Metabolic clearance rates of oxyntomodulin and glucagon in the rat: contribution of the kidney

The half-life (t1/2) and metabolic clearance rate (MCR) of exogenous natural porcine oxyntomodulin (porcine OXM) and the synthetic analog of rat oxyntomodulin, [Nle27]-OXM (rat OXM), were compared with that of glucagon in control, sham-operated and acutely nephrectomized rats using the primed-continuous infusion technique. The half-disappearance times for porcine OXM (8.2 +/- 0.5 min) and rat OXM (6.4 +/- 0.5 min) were 3-fold slower than that of glucagon (1.9 +/- 0.1 min). Acute bilateral nephrectomy significantly prolonged the half-disappearance time of rat OXM (8.2 +/- 0.7 min) and glucagon (3.6 +/- 0.4 min) compared with that of sham-operated animals (6.5 +/- 0.8 min and 2.5 +/- 0.2 min, respectively). The mean MCRs were similar for porcine and rat OXM (11.3 +/- 0.7 and 11.9 +/- 0.5 ml.kg-1.min-1) but were 3 times lower than that measured with glucagon (36 +/- 5 ml.kg-1.min-1). Bilateral nephrectomy reduced the MCR of OXM and glucagon by 38% and 34%, respectively. No significant increase in C-terminal glucagon immunoreactivity was noticed during infusion of either porcine or rat OXM, measured directly in plasma, with a specific C-terminal glucagon antiserum or after HPLC. In the course of the glucagon infusion, blood glucose was increased 2-fold, while the same dose of porcine OXM or of rat OXM induced only a small increase over the values in phosphate buffer-infused rats. 10 times higher doses of rat OXM were necessary to obtain a similar hyperglycemic effect. These results indicate that: (1) the metabolism of OXM is 3-fold slower than that of glucagon, (2) renal clearance contributed close to 35% of the overall metabolic plasma extraction for OXM and glucagon and (3) OXM, although effective at a higher dose, when compared with glucagon, displays a hyperglycemic effect probably through the glucagon receptors.     

Structure-function relationships in glucagon. Re-evaluation of glucagon-(1-21)

Glucagon-(1-21) was prepared fully synthetically as well as by carboxypeptidase A digestion of natural porcine glucagon. Neither of the two preparations had glucagon agonistic effects with regard to receptor binding or adenylate cyclase activation in purified rat liver plasma membranes. Nor did these preparations contain lipolytic activity in isolated free fat cells. A preliminary batch of glucagon-(1-21) prepared by carboxypeptidase A digestion did, however, contain 1-2% glucagon bioactivity. This activity was separated from glucagon-(1-21) by high-performance liquid chromatography and quantitatively recovered in four minor hind peaks which eluted close to but not in a position identical to the elution position of native glucagon.     

Effects of acute and chronic endotoxin treatment on glucagon and insulin receptors on rat liver plasma membranes

The effects of acute and chronic endotoxin treatment on the plasma levels of insulin and glucagon and their binding to rat liver plasma membranes were examined. Both acute and chronic endotoxin administration increased plasma glucagon levels and decreased the glucagon to insulin molar ratio. Acute, but not chronic, endotoxin decreased blood glucose and insulin levels. Glucagon binding was increased in membranes prepared from the acutely treated rats. However, in membranes obtained from rats treated chronically with endotoxin, only insulin binding was increased. The increases in the binding of both insulin and glucagon were the result of increases in receptor sites.     

Characterization of glucagon receptors in Golgi fractions of fetal rat liver

The present study was designed to determine if Golgi fractions from fetal rat liver contain glucagon receptors and to characterize the properties of such receptors. Purification patterns of liver plasma membranes and Golgi fractions from fetal and adult rats were similar, as verified by morphological and biochemical approaches. Glucagon binding was greater in plasma membranes of adult than fetal rats, while in Golgi fractions glucagon binding was similar in both groups. The modifications in in glucagon binding reflect changes in glucagon receptors. Glucagon association and glucagon receptor inactivation by liver membranes were similar in the two groups of animals, while glucagon degradation was lower in fetal than in adult rats.     

Differential acid stabilities of citraconylated amino groups of glucagon. Preparation of N alpha-citraconyl glucagon and evaluation of its biological properties

Acylation of the alpha- and epsilon-amino groups of histidine-1 and lysine-12 in glucagon with citraconic anhydride resulted in the formation of amide bonds which displayed different stabilities to hydrolysis under mild acid conditions. Treatment of N alpha,epsilon-dicitraconyl glucagon at pH 4.0 and room temperature regenerated the free epsilon-amino group within 16 h, while the citraconyl-alpha-amino group was stable. N alpha-Citraconyl glucagon was purified by anion-exchange chromatography and was a weak partial agonist in stimulating adenylate cyclase in rat liver plasma membranes. The derivative exhibited 1% of the biological potency and 35-40% of the maximal stimulation of glucagon. Binding affinity to plasma membranes was also reduced, but not to as great an extent as adenylate cyclase activity. Removal of the alpha-citraconyl group by treatment with 10 mM HCl at 40 degrees C restored full potency and stimulation to glucagon. These results suggest that the N-terminal histidine of glucagon is involved in both binding to plasma membranes and transduction of the signal to adenylate cyclase.     

Glucagon receptor binding, dissociation and degradation in rat liver plasma membranes studied by a microperifusion method

The association process of glucagon receptor binding in purified rat liver plasma membranes and prolonged incubation of the hormone-receptor complex at 30 degrees C did not result in degradation of bound labelled glucagon. In contrast, up to 95% of the non-membrane-bound labelled glucagon was degraded. The rate of spontaneous dissociation of the glucagon-receptor complex was slow, and amounted to about 0.1% per min of that bound. GTP greatly enhanced the rate of dissociation. Half the maximal dissociation of the complex was effected by 10(-5) mol/l of GTP under equilibrium binding conditions. At maximally effective concentrations of GTP, 80% of the glucagon-receptor complex was dissociated within 2 min. A microperifusion system for the perifusion of isolated plasma membranes was devised and used for the separation of labelled glucagon from the plasma membranes subsequent to a GTP-induced dissociation of the hormone-receptor complex. Rebinding of the dissociated peptide to fresh membranes showed that maximum binding ability was retained. The glucagon molecule was protected against degradation while bound to the receptor, indicating that the glucagon effector system is completely separate from the inactivating system(s) in isolated plasma membranes. Thus, the hormonal effect of glucagon could be exerted through the sequential interaction of each glucagon molecule with several receptors.     

Interactions of glucagon and related peptides with chicken adipose tissue.

The effect of glucagon, Vasoactive Intestinal Polypeptide (VIP), secretin and gut glucagon on the cyclic adenosine 3'5' monophosphate (cAMP) level, and on the specific binding of these 125I-peptides to the adipocyte plasma membrane was measured in chicken adipocytes and compared to the results obtained in rat adipocytes. The displacement of 125I-glucagon from its specific sites was observed with about the same concentration of unlabeled hormone in fat cell plasma membranes of both species. However, the rise in cAMP induced by glucagon was much higher in chicken than in rat adipocytes. In chicken fat cells unlike rat fat cells, the cAMP accumulation elicited by glucagon was maintained during at least 60 min even in the absence of theophylline. Theophylline at 1-10 mM potentiated the glucagon-stimulated cAMP levels in rat fat cells, but had only a slight effect, if any, in chicken adiposyces. Porcine VIP, secretin or gut glucagon exerted no detectable action on the cAMP level of chicken adipocytes. The lack of cAMP accumulation was in good agreement with the absence of binding of 125I-VIP and 125I-secretin by chicken plasma membranes. These findings suggest that: 1) the difference of glucagon effect in rat and chicken fat cells results from variations in the rate of degradation of cAMP rather than from differences in the specific binding of glucagon between the two species; 2) the use of chicken fat cells is suitable to discriminate between glucagon and structurally related peptides from mammals.     

Glucagon: structure-function relationships investigated by sequence deletions

A series of glucagon analogues, des-(1-4)-glucagon, des-(5-9)-glucagon, des-(10-15)-glucagon, des-(16-21)-glucagon, des-(22-26)-glucagon and des-(27-29)-glucagon, were prepared by condensation of synthetic fragments and characterized biologically and immunologically. Fully synthetic glucagon was also characterized. The potencies with regard to glucagon receptor binding in purified rat liver plasma membranes were, in decreasing order: synthetic glucagon 108%, des-(1-4)-glucagon 5.7%, des-(27-29)-glucagon 0.92%, des-(5-9)-glucagon 0.47%, des-(10-15)-glucagon 0.0028%, des-(16-21)-glucagon 0.0017% and des-(22-26)-glucagon 0.00060% relative to that of natural porcine glucagon. Des-(27-29)-glucagon was the only analogue that activated the adenylate cyclase in rat liver plasma membranes or stimulated the lipolysis in isolated free fat cells from rat epididymal fat pad. The potencies were 0.16% and 0.20% of that of glucagon, respectively. Des-(1-4)-glucagon was a glucagon antagonist in the adenylate cyclase assay. The immunoreactivities of the glucagon analogues were determined with two commonly used anti-glucagon sera, K 5563 and K 4023, directed towards the C-terminus and some segment in the sequence 2-23, respectively. In the K 5563 assay, des-(27-29)-glucagon and des-(22-26)-glucagon had potencies of 0.0009% and less than 0.09% of that of glucagon, respectively. The remaining analogues had potencies varying from 45% to 141% of that of glucagon. In the K 4023 assay, the analogues showed a non-linear dilution effect. The combined results indicate a partition within the glucagon molecule with regard to receptor binding and adenylate cyclase activation. The region 10-26 appears to be the most important for receptor binding, whereas 1-4 is essential for adenylate cyclase activation. The C-terminal segment 27-29 is important for the maintenance of full receptor binding but non-essential for adenylate cyclase activation.     

Hormone regulated enzyme activities of plasma membrane of decidual endometrium of the rat

Plasma membranes were purified from deciduoma of pseudopregnant rats and rat liver. Preparations contained 80% plasma membrane-derived material as based on electron microscope morphometry and analysis of enzyme markers. Several plasma membrane enzymes were tested for direct response to hormones. NADH-ferricyanide reductase of plasma membranes from both tissues was stimulated by glucagon and inhibited by insulin but was unresponsive to steroids. For steroids, responsiveness was limited to a reduction in NaF-stimulated adenylate cyclase activity by the steroid R5020. Thus, interaction of steroid hormones with plasma membranes, unlike that of glucagon and insulin, is not reflected in an altered activity of plasma membrane-bound dehydrogenases but may be exerted directly on adenylate cyclase.     

Distribution of adenylate cyclase among membrane fractions of rat liver.

Adenylate cyclase activity was detected in plasma membranes, Golgi apparatus, and endoplasmic reticulum from rat liver. Adenylate cyclase activities of purified membranes were determined biochemically by two methods. In one, the synthesis of radioactive cyclic AMP from ATalpha32P was monitored. In the other, the synthesis of cyclic AMP was quantitiated using a protein which specifically binds cyclic AMP. The enzyme activity was responsive to activation by both glucagon and sodium fluoride although differences in degree of activation were noted comparing plasma membrane, Golgi apparatus, and endoplasmic reticulum. Cytochemical studies, using both whole tissue and purified cell fractions and conducted in parallel, confirmed the biochemical results. Deposition of lead phosphate, enhanced by glucagon and NaF with samples incubated with appropriate substrates, was not restricted to plasma membranes of hepatocytes but was present in intracellular membranes as well. Adenylate cyclase of rat hepatocytes appears more widely distributed among internal membranes than previously recognized.     

A radioreceptor assay for insulin: direct measurement of dog pancreatic vein serum insulin.

A simple radioreceptor assay for insulin rat liver membranes as receptor sites, with sufficient specificity precision, and sensitivity to detect 10 ng or 276 muU/ml of serum insulin, has been developed. In the presence of standard porcine insulin at the concentration of 1.0 ng/tube, approximately 8% of 125I-porcine insulin was bound to the plasma membranes and ninety-five per cent of this binding was inhibited by 1.0 microgram of standard insulin per tube. Four animal insulins inhibited the binding of 125I-insulin while ACTH, glucagon, human growth hormone, and oxytocin were inert. Insulin values in dog pancreatic vein sera obtained during and after glucose loading and measured by the present radioreceptor assay agreed well with immunoreactive insulin. The ratio of IRI to the measurement by radioreceptor assay was 1.09 +/- 0.18 for the same sera.     

Studies on submaxillary gland immunoreactive glucagon.

Biologically active immunoreactive glucagon is present in submaxillary gland of rat, mouse, guinea pig and human and can be extracted by saline adjusted to pH 2.8 with HCl. Chromatography on Sephadex G-150 indicates its molecular weight to be 29,000. It has similar immunologic characteristics as pancreatic glucagon. It is biologically active and elevates plasma glucose and insulin when injected intraperitoneally into rats. Compared to pancreatic glucagon, the hyperglycemic effect persists much longer. It competes with pancreatic glucagon for binding to specific glucagon receptors of rat liver plasma membranes. It is stable to pH changes, however, urea dissociates it into several smaller molecular weight fragments including that of 3500. It appears to be an aggregate of smaller glucagon molecules and is not responsible for immunoreactive glucagon in totally eviscerated rats. $\text{In vitro}$, the submaxillary gland does not release immunoreactive glucagon in response to arginine or glucose.     

Immunological specificity and biological action of biliary glucagon-like substance in the rabbit

A glucagon-like substance named biliary IRG2000 whose molecular weight is approximately 2,000 was isolated by gel filtration from rabbit bile. This substance showed a strong crossreactivity as equivalent to 25.7 +/- 5.1 ng/ml of porcine glucagon in RIA with antiserum 30K specificity. Biliary IRG2000 brought about a significant increase and delayed the response of blood glucose level in coexistence with porcine glucagon, though it has no appreciable effect on the glucose level when administered singly to the mouse intraperitoneally. The response with the coexistence of these materials was far greater than when porcine glucagon was given alone. In Mortimore's type rat liver perfusion, a significant rise in glucose concentration in effluent was also observed when a mixture of biliary IRG2000 and porcine glucagon was perfused. The rate of 125I-glucagon degradation was found delayed in the presence of biliary IRG2000 when examined in the rat. Thus the increase and delayed response of glucose level in coexistence of porcine glucagon with biliary IRG2000 may be explained by a suppressive effect of glucagon degradation due to biliary IRG2000.     

Enzymatic,biophysical and ultrastructural changes of plasma membranes in chemical-induced rat hepatoma

Plasma membranes from liver of control rats or from chemical-induced hepatoma were prepared. The basal activity of adenylate cyclase was increased significantly in the rat plasma membranes of DEN-induced hepatoma compared to normal tissue. The glucagon-induced response on the cellular effector systems via guanine nucleotide-binding regulatory proteins (G proteins) was inhibited in hepatoma plasma membranes. These findings suggest that in hepatoma membranes, unlike normal hepatic membranes, the response to hormonal stimuli through regulatory G proteins results in a loss of response to glucagon, as well as to GTP plus glucagon or to GTPγS. However, the activating effects of forskolin, which catalyses the formation of cyclic AMP from ATP acting on the catalytic subunit, were to some extent retained. The methyltransferase-I behaved in the opposite direction to the adenylate cyclase, showing a decreased activity in hepatoma plasma membranes compared to control membranes. In contrast, the activity of the ecto-5′-nucleotidase was significantly increased in hepatoma. These enzymatic changes have been found to influence the membrane fluidity and to be responsible for the ultrastructural modifications of hepatoma plasma membranes which are induced by chemical carcinogens.     

N-ethylmaleimide uncouples the glucagon receptor from the regulatory component of adenylyl cyclase

125I-Glucagon binding to rat liver plasma membranes was composed of high- and low-affinity components. N-Ethylmaleimide (NEM) and several other alkylating agents induced a dose-dependent loss of high-affinity sites. This diminished the apparent affinity of glucagon receptors for hormone without decreasing the binding capacity of membranes. Solubilized hormone-receptor complexes were fractionated as high molecular weight (Kav = 0.16) and low molecular weight (Kav = 0.46) species by gel filtration chromatography; NEM or guanosine 5'-triphosphate (GTP) diminished the fraction of high molecular weight complexes, suggesting that NEM uncouples glucagon receptor-N-protein complexes. Exposure of intact hepatocytes to the impermeable alkylating reagent p-(chloromercuri)benzenesulfonic acid failed to diminish the affinity of glucagon receptors on subsequently isolated plasma membranes, indicating that the thiol that affects receptor affinity is on the cytoplasmic side of the membrane. Hormone binding to plasma membranes was altered by NEM even after receptors were uncoupled from N proteins by GTP. These data suggest that a sensitive thiol group that affects hormone binding resides in the glucagon receptor, which may be a transmembrane protein. Alkylated membranes were fused with wild-type or cyc- S49 lymphoma cells to determine how alkylation affects the various components of the glucagon-adenylyl cyclase system. Stimulation of adenylyl cyclase with fluoride, guanylyl 5'-imidodiphosphate, glucagon, or isoproterenol was observed after fusion of cyc- S49 cells [which lack the stimulatory, guanine nucleotide binding, regulatory protein of adenylyl cyclase (Ns)] with liver membranes alkylated with 1.5 mM NEM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of glucagon by dipeptidyl peptidase IV (CD26)

Glucagon is a 29-amino acid polypeptide released from pancreatic islet alpha-cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis. Despite its importance, there remains controversy about the mechanisms responsible for glucagon clearance in the body. In the current study, enzymatic metabolism of glucagon was assessed using sensitive mass spectrometric techniques to identify the molecular products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) yielded sequential production of glucagon(3-29) and glucagon(5-29). In human serum, degradation to glucagon(3-29) was rapidly followed by N-terminal cyclization of glucagon, preventing further DP IV-mediated hydrolysis. Bioassay of glucagon, following incubation with purified DP IV or normal rat serum demonstrated a significant loss of hyperglycemic activity, while a similar incubation in DP IV-deficient rat serum did not show any loss of glucagon bioactivity. Degradation, monitored by mass spectrometry and bioassay, was blocked by the specific DP IV inhibitor, isoleucyl thiazolidine. These results identify DP IV as a primary enzyme involved in the degradation and inactivation of glucagon. These findings have important implications for the determination of glucagon levels in human plasma.     

Glucagon1-6 binds to the glucagon receptor and activates hepatic adenylate cyclase.

A fragment of glucagon encompassing its first six NH2-terminal residues (His-Ser-Gln-Gly-Thr-Phe) binds to the glucagon receptor and stimulates adenylate cyclase activity in rat liver plasma membranes. Glucagon1-6 is a partial agonist since it stimulates, at saturating concentrations, to the extent of 75% of the maximal activity given by the native hormone. The binding affinity and potency of glucagon1-6 are 0.001% the native hormone. Discussed are the implications of these findings on the structure-function relationships required for the action of glucagon and for preparing clinically useful analogs of the hormone.     

Physicochemical and biological properties of glucagon-like polypeptides from porcine colon.

Polypeptide material displaying glucagon-like immunoreactivity was isolated from porcine colon using immunoaffinity chromatography. The immunoreactive material was tightly bound to high molecular weight proteins but was dissociated by 0.1% w/v sodium dodecyl sulphate solution into immunoreactive components of approximate molecular weights 12,000,8000,5000 and 3000. These components reacted at least 50 times more strongly with antibodies specific for the N-terminal region of glucagon than with antibodies specific for the C-terminal region of glucagon. While the 8000 and 3000 dalton fractions were homogeneous, the 12,000 and 5000 dalton fractions were resolved into multiple bands by isoelectric focusing. The 12,000 dalton fraction was devoid of glycogenolytic and lipolytic activity, was not insulin releasing and showed no ability to bind to receptor sites specific for glucagon on hepatic plasma membranes and to active hepatic adenylate cyclase. The 8000 and 5000 dalton components showed weak lipolytic activity. The possible significance of colonic glucagon-like immunoreactivity relative to pancreatic glucagon and immunoreactivity from other tissues is discussed.     

Characterization of glucagon receptors in Golgi fractions of rat liver: evidence for receptors that are uncoupled from adenylyl cyclase

Glucagon receptors have been identified and characterized in intermediate (Gi) and heavy (Gh) Golgi fractions from rat liver. At saturation, plasma membranes bound 3500 fmol of hormone/mg of membrane protein, while Gi and Gh bound 24 and 60 fmol of 125I-glucagon/mg of protein, respectively. Half-maximal saturation of binding to plasma membranes, Gi, and Gh occurred at approximately 4, 10, and 20 nM 125I-glucagon, respectively. Trichloroacetic acid precipitation of intact, but not degraded, glucagon was used to correct binding isotherms for hormone degradation. After such correction, half-maximal saturation of binding to plasma membranes, Gi, and Gh was observed in the presence of approximately 2, 7, and 14 nM hormone, respectively. After 90 min of dissociation in the absence of guanosine 5'-triphosphate (GTP), 86% of 125I-glucagon remained bound to plasma membranes, whereas only 42% remained bound to Golgi membranes. GTP significantly increased the fraction of 125I-glucagon released from plasma membranes but only slightly augmented the dissociation of hormone from Golgi fractions. 125I-Glucagon/receptor complexes solubilized from plasma membranes fractionated by gel filtration as high molecular weight (Kav = 0.16), GTP-sensitive complexes and lower molecular weight (Kav = 0.46), GTP-insensitive complexes. 125I-Glucagon complexes solubilized from Golgi membranes fractionated almost exclusively as the lower molecular weight species. The lower affinity of Golgi than plasma membrane receptors for hormone, the ability of glucagon to stimulate plasma membrane, but not Golgi membrane, adenylyl cyclase, and the near absence of high molecular weight, GTP-sensitive complexes in solubilized Golgi membranes demonstrate that plasma membrane contamination of Golgi fractions cannot account for the 125I-glucagon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dietary carbohydrates on insulin and glucagon receptors in a new model of noninsulin-dependent diabetes-SHR/N-corpulent rat

A new congenic strain of rat, the SHR/N-corpulent, provides a good model for noninsulin-dependent diabetes and was used in the present study. Corpulent rats as compared to their lean littermates are obese, hyperlipidemic, and severely hyperinsulinemic, and show an age-dependent loss of glucose tolerance. Mild fasting hyperglycemia is seen only in corpulent rats fed sucrose. Since dietary sucrose is more lipogenic than starch and since insulin and glucagon are involved in lipid and carbohydrate metabolism, we studied the effect of the type of dietary carbohydrate on insulin and glucagon levels and their receptors in lean and corpulent SHR/N rats. A significant phenotypic effect was observed (corpulent greater than lean) on plasma levels of triglyceride, cholesterol, and insulin. Dietary sucrose increased these parameters in corpulent rats but not in lean rats. Insulin and glucagon binding to liver plasma membranes was lower in corpulent rats than in lean; decreases were due to fewer receptors without a significant change in affinity. Thus, in corpulent rats, in addition to hyperinsulinemia, fewer glucagon receptors and their failure to be regulated by plasma glucagon levels appear to contribute to the hyperlipidemia. Furthermore, the hyperglycemia observed in sucrose-fed corpulent rats may be due to extreme resistance to insulin despite lower plasma glucagon and fewer glucagon receptors.