Liver Glucagon Receptors (GluR): Effect of Exercise and Fasting on Binding Characteristics, GluR-mRNA, and GluR Protein Content in Rats

Abstract

The purpose of the study was to investigate the effects of acute exercise and fasting on glucagon receptor (GluR) binding characteristics, GluR-mRNA, and protein content in rat liver. Liver homogenates were prepared and plasma membranes were purified by aqueous 2-phase affinity partitioning in rats fed at rest (control) and after 180 min of swimming exercise and 24 h of fasting (7 rats/group). Saturation curve of plasma membranes incubated with [¹²⁵I]-glucagon showed significant higher GluR density following exercise and fasting than in the control group (8.19±0.29 and 8.01±0.65 vs. 3.09±0.12 pmol/mg of proteins, respectively). When compared to control rats, GluR Kd was also higher following exercise and fasting (0.46±0.05 and 0.56±0.13 vs. 0.33±0.05 nM, respectively; significantly different for fasting only). Expression of GluR-mRNA and protein content were both significantly higher (~100% and ~90%, respectively) following the 24-h fast than in the control rats, but not following exercise. These results, in line with the literature showing an increased sensitivity of the liver to glucagon following exercise and fasting, indicate that an increased density of GluR on plasma membranes can be obtained by 2 complementary mechanisms: externalization of pre-existing GluR from intracellular pools operative in response to the prolonged exercise, and de novo synthesis of GluR operative only in response to fasting. The reduction in plasma insulin concentration and/or depletion of liver glycogen stores, which results from both prolonged exercise and fasting, could be involved in the control of these mechanisms.

Key words

de novo synthesis - externalization - glycogen - glucagon/insulin ratio - swimming

• References

• 1 Wasserman DH. Four grams of glucose. Am J Physiol Endocrinol Metab 2009; 296: E11-E21
• 2 Cherrington AD, Stevenson RW, Steiner KE. Glucagon as a regulator of hepatic glucose production in vivo. Biochem Soc Trans 1987; 15: 1023-1027
• 3 Ramnanan CJ, Edgerton DS, Kraft G, Cherrington AD. Physiologic action of glucagon on liver glucose metabolism. Diabetes Obes Metab 2011; 13 (Suppl. 01) 118-125
• 4 Brand CL, Jorgensen PN, Knigge U, Warberg J, Svendsen I, Kristensen JS, Holst JJ. Role of glucagon in maintenance of euglycemia in fed and fasted rats. Am J Physiol 1995; 269: E469-E477
• 5 Hendrick GK, Wasserman DH, Frizzell RT, Williams PE, Lacy DB, Jaspan JB, Cherrington AD. Importance of basal glucagon in maintaining hepatic glucose production during a prolonged fast in conscious dogs. Am J Physiol 1992; 263: E541-E549
• 6 Bonjorn VM, Latour MG, Belanger P, Lavoie JM. Influence of prior exercise and liver glycogen content on the sensitivity of the liver to glucagon. J Appl Physiol 2002; 92: 188-194
• 7 Wagle SR. Studies on the differential response to glucagon concentration on gluconeogenesis in isolated hepatocytes containing high and low glycogen. Biochem Biophy Res Commun 1974; 59: 1366-1372
• 8 Caro JF, Amatruda JM. The effects of fasting on glucagon binding and action in isolated rat hepatocytes. Metabolism 1980; 29: 732-738
• 9 Charbonneau A, Melancon A, Lavoie C, Lavoie JM. Alterations in hepatic glucagon receptor density and in Gsalpha and Gialpha2 protein content with diet-induced hepatic steatosis: effects of acute exercise. Am J Physiol Endocrinol Metab 2005; 289: E8-E14
• 10 Charbonneau A, Unson CG, Lavoie JM. High-fat diet-induced hepatic steatosis reduces glucagon receptor content in rat hepatocytes: potential interaction with acute exercise. J Physiol 2007; 579: 255-267
• 11 Burcelin R, Mrejen C, Decaux JF, De Mouzon SH, Girard J, Charron MJ. In vivo and in vitro regulation of hepatic glucagon receptor mRNA concentration by glucose metabolism. J Biol Chem 1998; 273: 8088-8093
• 12 Fouchereau-Peron M, Rancon F, Freychet P, Rosselin G. Effect of feeding and fasting on the early steps of glucagon action in isolated rat liver cells. Endocrinology 1976; 98: 755-760
• 13 Broer Y, Freychet P, Rosselin G. Insulin and glucagon-receptor interactions in the genetically obese Zucker rat: studies of hormone binding and glucagon-stimulated cyclic AMP levels in isolated hepatocytes. Endocrinology 1977; 101: 236-249
• 14 Kregel KC, Allen DL, Booth FW, Fleshner MR, Henriksen EJ, Musch TI, O’Leary DS, Parks CM, Poole DC, Ra’anan AW, Sheriff DD, Sturek MS, Toth LA. Resource Book for the Design of Animal Exercise Protocols. Bethesda, MD: American Physiological Society; 2006: 35-41
• 15 Baar K, Wende AR, Jones TE, Marison M, Nolte LA, Chen M, Kelly DP, Holloszy JO. Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1. Faseb J 2002; 16: 1879-1886
• 16 Passonneau JV, Lauderdale VR. A comparison of three methods of glycogen measurement in tissues. Anal Biochem 1974; 60: 405-412
• 17 Persson A, Jergil B. Purification of plasma membranes by aqueous two-phase affinity partitioning. Anal Biochem 1992; 204: 131-136
• 18 Legare A, Drouin R, Milot M, Massicotte D, Peronnet F, Massicotte G, Lavoie C. Increased density of glucagon receptors in liver from endurance-trained rats. Am J Physiol Endocrinol Metab 2001; 280: E193-E196
• 19 Frandsen EK, Thim L, Moody AJ, Markussen J. Structure-function relationships in glucagon. Re-evaluation of glucagon-(1-21). J Biol Chem 1985; 260: 7581-7584
• 20 Cadrin M, Hovington H, Marceau N, McFarlane-Anderson N. Early perturbations in keratin and actin gene expression and fibrillar organisation in griseofulvin-fed mouse liver. J Hepatol 2000; 33: 199-207
• 21 Unson CG, Cypess AM, Wu CR, Goldsmith PK, Merrifield RB, Sakmar TP. Antibodies against specific extracellular epitopes of the glucagon receptor block glucagon binding. Proc Natl Acad Sci USA 1996; 93: 310-315
• 22 Carruthers CJ, Unson CG, Kim HN, Sakmar TP. Synthesis and expression of a gene for the rat glucagon receptor. Replacement of an aspartic acid in the extracellular domain prevents glucagon binding. J Biol Chem 1994; 269: 29321-29328
• 23 Jelinek LJ, Lok S, Rosenberg GB, Smith RA, Grant FJ, Biggs S, Bensch PA, Kuijper JL, Sheppard PO, Sprecher CA. Expression cloning and signaling properties of the rat glucagon receptor. Science 1993; 259: 1614-1616
• 24 Authier F, Desbuquois B. Glucagon receptors. Cell Mol Life Sci 2008; 65: 1880-1899
• 25 Authier F, Desbuquois B, De Galle B. Ligand-mediated internalization of glucagon receptors in intact rat liver. Endocrinology 1992; 131: 447-457
• 26 Authier F, Janicot M, Lederer F, Desbuquois B. Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment. Biochem J 1990; 272: 703-712
• 27 Krilov L, Nguyen A, Miyazaki T, Unson CG, Bouscarel B. Glucagon receptor recycling: role of carboxyl terminus, beta-arrestins, and cytoskeleton. Am J Physiol Cell Physiol 2008; 295: C1230-C1237
• 28 Krilov L, Nguyen A, Miyazaki T, Unson CG, Williams R, Lee NH, Ceryak S, Bouscarel B. Dual Mode of glucagon receptor internalization: Role of PKCalpha, GRKs and beta-arrestins. Exp Cell Res 2011; 317: 2981-2994
• 29 Merlen C, Fabrega S, Desbuquois B, Unson CG, Authier F. Glucagon-mediated internalization of serine-phosphorylated glucagon receptor and Gsalpha in rat liver. FEBS Lett 2006; 580: 5697-5704
• 30 Premont RT, Iyengar R. Glucagon-induced desensitization of adenylyl cyclase in primary cultures of chick hepatocytes. Evidence for multiple pathways. J Biol Chem 1988; 263: 16087-16095
• 31 Cahill GF. Glucagon action in isolated perfused liver. Adv Enzyme Regul 1965; 3: 195-201
• 32 Abrahamsen N, Lundgren K, Nishimura E. Regulation of glucagon receptor mRNA in cultured primary rat hepatocytes by glucose and cAMP. J Biol Chem 1995; 270: 15853-15857
• 33 Balks HJ, Jungermann K. Regulation of peripheral insulin/glucagon levels by rat liver. Eur J Biochem 1984; 141: 645-650
• 34 Wasserman DH, Lacy DB, Bracy DP. Relationship between arterial and portal vein immunoreactive glucagon during exercise. J Appl Physiol 1993; 75: 724-729