Both SL and ML were reduced 20% by the highest dose (100 ng/mL) of cGLU. The reductions of lipolysis observed upon preincubation with 100 ng/mL cGLU (Figure 2) were lower in magnitude than those observed with 100 ng/mL pGLU (Figure 1). This suggested that cGLU had a lower biological potency than pGLU; however, the dose-response experiment with cGLU (Figure 2) was conducted after the dose-response experiment with pGLU (Figure 1). To directly compare the effects of preincubation with pGLU and cGLU on lipolysis, adipocytes were incubated for 24 h without GLU (control) or with 100 ng/mL cGLU or 100 ng/mL pGLU before removal of hormones by washing and measurement of lipolysis
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FIGURE 3. Comparison of the effect of preincubation with porcine glucagon (pGLU) and chicken glucagon (cGLU) on lipolysis by broiler adipocytes in primary culture. Adipocytes were incubated without GLU (control) or with 100 ng/mL cGLU or 100 ng/ mL pGLU for 24 h before removal of hormones by washing and assessment of basal lipolysis (BL), physiological lipolysis (PL), submaximal lipolysis (SL), and maximal lipolysis (ML) with pGLU. Each bar is the average of four replicates. Bars within a cluster with no common letters differ (P < .05).
(Figure 3). Both cGLU and pGLU reduced BL 75%. In general, pretreatment with pGLU reduced PL, SL, and ML to a greater extent than pretreatment with cGLU but only ML was statistically lower from pGLU-treated adipocytes; ML was reduced 55% by cGLU and 70% by pGLU. To compare the acute effects of pGLU and cGLU on lipolysis, adipocytes were preincubated for 24 h without GLU (control) or with 100 ng/mL cGLU before removal of cGLU by washing and assessment of lipolysis within each culture treatment using both cGLU and pGLU (Figure 4). Similar to previous experiments (Figures 2 and 3), preincubation with 100 ng/mL cGLU reduced PL, SL, and ML but did not alter BL. Within each culture
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FIGURE 4. Comparison of the acute effects of porcine glucagon (pGLU) and chicken glucagon (cGLU) on lipolysis by broiler adipocytes in primary culture. Adipocytes were incubated with 0 (control) or 100 ng/mL cGLU (cGLU-treated) for 24 h before removal of cGLU by washing and measurement of basal lipolysis 03L), physiological lipolysis (PL), submaximal lipolysis (SL), and maximal lipolysis (ML) within each culture treatment with both cGLU and pGLU. Each bar is the average of four replicates. Bars within a cluster with no common letters differ (P < .05).
treatment, PL was 60% higher and SL was 30% higher when pGLU was used to assess lipolysis. As expected, ML within culture treatment was similar when measured with cGLU or pGLU. All types of lipolysis decreased rapidly after addition of 100 n g / m L pGLU to cultures of adipocytes (Figure 5). Significant reductions in lipolysis were noted after .5 h of incubation with pGLU. These reductions were maximal by 4 h. Maximal lipolysis of control adipocytes did not change during the 24-h incubation. However, BL decreased and SL increased over time in control adipocytes. Washing control adipocytes after 4 h of incubation did not alter BL or ML 20 h later but increased SL 40% (Figure 5). The depression of BL observed after 4 h incubation with 100 n g / m L pGLU was completely reversed 20 h after removal of pGLU by washing. However, the reductions of SL and ML of GLU-treated adipocytes noted at 4 h of incubation were
The results of the current study indicate that in addition to acute stimulation, GLU regulation of lipolysis also involves attenuation of its ability to stimulate triglyceride mobilization; a process known as desensitization. Both pGLU and cGLU exhibited similar effects on lipolysis. Preincubation of adipocytes for 24 h with high doses (10 to 100 ng/mL) of pGLU or cGLU reduced lipolysis. In addition, both types of GLU acutely stimulated lipolysis from control and desensitized adipocytes. However, pGLU exhibited a higher biological potency than cGLU. At the same dose in culture, pGLU reduced ML to a greater extent (Figure 3), whereas in the lipolysis assay PL and SL were higher when assessed with pGLU (Figure 4). McCumbee and Hazelwood (1978) and Shchenkova et al. (1987) also found that cGLU exhibits lower biological potency than mammalian GLU for acutely stimulating lipolysis from chicken adipocytes. These results are surprising because the structure of cGLU differs from pGLU by only one amino acid (Pollock and Kimmel, 1975). Chicken GLU contains a serine but pGLU has an asparagine at position 28 of GLU, a 29 amino acid length polypeptide. Further evidence that a single amino acid sequence difference in GLU can alter its biological potency comes from studies using synthetic GLU analogs. Substitution of aspartate or glutamate for glutamine at position 3 to produce [Asp3]-GLU and [Glu3]-GLU results in GLU analogs that stimulate adenylate cyclase in rat liver membranes with only 2.5% the potency of native GLU (Andreu and Merrifield, 1987). In addition, [Asp 3 ]-GLU has 125-fold and [Glu3]-GLU has 70-fold lower affinity than native GLU for binding to the rat liver GLU receptor. Other GLU analogs (i.e., [Glu9]-GLU, and [D-Phe4]-GLU) also exhibit lower biological potency and binding affinity than native GLU in liver prepara-
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FIGURE 5. Time course and reversibility of the effect of porcine glucagon (pGLU) on lipolysis by broiler adipocytes in primary culture. Adipocytes A variety of evidence suggests that the were incubated without GLU (Control; •), or with rapid phase of GLU-induced desensitiza100 ng/mL pGLU (•) for .5,4, or 24 h before removal of pGLU by washing and measurement of lipolysis tion of adenylate cyclase involves an with pGLU. In addition, after incubation for 4 h, a uncoupling of signal transduction between control (O) and pGLU-treated (•) culture in each the GLU receptor and the stimulatory replicate (n = 3) were washed under sterile condi- guanine nucleotide binding protein (Gs) tions and reincubated in culture for an additional 20 that activates adenylate cyclase (Heyworth h before measurement of lipolysis. Points within a time of incubation with no common letters differ (P < and Houslay, 1983; Noda et ah, 1984; Murphy et ah, 1987; Attramadal et ah, .05).
tions (Unson et ah, 1987; Hagopian et ah, 1987). In fact, circular dichroism analysis of [Glu9]-GLU revealed an increase in ochelix and a decrease in P-sheet conformation (Unson et ah, 1987). Thus, the altered biological potencies of GLU differing by one amino acid may result from conformational changes in the molecule that alter its binding affinity for the GLU receptor. Recently, Oscar (unpublished data) found that cGLU has a threefold lower affinity than pGLU for binding to cell-surface GLU receptors of primary cultured broiler adipocytes. This funding is consistent with the data for GLU analogs and indicates that the lower biological potency of cGLU
1988). Compounds, such as [1-N-ottrinitrophenylhistidine,12-homo-arginine]GLU (TH-GLU), angiotensin, and vasopressin, which stimulate inositol phospholipid metabolism but not cyclic adenosine monophosphate (cAMP) production, mimic GLU-induced desensitization (Murphy et ah, 1987). Furthermore, Wakelam et al. (1986) found that hepatocytes contain two populations of GLU receptors: one that stimulates adenylate cyclase and one that stimulates inositol phospholipid metabolism. Thus, it is proposed that GLU activation of protein kinase C via phosphatidylinositol-4,5bisphosphate breakdown results in phosphorylation of either the GLU receptor or G s or both to uncouple them from adenylate cyclase (Murphy et ah, 1987).
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in avian adipocytes results from its lower affinity for the GLU receptor. Glucagon-induced desensitization of lipolysis by broiler adipocytes occurred rapidly and was 70% reversible 20 h after induction for 4 h (Figure 5). Similar to results of the present study, GLU-induced desensitization of its metabolic effects in hepatocytes (Gurr and Ruh, 1980; Iyengar et ah, 1980; Heyworth and Houslay, 1983; Noda et ah, 1984; Murphy et ah, 1987), immature Sertoli cells (Attramadal et ah, 1988), and canine kidney cell line MDCK (Rich et ah, 1984) occurs rapidly and is reversible (DeRubertis and Craven, 1976; Gurr and Ruh, 1980). The rapid induction of desensitization of GLU-stimulated adenylate cyclase activity by GLU in liver is not accompanied by an immediate reduction in GLU binding (Santos and Blasquez, 1982; Heyworth and Houslay, 1983). However, down-regulation of GLU receptors is observed upon longer induction of desensitization (Santos a n d Blasquez, 1982; Noda et ah, 1984), which indicates that GLU receptors are involved in the mediation of desensitization in liver.
DESENSinZATION OF ADIPOCYTE LIPOLYSIS
Contrary to studies in rat hepatocytes, a cAMP analog was able to mimic GLUinduced desensitization of GLUstimulated adenylate cyclase activity in the canine kidney cell line MDCK (Rich et ah, 1984). In addition, GLU-induced desensitization of adenylate cyclase in MDCK cells was attributed to an increase in level or activity of Gj, rather than to an alteration in the level or activity of Gs. Thus, the mechanism of GLU-induced desensitization may differ between tissues and cell types. The mechanism by which GLU induces desensitization of lipolysis in broiler adipocytes is unknown but may involve changes in both GLU receptor and G protein metabolism.
Andreu, D., and R. B. Merrifield, 1987. Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs. Eur. J. Biochem. 164585-590. Attramadal, H., L. Eikvar, and V. Hansson, 1988. Mechanisms of glucagon-induced homologous and heterologous desensitization of adenylate cyclase in membranes and whole Sertoli cells of the rat. Endocrinology 123:1060-1068. Cogburn, L. A., S. S. Liou, A. L. Rand, and J. P. McMurtry, 1989. Growth, metabolic and endocrine responses of broiler cockerels given daily subcutaneous injection of natural or biosynthetic chicken growth hormone. J. Nutr. 119: 1213-1222. DeRubertis, F. R., and P. Craven, 1976. Reduced sensitivity of the hepatic adenylate cyclasecyclic AMP system to glucagon during sustained hormonal stimulation. J. Clin. Invest. 57: 435-443. Eggstein, M., and E. Kuhlmann, 1974. Triglycerides and glycerol. Determination after alkaline hydrolysis. Pages 1825-1831 in: Methods of Enzymatic Analysis. H. U. Bergmeyer, ed. Academic Press Inc., New York, NY. Gurr, J. A., and T. A. Ruh, 1980. Desensitization of primary cultures of adult rat liver parenchymal cells to stimulation of adenosine 3',5'monophosphate production by glucagon and epinephrine. Endocrinology 107:1309-1319. Hagopian, W. A., H. S. Tager, B. Gysin, D. Trivedi, and V. J. Hruby, 1987. Interactions of glucagon and glucagon analogs with isolated canine hepatocytes. J. Biol. Chem. 262:15506-15513. Heyworth, C. M., and M. D. Houslay, 1983. Challenge of hepatocytes by glucagon triggers a rapid modulation of adenylate cyclase in isolated membranes. Biochem. J. 21453-98. Iyengar, R., P. W. Mintz, T. L. Swartz, and L. Birnbaumer, 1980. Divalent cation-induced desensitization of glucagon-stimulable adenylyl cyclase in rat liver plasma membrane. J. Biol. Chem. 255:11875-11882.
Labarca, C , and K. Paigen, 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344-352. Leclercq, B., 1984. Adipose tissue metabolism and its control in birds. Poultry Sci. 632044-2054. McCumbee, W. D-, and R. L. Hazelwood, 1978. Sensitivity of chicken and rat adipocytes and hepatocytes to isologous and heterologous pancreatic hormones. Gen. Comp. Endocrinol. 34: 421-427. Murphy, G. J., V. J. Hruby, D. Trivedi, M.J.O. Wakelam, and M. D. Houslay, 1987. The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism. Biochem. J. 24339-46. Noda, C , F. Shinjyo, A. Tomomura, S. Kato, T. Nakamura, and A. Ichihara, 1984. Mechanism of heterologous desensitization of the adenylate cyclase system by glucagon in primary cultures of adult rat hepatocytes. J. Biol. Chem. 259: 7747-7754. Oscar, T. P., 1991. Glucagon-stimulated lipolysis of primary cultured broiler adipocytes. Poultry Sci. 70:326-332. Plas, C , and J. Nunez, 1975. Glycogenolytic response to glucagon of cultured fetal hepatocytes. J. Biol. Chem. 250:5304-5311. Pollock, H. G., and J. R. KimmeL 1975. Chicken glucagon. Isolation and amino acid sequence. J. Biol. Chem. 250:9377-4380. Rich, K. A., J. Codina, G. Floyd, R. Sekura, J. D. Hildebrandt, and R. Iyengar, 1984. Glucagoninduced heterologous desensitization of the MDCK cell adenylyl cyclase. J. Biol. Chem. 259: 7893-7901. Santos, A., and E. Blasquez, 1982. Direct evidence of a glucagon-dependent regulation of the concentration of glucagon receptors in the liver. Eur. J. Biochem. 121:671-677. SAS Institute, 1985. SAS® User's Guide: Statistics. Version 5 Edition. SAS Institute Inc., Cary, NC. Shchenkova, I. M., M. N. Pertseva, L. V. Dmitrenko, D. I. Ostrovsky, L. P. Soltitskaya, and L. A. Kuznetsova, 1987. Immunological properties and biological activity of chicken glucagon. Biokhimiya 52:359-367. Sibley, D. R., and R. J. Lefkowitz, 1985. Molecular mechanisms of receptor desensitization using the ^-adrenergic receptor-coupled adenylate cyclase system as a model. Nature (Lond.) 317: 124-129. Soman, V., and P. Felig, 1978. Regulation of the glucagon receptor by physiological hyperglucagonaemia. Nature (Lond.) 272:829-832. Unson, C. G., D. Andreu, E. M. Gurzenda, and R. B. Merrifield, 1987. Synthetic peptide antagonists of glucagon. Proc. Natl. Acad. Sci. USA 84: 4083-4087. Wakelam, M.J.O., G. J. Murphy, V. J. Hruby, and Miles D. Houslay, 1986. Activation of two signal-transduction systems in hepatocytes by glucagon. Nature (Lond.) 323:68-71.
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REFERENCES
1021
1992 Poultry Science 71:1015-1021
INTRODUCTION
glucagonemia induced by exogenous GLU administration is associated with a reducThe phenomenon by which the biologi- tion of target tissue responsiveness to cal action of a hormone on a cell decreases GLU (DeRubertis and Craven, 1976; Sogradually over time is known as desensiti- man and Felig, 1978). zation (Sibley and Lefkowitz, 1985). A It is well established that GLU acutely number of in vitro studies indicate that stimulates lipolysis from avian adipocytes glucagon (GLU) induces desensitization of (Leclercq, 1984). However, it has not been its metabolic effects in rat hepatocytes determined whether GLU induces desen(Plas and Nunez, 1975; Gurr and Ruh, sitization of its lipolytic effect in avian 1980; Iyengar et a\., 1980; Santos and adipocytes. Induction of desensitization by Blasquez, 1982; Heyworth and Houslay, GLU in hepatocytes results in down1983; Noda et ah, 1984; Murphy et ah, regulation of GLU receptors (Santos and 1987), canine kidney cell line MDCK (Rich Blasquez, 1982; Noda et ah, 1984), whereas et ah, 1984), and rat Sertoli cells (Attrama- in the canine kidney cell line MDCK dal et ah, 1988). Likewise, in vivo hyper- desensitization is associated with increased activity of the inhibitory guanine nucleotide binding protein (Gi) (Rich et ah, 1984). Thus, GLU-induced desensitization Received for publication October 24, 1991. of its metabolic effects in vitro may Accepted for publication January 15, 1992. 1 Scientific Article Number 2288, West Virginia Uni- provide a useful model system for investiversity Agricultural and Forestry Experiment Station. gating the role of cellular proteins in the Present address: USDA, Agricultural Research regulation of GLU biological action. Service, Poultry Research Laboratory, Georgetown, Therefore, the present study was underDE 19947. 1015
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ABSTRACT Continuous exposure of cells to a hormonal stimulus results in attenuation of the hormone's effects on the cell; a process known as desensitization. The present study was undertaken to determine whether glucagon (GLU) induces desensitization of its lipolytic effect in adipocytes isolated from the abdominal fat of market-age broilers. Preincubation of adipocytes with 10 to 100 n g / m L of porcine GLU (pGLU) or chicken GLU (cGLU) for 24 h reduced (P < .05) GLU-stimulated lipolysis. However, pGLU decreased (P < .05) lipolysis to a greater extent than cGLU. Maximal lipolysis was reduced 70% by pGLU and 55% by cGLU. Chicken GLU also exhibited lower biological potency for acutely stimulating lipolysis from control and cGLU-treated adipocytes. Glycerol release from control adipocytes incubated for 1 h with .3 n g / m L of cGLU or pGLU was 26 and 42 n m o l / h per 3% cells, respectively. The GLU-induced decrease in lipolysis occurred rapidly and was partially reversible. The results of the present study indicated that GLU induced desensitization of its lipolytic effect in broiler adipocytes. {Key words: broiler adipocytes, chicken glucagon, porcine glucagon, lipolysis, desensitization)
1016
OSCAR
Isolation and Culture of Adipocytes
Lipolysis Assay
Adipocytes were isolated under sterile conditions and maintained in primary culture using the method of Oscar (1991). In brief, broilers w e r e euthanatized by asphyxiation with carbon dioxide. Adipose tissue (2 g per digest) was minced in plastic containers and then digested with sterile digestion medium (SDM) for 1 h at 37 C in an orbital shaking (150 opm) water bath. The SDM contained Dulbecco's MEM supplemented with 10 m M glucose, 25 mM
To remove GLU before measurement of lipolysis, adipocytes were washed with 15, 10, and 6 mL of washing medium (WM). The WM was identical in composition to AM except that it contained 1% BSA rather than 3% BSA. In addition, a 20-min incubation at 37 C was included after the first two w a s h steps to facilitate removal of adipocyte-associated GLU. After washing, adipocytes were resuspended in AM. The packed cell volume of the suspension -was determined by centrifugation in microhematocrit tubes (Oscar, 1991). Adipocytes (total volume of 9 uL or a final concentration of 3% adipocyte volume per assay volume) were assayed for lipolysis by incubation for 1 h at 37 C in .3 mL of
MATERIALS AND METHODS Materials Porcine GLU (pGLU) was a gift from Eli Lilly.3 Chicken GLU (cGLU) was prepared by Litron Laboratories 4 and was a gift from John P. McMurtry. 5 Tissue culture ingredients were obtained as previously described (Oscar, 1991). Female broilers (Vantress x Cobb), 49 to 63 days old, were used as the source of abdominal adipose tissue.
Glucagon Solutions
^ l i Lilly and Co., Indianapolis, IN 46200. Litron Laboratories, Rochester, NY 14620. ^JSDA, Agricultural Research Service, Beltsville, MD 20705. 4
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Chicken GLU and pGLU were dissolved in .001 NHC1, pH 2.5 and the concentration of GLU (.2 to .3 mg/mL) in these solutions was determined spectrophotometrically (Pollock and Kimmel, 1975). Stock solutions of GLU (.05 mg/mL) were prepared by diluting the acidified solutions of pGLU and cGLU with assay medium (AM). Assay medium was composed of phenol red and pyruvate-free Dulbecco's modified Eagle's medium (MEM) supplemented with 10 mM glucose, 25 mM 4-(2-hydroxyethyl)-lpiperazine-ethanesulfonic acid (HEPES), and 3% BSA with a pH of 7.6 at 22 C. Aliquots (.2 mL) of the GLU stock solutions were stored at -20 C.
HEPES, 3% BSA, 250 units/mL collagenase, and .125% trypsin; p H 7.6 at 22 C. To obtain enough adipocytes for a replicate of an experiment, one digest was conducted for each treatment. After 1 h of digestion, 26 mL of sterile incubation medium (SIM) was added to each digest container. The SIM was composed of Dulbecco's MEM supplemented with 10 mM glucose, 25 mM HEPES, antibiotics (penicillin, streptomycin, and neomycin), and 1% BSA; p H 7.6 at 22 C. To remove undigested tissue, the diluted digestion mixture was filtered through polypropylene mesh and collected into separate 50-mL polypropylene centrifuge tubes. Once the adipocytes had floated to form a layer, the infranatant that contained digestion enzymes and stromal-vascular cells was removed and the adipocytes were resuspended in 30 mL of SIM. This washing procedure was repeated three more times except that the adipocytes were resuspended in 20,10, and 3 mL of SIM. After the final wash step, the adipocyte suspensions were combined to produce a single preparation of adipocytes. Adipocytes were cultured at 37 C in 50-mL polypropylene centrifuge tubes that contained 30 mL of SIM under an initial atmosphere of air. After 24 h in culture, pGLU or cGLU (0 to 100 ng/mL) were added and adipocytes were incubated for .5 to 24 h before removal of GLU by washing and assessment of lipolysis.
taken to determine whether GLU induces desensitization of lipolysis by broiler adipocytes maintained in primary culture.
DESENSITIZATION OF ADIPOCYTE LIPOLYSIS
(Figure 1). Likewise, preincubation with 10 n g / m L pGLU did not alter BL or SL but reduced ML 10%. Submaximal lipolysis was 55% lower and ML was 40% lower from adipocytes pretreated with 100 n g / mL pGLU, whereas BL was similar between control adipocytes and those incubated with 100 n g / m L pGLU. In a subsequent experiment (results not shown), similar reductions of SL and ML were noted when adipocytes were preincubated with 25 or 50 n g / m L pGLU for 24 h. To determine whether preincubation with the homologous hormone would also reduce lipolysis, adipocytes were incubated for 24 h with 0, 1, 10, or 100 n g / mL cGLU before removal of cGLU by washing and assessment of lipolysis (Figure 2). Similar to results with 1 n g / m L pGLU, preincubation of adipocytes with 1 n g / m L cGLU did not alter lipolysis. In
Statistical Analysis Treatment effects on characteristics of lipolysis were examined by ANOVA for a randomized complete block design using the General Linear Models (GLM) procedure of base SAS® software (SAS Institute, 1985). Each experiment consisted of three to five replicates with each replicate involving the preparation of a single batch of adipocytes from one donor bird. When a significant (P < .05) treatment effect was observed, least square means were compared using the PDIFF option (uses repeated t tests) of the GLM procedure of SAS® (SAS Institute, 1985).
RESULTS Preincubation of adipocytes with 1 n g / mL pGLU for 24 h did not alter lipolysis
1 10 pGLU (ng/mL)
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FIGURE 1. Effect of porcine glucagon (pGLU) on lipolysis by broiler adipocytes in primary culture. Adipocytes were incubated with 0,1, 10, or 100 ng/ mL pGLU for 24 h before removal of pGLU by washing and assessment of basal lipolysis (•), submaximal lipolysis (•), and maximal lipolysis (•) with pGLU. Each point is the average of four replicates. Points within a line with no common letters differ (P < .05).
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AM that contained 0 to 10 n g / m L pGLU or cGLU (Oscar, 1991). Glycerol released into the medium was used as the index of lipolysis and was measured using the glycerol kinase method of Eggstein and Kuhlmann (1974). Four characteristics of lipolysis were examined in the present study. 1) Basal lipolysis (BL) was the amount of glycerol release that occurred in the absence of hormonal stimulation. 2) Maximal lipolysis (ML) was the amount of lipolysis in the presence of 10 n g / m L GLU, a dose of GLU that stimulates lipolysis maximally in primary cultured broiler adipocytes (Oscar, 1991). 3) Submaximal lipolysis (SL) was the quantity of glycerol release in the presence of a less than maximal dose of GLU (1 n g / mL). 4) Physiological lipolysis (PL) was the amount of lipolysis in the presence of .3 n g / mL GLU, which is a dose that is within the normal range of plasma GLU concentrations in broilers (Cogburn et ah, 1989). The DNA content of adipocytes used to assess lipolysis was determined using the fluorometric method of Labarca and Paigen (1980) with calf thymus DNA as the standard. Preincubation of adipocytes with GLU did not alter the amount of DNA per 9 uL or 3% adipocytes (results not shown). Therefore, lipolysis results were expressed as glycerol release in nanomoles per hour per 3% adipocytes.
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FIGURE 2. Effect of chicken glucagon (cGLU) on lipolysis by broiler adipocytes in primary culture. Adipocytes were incubated with 0,1, 10, or 100 ng/ mL cGLU for 24 h before removal of cGLU by washing and assessment of basal lipolysis (•), submaximal lipolysis (•), and maximal lipolysis (A) with porcine GLU (pGLU). Each point is the average of five replicates. Points within a line with no common letters differ (P < .05).
addition, BL was not affected by pretreatment with cGLU. However, SL was decreased 15% and ML was nonsignificantly (P < .10) decreased 10% by preincubation with 10 ng/mL cGLU. Both SL and ML were reduced 20% by the highest dose (100 ng/mL) of cGLU. The reductions of lipolysis observed upon preincubation with 100 ng/mL cGLU (Figure 2) were lower in magnitude than those observed with 100 ng/mL pGLU (Figure 1). This suggested that cGLU had a lower biological potency than pGLU; however, the dose-response experiment with cGLU (Figure 2) was conducted after the dose-response experiment with pGLU (Figure 1). To directly compare the effects of preincubation with pGLU and cGLU on lipolysis, adipocytes were incubated for 24 h without GLU (control) or with 100 ng/mL cGLU or 100 ng/mL pGLU before removal of hormones by washing and measurement of lipolysis
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FIGURE 3. Comparison of the effect of preincubation with porcine glucagon (pGLU) and chicken glucagon (cGLU) on lipolysis by broiler adipocytes in primary culture. Adipocytes were incubated without GLU (control) or with 100 ng/mL cGLU or 100 ng/ mL pGLU for 24 h before removal of hormones by washing and assessment of basal lipolysis (BL), physiological lipolysis (PL), submaximal lipolysis (SL), and maximal lipolysis (ML) with pGLU. Each bar is the average of four replicates. Bars within a cluster with no common letters differ (P < .05).
(Figure 3). Both cGLU and pGLU reduced BL 75%. In general, pretreatment with pGLU reduced PL, SL, and ML to a greater extent than pretreatment with cGLU but only ML was statistically lower from pGLU-treated adipocytes; ML was reduced 55% by cGLU and 70% by pGLU. To compare the acute effects of pGLU and cGLU on lipolysis, adipocytes were preincubated for 24 h without GLU (control) or with 100 ng/mL cGLU before removal of cGLU by washing and assessment of lipolysis within each culture treatment using both cGLU and pGLU (Figure 4). Similar to previous experiments (Figures 2 and 3), preincubation with 100 ng/mL cGLU reduced PL, SL, and ML but did not alter BL. Within each culture
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FIGURE 4. Comparison of the acute effects of porcine glucagon (pGLU) and chicken glucagon (cGLU) on lipolysis by broiler adipocytes in primary culture. Adipocytes were incubated with 0 (control) or 100 ng/mL cGLU (cGLU-treated) for 24 h before removal of cGLU by washing and measurement of basal lipolysis 03L), physiological lipolysis (PL), submaximal lipolysis (SL), and maximal lipolysis (ML) within each culture treatment with both cGLU and pGLU. Each bar is the average of four replicates. Bars within a cluster with no common letters differ (P < .05).
treatment, PL was 60% higher and SL was 30% higher when pGLU was used to assess lipolysis. As expected, ML within culture treatment was similar when measured with cGLU or pGLU. All types of lipolysis decreased rapidly after addition of 100 n g / m L pGLU to cultures of adipocytes (Figure 5). Significant reductions in lipolysis were noted after .5 h of incubation with pGLU. These reductions were maximal by 4 h. Maximal lipolysis of control adipocytes did not change during the 24-h incubation. However, BL decreased and SL increased over time in control adipocytes. Washing control adipocytes after 4 h of incubation did not alter BL or ML 20 h later but increased SL 40% (Figure 5). The depression of BL observed after 4 h incubation with 100 n g / m L pGLU was completely reversed 20 h after removal of pGLU by washing. However, the reductions of SL and ML of GLU-treated adipocytes noted at 4 h of incubation were
The results of the current study indicate that in addition to acute stimulation, GLU regulation of lipolysis also involves attenuation of its ability to stimulate triglyceride mobilization; a process known as desensitization. Both pGLU and cGLU exhibited similar effects on lipolysis. Preincubation of adipocytes for 24 h with high doses (10 to 100 ng/mL) of pGLU or cGLU reduced lipolysis. In addition, both types of GLU acutely stimulated lipolysis from control and desensitized adipocytes. However, pGLU exhibited a higher biological potency than cGLU. At the same dose in culture, pGLU reduced ML to a greater extent (Figure 3), whereas in the lipolysis assay PL and SL were higher when assessed with pGLU (Figure 4). McCumbee and Hazelwood (1978) and Shchenkova et al. (1987) also found that cGLU exhibits lower biological potency than mammalian GLU for acutely stimulating lipolysis from chicken adipocytes. These results are surprising because the structure of cGLU differs from pGLU by only one amino acid (Pollock and Kimmel, 1975). Chicken GLU contains a serine but pGLU has an asparagine at position 28 of GLU, a 29 amino acid length polypeptide. Further evidence that a single amino acid sequence difference in GLU can alter its biological potency comes from studies using synthetic GLU analogs. Substitution of aspartate or glutamate for glutamine at position 3 to produce [Asp3]-GLU and [Glu3]-GLU results in GLU analogs that stimulate adenylate cyclase in rat liver membranes with only 2.5% the potency of native GLU (Andreu and Merrifield, 1987). In addition, [Asp 3 ]-GLU has 125-fold and [Glu3]-GLU has 70-fold lower affinity than native GLU for binding to the rat liver GLU receptor. Other GLU analogs (i.e., [Glu9]-GLU, and [D-Phe4]-GLU) also exhibit lower biological potency and binding affinity than native GLU in liver prepara-
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FIGURE 5. Time course and reversibility of the effect of porcine glucagon (pGLU) on lipolysis by broiler adipocytes in primary culture. Adipocytes A variety of evidence suggests that the were incubated without GLU (Control; •), or with rapid phase of GLU-induced desensitiza100 ng/mL pGLU (•) for .5,4, or 24 h before removal of pGLU by washing and measurement of lipolysis tion of adenylate cyclase involves an with pGLU. In addition, after incubation for 4 h, a uncoupling of signal transduction between control (O) and pGLU-treated (•) culture in each the GLU receptor and the stimulatory replicate (n = 3) were washed under sterile condi- guanine nucleotide binding protein (Gs) tions and reincubated in culture for an additional 20 that activates adenylate cyclase (Heyworth h before measurement of lipolysis. Points within a time of incubation with no common letters differ (P < and Houslay, 1983; Noda et ah, 1984; Murphy et ah, 1987; Attramadal et ah, .05).
tions (Unson et ah, 1987; Hagopian et ah, 1987). In fact, circular dichroism analysis of [Glu9]-GLU revealed an increase in ochelix and a decrease in P-sheet conformation (Unson et ah, 1987). Thus, the altered biological potencies of GLU differing by one amino acid may result from conformational changes in the molecule that alter its binding affinity for the GLU receptor. Recently, Oscar (unpublished data) found that cGLU has a threefold lower affinity than pGLU for binding to cell-surface GLU receptors of primary cultured broiler adipocytes. This funding is consistent with the data for GLU analogs and indicates that the lower biological potency of cGLU
1988). Compounds, such as [1-N-ottrinitrophenylhistidine,12-homo-arginine]GLU (TH-GLU), angiotensin, and vasopressin, which stimulate inositol phospholipid metabolism but not cyclic adenosine monophosphate (cAMP) production, mimic GLU-induced desensitization (Murphy et ah, 1987). Furthermore, Wakelam et al. (1986) found that hepatocytes contain two populations of GLU receptors: one that stimulates adenylate cyclase and one that stimulates inositol phospholipid metabolism. Thus, it is proposed that GLU activation of protein kinase C via phosphatidylinositol-4,5bisphosphate breakdown results in phosphorylation of either the GLU receptor or G s or both to uncouple them from adenylate cyclase (Murphy et ah, 1987).
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in avian adipocytes results from its lower affinity for the GLU receptor. Glucagon-induced desensitization of lipolysis by broiler adipocytes occurred rapidly and was 70% reversible 20 h after induction for 4 h (Figure 5). Similar to results of the present study, GLU-induced desensitization of its metabolic effects in hepatocytes (Gurr and Ruh, 1980; Iyengar et ah, 1980; Heyworth and Houslay, 1983; Noda et ah, 1984; Murphy et ah, 1987), immature Sertoli cells (Attramadal et ah, 1988), and canine kidney cell line MDCK (Rich et ah, 1984) occurs rapidly and is reversible (DeRubertis and Craven, 1976; Gurr and Ruh, 1980). The rapid induction of desensitization of GLU-stimulated adenylate cyclase activity by GLU in liver is not accompanied by an immediate reduction in GLU binding (Santos and Blasquez, 1982; Heyworth and Houslay, 1983). However, down-regulation of GLU receptors is observed upon longer induction of desensitization (Santos a n d Blasquez, 1982; Noda et ah, 1984), which indicates that GLU receptors are involved in the mediation of desensitization in liver.
DESENSinZATION OF ADIPOCYTE LIPOLYSIS
Contrary to studies in rat hepatocytes, a cAMP analog was able to mimic GLUinduced desensitization of GLUstimulated adenylate cyclase activity in the canine kidney cell line MDCK (Rich et ah, 1984). In addition, GLU-induced desensitization of adenylate cyclase in MDCK cells was attributed to an increase in level or activity of Gj, rather than to an alteration in the level or activity of Gs. Thus, the mechanism of GLU-induced desensitization may differ between tissues and cell types. The mechanism by which GLU induces desensitization of lipolysis in broiler adipocytes is unknown but may involve changes in both GLU receptor and G protein metabolism.
Andreu, D., and R. B. Merrifield, 1987. Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs. Eur. J. Biochem. 164585-590. Attramadal, H., L. Eikvar, and V. Hansson, 1988. Mechanisms of glucagon-induced homologous and heterologous desensitization of adenylate cyclase in membranes and whole Sertoli cells of the rat. Endocrinology 123:1060-1068. Cogburn, L. A., S. S. Liou, A. L. Rand, and J. P. McMurtry, 1989. Growth, metabolic and endocrine responses of broiler cockerels given daily subcutaneous injection of natural or biosynthetic chicken growth hormone. J. Nutr. 119: 1213-1222. DeRubertis, F. R., and P. Craven, 1976. Reduced sensitivity of the hepatic adenylate cyclasecyclic AMP system to glucagon during sustained hormonal stimulation. J. Clin. Invest. 57: 435-443. Eggstein, M., and E. Kuhlmann, 1974. Triglycerides and glycerol. Determination after alkaline hydrolysis. Pages 1825-1831 in: Methods of Enzymatic Analysis. H. U. Bergmeyer, ed. Academic Press Inc., New York, NY. Gurr, J. A., and T. A. Ruh, 1980. Desensitization of primary cultures of adult rat liver parenchymal cells to stimulation of adenosine 3',5'monophosphate production by glucagon and epinephrine. Endocrinology 107:1309-1319. Hagopian, W. A., H. S. Tager, B. Gysin, D. Trivedi, and V. J. Hruby, 1987. Interactions of glucagon and glucagon analogs with isolated canine hepatocytes. J. Biol. Chem. 262:15506-15513. Heyworth, C. M., and M. D. Houslay, 1983. Challenge of hepatocytes by glucagon triggers a rapid modulation of adenylate cyclase in isolated membranes. Biochem. J. 21453-98. Iyengar, R., P. W. Mintz, T. L. Swartz, and L. Birnbaumer, 1980. Divalent cation-induced desensitization of glucagon-stimulable adenylyl cyclase in rat liver plasma membrane. J. Biol. Chem. 255:11875-11882.
Labarca, C , and K. Paigen, 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344-352. Leclercq, B., 1984. Adipose tissue metabolism and its control in birds. Poultry Sci. 632044-2054. McCumbee, W. D-, and R. L. Hazelwood, 1978. Sensitivity of chicken and rat adipocytes and hepatocytes to isologous and heterologous pancreatic hormones. Gen. Comp. Endocrinol. 34: 421-427. Murphy, G. J., V. J. Hruby, D. Trivedi, M.J.O. Wakelam, and M. D. Houslay, 1987. The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism. Biochem. J. 24339-46. Noda, C , F. Shinjyo, A. Tomomura, S. Kato, T. Nakamura, and A. Ichihara, 1984. Mechanism of heterologous desensitization of the adenylate cyclase system by glucagon in primary cultures of adult rat hepatocytes. J. Biol. Chem. 259: 7747-7754. Oscar, T. P., 1991. Glucagon-stimulated lipolysis of primary cultured broiler adipocytes. Poultry Sci. 70:326-332. Plas, C , and J. Nunez, 1975. Glycogenolytic response to glucagon of cultured fetal hepatocytes. J. Biol. Chem. 250:5304-5311. Pollock, H. G., and J. R. KimmeL 1975. Chicken glucagon. Isolation and amino acid sequence. J. Biol. Chem. 250:9377-4380. Rich, K. A., J. Codina, G. Floyd, R. Sekura, J. D. Hildebrandt, and R. Iyengar, 1984. Glucagoninduced heterologous desensitization of the MDCK cell adenylyl cyclase. J. Biol. Chem. 259: 7893-7901. Santos, A., and E. Blasquez, 1982. Direct evidence of a glucagon-dependent regulation of the concentration of glucagon receptors in the liver. Eur. J. Biochem. 121:671-677. SAS Institute, 1985. SAS® User's Guide: Statistics. Version 5 Edition. SAS Institute Inc., Cary, NC. Shchenkova, I. M., M. N. Pertseva, L. V. Dmitrenko, D. I. Ostrovsky, L. P. Soltitskaya, and L. A. Kuznetsova, 1987. Immunological properties and biological activity of chicken glucagon. Biokhimiya 52:359-367. Sibley, D. R., and R. J. Lefkowitz, 1985. Molecular mechanisms of receptor desensitization using the ^-adrenergic receptor-coupled adenylate cyclase system as a model. Nature (Lond.) 317: 124-129. Soman, V., and P. Felig, 1978. Regulation of the glucagon receptor by physiological hyperglucagonaemia. Nature (Lond.) 272:829-832. Unson, C. G., D. Andreu, E. M. Gurzenda, and R. B. Merrifield, 1987. Synthetic peptide antagonists of glucagon. Proc. Natl. Acad. Sci. USA 84: 4083-4087. Wakelam, M.J.O., G. J. Murphy, V. J. Hruby, and Miles D. Houslay, 1986. Activation of two signal-transduction systems in hepatocytes by glucagon. Nature (Lond.) 323:68-71.
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REFERENCES
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