Glucagon and Its Receptor in Various Tissues

Glucagon and Its Receptor in Various Tissues

JEAN CHRISTOPHE" Department oj Experimental Surge?

Medical School Universith Lihre de Bruxelles, CP 500

40 Avenue J. Wybrun, B-1070 Brussels, Belgium

STRUCTURE/ACTIVITY RELATIONSHIP OF GLUCAGON AND GLUCAGON ANALOGS

Glucagon contributes to the pathogenesis of diabetes, so a potent peptidase- resistant and long-acting glucagon antagonist could be potentially useful in the treatment of this disease, by alleviating high gluconeogenesis. This aim is, however, difficult to achieve because the data so far obtained (in Brussels, Copenhagen, Munich, New Orleans, New York, Tucson, etc.) indicate that the entire sequence of glucagon is biologically important: the C-terminal moiety for binding and the N-terminal moiety for selective binding and effector coupling.

A proper a-helix conformation in the far C-terminal (22-29) is indeed needed for binding, but specific requirements are less stringent in this area than for the parent peptide truncated glucagon-like peptide 1 (tCLP-I), when it binds to its own receptor. For instance, replacing Trp2.5, Met27, and Thr29 in combination with Phe25, Leu27, and Thr29-NH2 increases the affinity of glucagon (in vivo also). [Va127, Lys28, Glu29, Arg30]glucagon is relatively well recognized despite four changes. However, substitution with nonaromatic Gly2.5 sharply decreases the affinity.','

In the central part of glucagon, the basic groups in positions 17 and 18 favor binding: [Lysl7,18,Glu2l]glucagon, designed for enhancing the beginning of the amphipathic a-helix, exhibits increased binding potency.'.' Ser8, TyrlO, Serl I , Lysl2, and AsplS contribute to binding. Reducing the p-turn potential in 15- 18 or replacing Lysl2 by Argl2 is detrimental.

Inadequate substitutions in the first p turn (the 2-4 triad) destroy selective recogni- tion. The use of ~ - G l n 3 or Phe4 yields partial agonists. On the other hand, a superago- nist is even obtained when the p-turn is stabilized by a Gly4 3 ~ - P h e 4 substitution: [~-Phe4]ghcagon is five times more potent than glucagon in vitro' as well as on blood glucose levels in the rat.

It appears that His1 cooperates with Asp9 and Serl6 for adenylyl cyclase stimulation: the protonated Hisl-imidazole in this triad is stabilized by Asp9 and probably deprotonated by Serl6, when the hormone is in the presence of a complementary site in the receptor protein. Asp21 is at best a poor surrogate, so any substitution of Asp9 severely diminishes the transmission of the biological The importance of His1 is examplified by the fact that several desHisl derivatives, such as desHisl-

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32 ANNALS NEW YORK ACADEMY OF SCIENCES

glucagon and desHis 1 [Glu9]glucagon-amide, show very low agonist activity and block the hyperglycemic effect of glucagon administered to rabbits, or reduce the hyperglycemia produced by endogenous glucagon in streptozotocin diabetic rats. Other desHisl derivatives, such as desHisl[~-Phe4, Tyr5, Argl2, Lys17,18, Glu2llg- lucagon, desHisl [Nle9, Alal 1, Alal6]glucagon, and cyclic desHisl-cyclo[Glu9,Lysl- 2lglucagon-amide are full antagonists in the adenylyl cyclase a s ~ a y . ~ . ~ , ~ - ' ' [ 1 -IT- trinitrophenylHis1, homoArg12]glucagon is also a potent antagonist in vitro and lowers blood glucose in rats made diabetic with streptozotocin.12

CLONING OF RAT AND HUMAN GLUCAGON RECEPTORS

In 1993 we used a low stringency homology approach and sequenced four introns (no. 4, 5, 6, and 11) in addition to the ORF,'3.'4 while Jelinek et all5 utilized an expression cloning strategy revealing directly (only) the functional receptor. Both approaches yielded an ORF of 1455 nucleotides encoding a G-protein coupled protein with seven transmembrane (TM) segments. The deduced 485 amino acid protein (including the 22 amino acid signal sequence) discloses 47-57% full identity in the core TMI to TM7 sequence between this receptor and rat receptors for tGLP-I, secretin, vasoactive intestinal peptide (VIP), and pituitary adenylyl cyclase-activating peptide (PACAP). The degree of conservation is exceptional in TM3, TM6, and TM7.

In the relatively long N-terminal (143 amino acids), some residues are also well conserved. This is the case for Cys59, Asp64, Cys68, and Trp69. Asp64 is absolutely required for glucagon binding despite the proximity of the glycosylation site in A ~ n 6 0 . I ~ Since a similar observation can be made with the growth-hormone-releasing factor (GRF) receptor, Asp64 may in fact contribute to the general organization of the N-terminal. In this respect the N-terminal also contains eight Cys residues. Neglecting those two present in the 22 amino acid signal sequence, the six others are well conserved and may likewise contribute three disulfide bridges to the general structure. The four Asn-linked glycosylation sites (Asn-Ser/Thr) accept four N-linked oligosaccharide chains accounting for about 7 kDa altogether and may be a prerequisite for receptor deployment and function. Besides, highly specific features are also present in the N-terminal and in extracellular loops E l and E3, which make three obvious sites for privileged cooperation with glucagon. In addition E l and E2 may be linked by a disulfide bond.

On the intracellular side, two consensus sequences for phosphorylation are present in the intracellular loop 12. The I3 loop is relatively short and has a C-terminal Arg- Leu-Ala-Arg consensus motif that may cooperate with the 80 amino acid C-terminal in activating G proteins. In this specific C-terminal domain, Ser432 is a potential phosphorylation site for PKC (and desensitization?), and one of the three Cys residues could conceivably be palmitoylated and anchor the receptor to the membrane, thereby creating 14, a fourth (temporary?) loop.

When COSGs1 cells rich in Gs are transiently transfected with a pCDM8 plasmid containing the rat cDNA, their membranes bind glucagon selectively (IC50 4 nM). The competition curve extends over more than three logarithms, suggesting two states of receptors in the absence of GTP.I4 When ordinary COS cells are transfected, tGLP-I is the only ligand capable of competing for binding, but with an IC50

CHRISTOPHE: GLUCAGON & ITS RECEPTOR 33

1000-fold higher (10 FM) than that of glucagon ( 1 1 nM).'" In transfected COSGsI membranes, glucagon stimulates adenylyl cyclase 2.6-fold in the presence of 10 p M GTP, with a Kd at 10 nM and a monophasic sigmoid curve, suggesting that all receptors are in a low affinity state, when Gs is occupied by GTP.I4 I f the rat cDNA is expressed i n BKH kidney cells, glucagon causes a [Ca?+], increase through a second effector system" (see below).

The human glucagon receptor cloned in 1994 from a liver cDNA library contains 477 amino acids. This human receptor is 82% identical to the rat glucagon receptor, the human receptor being slightly shorter and showing lowest identity at the C- terminal. The membranes of COS-7 cells transfected with the human receptor display the following binding selectivity: glucagon > oxyntomodulin (glucagon (1 -37)) > tGLP- I .17.1x Chimeric receptors constructed with various parts of this receptor and the tGLP- 1 receptor, when expressed in COS-7 cells, allow high-affinity glucagon binding, provided that the following noncontiguous areas of the glucagon receptor are present: the second moiety of the N-terminal, E l , TM3, TM4, TM6, and perhaps E3.I9

THE INTRONEXON AND PROMOTER ORGANIZATION IN RAT AND HUMAN GLUCAGON RECEPTOR GENES

Southern blot analysis of rat genomic DNA, based on digestion with six enzymes and high stringency hybridization of genetic fragments with the cloned glucagon receptor cDNA, strongly suggests a single rat glucagon receptor ~OCUS.~ ' ' When rat genomic DNA and the cloned intronless receptor cDNA are PCR-amplified in parallel, using primers based on exonic sequences, it appears that the glucagon receptor gene has a coding DNA spanning -4 kb, and contains 12 exons and 11 introns?"-?? (see sequence data in Genbank, accession no. L31.574). The first four introns (of 100, 897. 8 I , and 95 kb, respectively) are present in the coding area for the signal peptide and the N-terminal. Then follows intron 5 (89 bp interrupting coding between TM1 and 11); intron 6 (85 bp interrupting coding in El ) ; intron 7 (71 bp interrupting coding in TM4); intron 8 (227 bp interrupting coding in E2); intron 9 (289 bp interrupting coding in TMS); intron 10 ( I 15 bp interrupting coding in 13); and finally intron 1 I (301 bp interrupting coding at the beginning of the C-terminal). Thus, the exonhntron organization of the rat glucagon receptor gene, like that of the parent human glucagon receptor,!? human type I VIP receptor,I4 mouse parathyroid hormone receptor." and niouse GRF receptor,'b show a highly fragmented exonic structure (with 12 exons encoding domains in the 21-7K amino acid range), this shuffling being due to the spreading of numerous introns over the entire 5'-3' region of the gene. There is no obvious relationship of the exonhntron positioning to the general organization of the receptor itself.

The human glucagon receptor gene present in chromosome 17 (band 425)'' contains 13 introns, the first large one ( 5 kbp) being located in the 5' untranslated region. Besides, the promoter region has no TATA box and contains various cis- acting elements, including multiple sequences for SP- 1, one for AP-2 (that responds to both protein kinases PKC and PKA), and one for specific liver factor LF-A1.17,27 By comparison the gene encoding the parent human type I VIP (i.e., type I1 PACAP) receptor, present i n chromosome 3 (band p 22), is composed of 12 introns (in the


0.3-6.1 kb range) and 13 exons (in the 42-1400 bp range). Its promoter region contains binding sequences for i.a. SP-1, AP-2, CRE, and retinoic acid response element.24

GENE EXPRESSION AND mRNA POLYMORPHISM OF RAT GLUCAGON RECEPTORS IN VARIOUS TISSUES

The relative concentration of rat glucagon receptor mRNA, tested with a RNase protection assay, and as compared to total tissue RNA, is highest in liver (100%) and kidney (34%), lower in heart (1 1 %), adrenal glands (lo%), and adipose tissue (5%), very low in brain, stomach, and duodenum, and absent in lung (where tGLP- 1 receptors are well represented).28

Using a primer covering the 5’ coding end in the mature rat liver receptor cDNA, a comparison can be made with the cDNAs of other tissues and with genomic DNA. Substantial mRNA polymorphism is revealed by RT-PCR, indicating the emergence of variously shortened mRNAs in this 5’ coding end: the largest band of approximately 900 bp keeps two introns (4 and 5) ; the 800 bp band keeps one intron ( 3 , 4, or 5); the regular 700 bp band corresponds to the fully mature cDNA; and three smaller cDNA fragments of 600 bp, 500 bp, and 400 bp correspond to “in block” splicing of early introns intertwined with exons 2 andlor 5, whose transcription ends with a stop codon before TM1. The 800-bp band and the mature 700-bp band are prominent in liver cDNA, the 700-bp band predominates in kidney, adrenal gland, and adipose tissue, and all bands are more evenly represented in heart cDNA.

When a primer covering the 3‘ coding end is used, the local situation is much simpler: a single intronless canonic cDNA fragment, of the expected size, is systemati- cally revealed, indicating that intron 11, present after the ORF coding for TM7 in genomic DNA, is precociously spliced out and does not provide an alternatively spliced form with possibly important consequences.20.21.2* To conclude, the mature rat glucagon receptor mRNA derives from a single gene after the full splicing out of 1 1 introns: after multiple and tissue-specific alternative splicing of intronic sequences, and much “wasting” at the 5’ end, the maturation ends up with only one intronless OW, that is translated into the functional receptor. This holds true for the liver, kidney, heart, adipose tissue, and possibly other tissues as well.

BIOLOGICAL STATES OF HEPATIC GLUCAGON RECEPTORS AND LOCAL FATES OF GLUCAGON

Receptor Density and Spareness as an Amplification Mechanism Toward Cyclic AMP (CAMP)

The density of glucagon receptors is low in rat fetal liver, that is, under proliferative conditions and at a time of transplacental glucose supply.29 Besides, on days 18-20 of late gestation, hepatic glucagon-sensitive CAMP production is still inefficient. In neonates, the brutal shift to low-carbohydrate, high-fat consumption is appropriately accompanied by better coupling to adenylyl cyclase, so that glucagon can now


stimulate gluconeogenesis. In addition, the density of glucagon receptors increases gradually, to reach adult levels at day 28 postnatally.?’

In isolated hepatocytes and liver plasma membranes from adult rat and mouse, only 10% of a single class of low-affinity receptors need to be occupied for full adenylyl cyclase stimulation.3” In hepatocytes and liver plasma membranes from dog, which are richer in receptors, a small minority ( 1 %) of high-affinity receptors” can be occupied to about 99%, and could be theoretically responsible for the CAMP elevation inducing glycogenolysis at fasting plasma glucagon concentration (oscillat- ing around 0.03 nM): in dog even more clearly than in rodents, the spareness of glucagon receptors is one clue for amplification. Besides, final biological responses also depend on the compartmentalization, stoichiometry, and subtypes of all compo- nents operating in this plasma membrane mechanism (see the section on two effector systems).

Desensitization (Uncoupling), Internalization, and Upregulation of the Receptor

In primary cultures of chick hepatocytes glucagon induces, within 5 min, complete homologous desensitization, that is, the uncoupling of receptors from adenylyl cyclase at the cell surface. Following the removal of glucagon, several receptors recouple within 20 min.”

Receptor uncoupling can also be followed by receptor internalization via coated pits and coated vesicles, together with the ligand. In vivo the resulting glucagon- binding activity accumulating in the Golgi -endosoma1 fractions of rat hepatocytes reaches a maximum by 12-24 min and undergoes reversal in 2 h. During this process, receptors are partly uncoupled, remaining at best moderately sensitive to GTP.33.34 These alterations may reflect in part the transient phosphorylation of receptors by a PKC isoform that is activated by diacylglycerol (DAG) generated when glucagon stimulates, in parallel with adenylyl cyclase, the second signaling system” (see the subsection on cross-talk between second messengers).

The opposite phenomenon, that is, the upregulation of hepatic glucagon receptors observed in genetically diabetic db/db mice (a 3-fold elevation) occurs at a posttran- scriptional level, since mRNA levels remain normal.”

Intracellular as Opposed to Extracellular Hepatic Glucagon Degradation

The transfer of glucagon with its receptor to endosomes-lysosomes leads to ligand degradation. This is linked to an ATP-dependent acidification of liver endosomes to pH 6-7.’’ Glucagon can also be degraded by a relatively unspecific thiolmetalloendo- peptidase, located at the rat hepatocyte surface and acting at neutral pH. The cleavage of the Arg 17-Arg 18 basic doublet and elimination of Arg 1 8 lead to the extracellular release of glucagon( 19-29), a C-terminal fragment capable of regulating a cell surface Ca?+ carrier operating in the presence of Py and as subunits of G proteins at the inner s ~ r f a c e . ” ~ ~ ~


TWO EFFECTOR SYSTEMS IN LIVER PLASMA MEMBRANES

The occupation of glucagon receptors by agonists activates two signaling path- ways: (1) it stimulates adenylyl cyclase (through Gs dissociation), and (2) it increases free [Ca2+Ii, as initiated by elevated phosphatidylinositol (PI) turnover due to inositol- triphosphate (Ins-P3) production by activated phospholipase C (PI-PLC).

Multimeric G Proteins

In rat liver membranes, the average mass of the transduction system, as estimated with mild detergents or by target irradiation analysis, is about 800 kDa prior to activation (i.e., in the absence of glucagon and GTP). This is much larger than if the receptor, Gs, and adenylyl cyclase were in 1: 1 : 1 association. Conceivably, the free high-affinity glucagon receptor, when moving by random diffusion, precouples with the borders of patches of multimeric Gs (8- 10 monomers) that are not associated with the cytoskeletal network. When exposed to glucagon, GTP (or a nonhydrolyzable analog) and Mg2t in combination, multimeric Gs disaggregates laterally and yields a smaller structure composed of one occupied receptor and only one Gs. This limited complex is now doubly associated with the plasma membrane through (1) the isopreny- lated y subunit of the aPy heterotrimeric Gs, and (2) through adenylyl cyclase (any given isoform present at the inner surface), which interacts with the fatty acylated a s subunit of Gs.39*40 The affinity of the glucagon-receptor complex is then reduced (e.g., the Kd for glucagon increases from 3 nM in the absence of GTP to 19 nM in its pre~ence.~') In parallel, Mg2t induces the dissociation of Gs into as and Py. The active as-adenylyl cyclase complex subsequently operates as long as a s has not hydrolyzed GTP to GDP + Pi. This scheme provides for four eventualities:

1. The initial G protein multimer can be composed of two (or more) species of G proteins, susceptible to simultaneous activation when glucagon receptors of a single type are occupied (see the following subsection).

there may remain enough GTP-free Gs to permit the presence of a fair amount of the high-affinity glucagon receptor-(GTP-free) Gs complex, even in the presence of GTP. This might allow for amplification, and facilitates the stimulation of the effector system at low physiological concentrations of glucagon (around 0.03 nM).

3. When the glucagon receptor to Gs ratio is lower (which may be the case in heart), both the potency of glucagon (in terms of Kact on adenylyl cyclase) and its efficacy (expressed as maximal enzyme activation) are low.

4. A full antagonist, such as desHis1 [Glu9]glucagon-amide, does not initiate the system. It can only bind to intact hepatocytes according to a single binding isotherm (whereas glucagon binds with two dissociation constants), and dissociates completely (whereas glucagon does n ~ t ) . ~ . ~ '

2. When the Gs concentration is very high (e.g., in

Cross-Talk Between Second Messengers Through a Single Receptor

In addition to its main effect on adenylyl cyclase, glucagon activates the PLC/ Ins-P3/DAG system through PI-PLCP and Gaq (or Ga l l ) , leading to DAG and


Ins-P3 production. [Ca2+], is mobilized from an intracellular pool at low glucagon concentration, and across the plasma membrane at higher hormone concentration, so that [Ca'+], oscillations may develop.?'.'s Transient phosphorylations of the endoplas- mic Ins-P3 receptor and of Ca?' carriers, as well as CaIt activation of type I adenylyl cyclase or CAMP-phosphodiesterase, could be instrumental in the synergism between CAMP and PI This complex interplay is further illustrated in rat liver when pretreatment with 12-0-tetradecanoyl-phorbol-13-acetate uncouples the glucagon receptor from adenylyl cyclase (by phosphorylation through PKC at Ser432 in the receptor?)."

In addition to its own dual role, glucagon can potentiate the effects of vasopressin, angiotensin 11, and phenylephrine (an (Y I -adrenergic agonist) on the second signal pathway, so that [Ca*+], oscillations and Ca?' inflow are greater than with either glucagon alone or with those strictly Ca2+-mobilizing agonists alone.J7

PHYSIOLOGICAL ROLES EXERTED THROUGH GLUCAGON RECEPTORS

Glucagon receptors and/or the corresponding cDNA are identified in a restricted number of tissues and cells, including the liver, kidney, heart, adipose tissue, pancreatic islets, stomach, intestine, and some brain areas, but not in lung where tGLP-1 receptors are present. Species differences may be encountered (see below).

The Liver- Kidney axis

Glucagon exerts rapid and delayed effects on the liver, whereby it provokes a coordinated CAMP- and Ca'+-dependent increase in glycogenolysis, glycolysis, glucose cycling, gluconeogenesis, and ketogenesis (reviewed in reference 48). Glucagon also stimulates hepatic proteolysis in lysosomes and inhibits protein synthesis so that glucogenic amino acids contribute easily to gluconeogenesis. This coincides with the disposal of nitrogen residues through hepatic urea synthesis and kidney e~cre t ion .~ '

One-third of the increase in urea excretion results from a rise in renal glomerular filtration rate and two-thirds from a parallel reduction in the reabsorption of urea and water in the proximal tubule. This kidney contribution depends on the diuretic action of liver-borne adenosine on the glomerulae and on CAMP produced in proximal tubular cells.Jy~s" Besides, glucagon stimulates adenylyl cyclase in the thick ascending limb of Henle's loop, the distal convoluted tubule, and the collecting whereby the hormone ( 1 ) regulates local gluconeogenesis and (2) enhances the reabsorption of Na', Kt, Cl-, Mg", and Ca2t and inhibits that of bicarbonate."

Which Zntraislet or/p/S-Cell Connections?

Glucagon potentiates glucose-induced insulin secretion during fasting, possibly by inhibiting ATP-dependent K' channels when the f3y complex, dissociated from Gs, associates (deactivates) c-wi On the other hand, glucagon receptor mRNA expression intensifies in cultured rat islets submitted to increasing levels of


glucose (+200%) or to somatostatin-28 (+75%), but decreases by 50-75% in the presence of forskolin plus IBMX, or dexamethasone. This indicates that various mechanisms alter the sensitivity to glucagon by acting on the mRNA stability or gene transcription of its receptor. The indentity of the islet cells at stake needs to be ~larified.~'

In Adrenals and Selected Brain Areas

It is desirable to document the role exerted by glucagon receptors in adrenals.?* Glucagon infused into the portal vein of rat produces a satiety signal that is

transmitted by the hepatic branch of the abdominal vagus to the sensory nucleus of the solitary tract involved in gustation in brain, thereby reducing spontaneous eating." Besides, the role exerted directly by glucagon and tGLP-I on adenylyl cyclase in the thalamus-hypothalamus remains to be ascertained.s9

Species Differences among Adipocytes

Glucagon activates adenylyl cyclase and induces a lipolytic response in rat adipocytes.60 In adrenalectomized6' or streptozotocin-diabetic rats,62 the marked reduction in the number of glucagon receptors may reflect a downregulation by hyperglucagonemia. The presence of glucagon receptors has been observed in a human liposar- coma,"3 but not in normal human adipocytes, and the in vivo lipolytic effect of glucagon in man is debatable.

Glucagon Receptors in Stomach and Intestine

Glucagon inhibits gastric acid secretion in man, presumably through an indirect effect via somatostatin-secreting cells.64 Glucagon exerts a spasmolytic function on dog intestinal smooth muscle cells.65 It also increases threefold the mRNA for the catalytic al subunit of Na, K-ATPase in the rat small intestinal mucosa.66

Heart Glucagon Receptors

Glucagon displays positive inotropic and chronotropic effects on ventricular and atrial preparations from rat, guinea-pig, dog, cat, and frog through CAMP.^^"^ Rat cardiac receptors are less sensitive to glucagon in vitro, as compared to rat hepatic receptors, and also less efficiently coupled to adenylyl cyclase. This is even more the case in atrial membranes than in ventricular membranes. As a consequence, several glucagon analogs behave only as partial agonists in heart membranes while acting as full agonists in liver membranes,2 thus reflecting a lower cardiac receptor density not a difference in primary receptor structure (see earlier).


SUMMARY AND PERSPECTIVES

The rat glucagon receptor gene contains 12 exons, 7 of which code for the TM domain. In rat liver, the gene is transcribed into several pre-mRNAs, variously shortened at the 5' end. One mature intronless mRNA, after the splicing out of the I 1 introns, is translated into the functional glucagon receptor.

We detected by PCR the expression of the same glucagon receptor in rat heart, islets (p cells'?), stomach, kidney, and adipocytes, suggesting that one gene allows the expression of only one type of glucagon receptor product, in terms of amino acid sequence. This G-protein-coupled protein with seven transmembrane (TM) segments activates adenylyl cyclase and phospholipase C.

Six lines of research are now obvious:

I . To examine the bearing of posttranslational processing of this receptor by glycosylation, phosphorylation, and palmitoylation.

2. In the absence of X-ray structure of the glucagon/receptor complex, further binding and functional data will reveal specific roles for each extra- and intracellular domain of the receptor. For instance, in stably transfected CHO cells, the interaction of glucagon with the receptor-effector complex can be analyzed after partial deletions or point mutations (to alter charges), or in chimeric constructions where a fragment of the glucagon receptor is substituted by the corresponding fragment of the parent tGLP-1 receptor. Mutagenesis of extracellular Asn and Cys residues will reveal the importance of glycosylation and disulfide bridges as prerequisites for receptor function. This evaluation requires the use of specific antipeptide antibodies (e.g., against the extreme C-terminal) to see whether a given mutation is not responsible for a mere three-dimensional (3-D) delocalization and general instability (inactivity) of the receptor synthesized by CHO cells.

3. A better understanding on how Hisl, Gly4, Asp9, Lysl2, and Serl6 in glucagon are sterically involved in effector coupling will give further clues in our search for long-acting peptidase-resistant glucagon antagonists.

4. The regulation of receptor mRNA transcription by the promoter depends on cis-acting enhancers, such as CRE and tissue-specific elements, which are regulated positively or negatively by transacting transcription factors, and cofactors reacting to either CAMP, phosphorylation, hormones (corticosterone, insulin), or nutrients (glucose, polyunsaturated fatty acids). Expression assays and transgenic mouse tech- nology could be used to identify these gene-regulatory elements and the cell-specific transcription factors that control the limited tissue distribution of this receptor.

5 . The expression of glucagon receptors in cultured islets, in separate cell types, and in insulinoma and glucagonoma cell lines, in the presence of nutrients (glucose, butyrate, polyunsaturated fatty acids, etc.), and/or dexamethasone will specify the contributions of glucagon receptors to secretory responses, (auto) feedback mechanisms, and prohormone gene expressions i n p, a, and 6 cells. For instance, the precise cellular localization of the receptor mRNA, by in siru hybridization procedures, could delineate whether p and 6 cells express glucagon receptors in response to glucagon secreted by a cells in the same islets.

6. The potential of multiple introns must be evaluated in all tissues concerned at each stage of development. Appropriate primers also allow a quantitative PCR assay of mRNA levels for glucagon receptors, under various pathological conditions


such as genetic obesity or hypertension in rodents, where changes in receptor number may reflect alterations in transcription rate and/or mRNA stability. In human physiopathology, starvation, malnutrition, neonatal undernutrition, sepsis, surgical stress, diabetes (types 1 and 2) , and adrenal disorders need also to be evaluated for the tissue expression of this receptor.

ACKNOWLEDGMENTS

I thank S. Mulongo and N. Van Laer for their skillful contribution in preparing this manuscript.

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DISCUSSION OF THE PAPER

HUBERT VAUDRY (INSERM, Mont-Saint-Aignan, France): I have a question related to the presence of glucagon receptors in the adrenal gland. You know that both catecholamines produced by the adrenal medulla and the glucocorticoids produced by the cortex are important for the carbohydrate metabolism. Where are the glucagon receptors in the adrenals?

JEAN CHRISTOPHE (Universitk Libre de Bruxelles, Belgium): I wish I could answer that. The only thing we did in Brussels was to observe the presence of messenger RNAs in adrenals, but this is a preliminary response, and I wish I knew more on the secretion of catecholamines under glucagon.

GABRIEL ROSSELIN (INSERM, Paris, France): Regarding the coupling of the glucagon receptor to G-proteins, could you expand on what could be the critical part of the intracellular loop or the C-terminal in that coupling?

CHRISTOPHE: For G-proteins, let us say a biochemist asked two people with huge computer programs to compare the third intracellular loop and the C-terminal for the glucagon receptor and whatever receptor you like. If you then ask them to tell you what all those C-terminals and third intracellular loops have in common, when


observing an activation or inhibition of adenylate cyclase, they will tell you they don’t know.

VAUDRY: I have a very short question related to physiopathology. Has any mutation of glucagon receptors been related or reported to be linked with diabetes?

CHRISTOPHE: Well, hyperglucagonemia has been described in STZ-treated rats. Therefore in uncontrolled diabetic patients, you might well have hyperglucagonemia. I wouldn’t dare to say that you have a systematic hyperglucogonemia in diabetes type 11, and the idea of using a glucagon antagonist would probably be useful only in cases of acute diabetic disorder, not as a chronic treatment. Besides, no mutation of the glucagon receptor has so far been reported in the case of diabetes.

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Glucagon and Its Receptor in Various Tissues