Low Concentrations Of Glucagon Research Articles

Review: Influence of postabsorptive metabolism on essential amino acid partitioning in lactating dairy cows

In the lactating cow, essential amino acids (EAAs) absorbed from the gut are partitioned to mammary and extra-mammary tissues via blood plasma circulation. There is also entry of EAA into plasma from the breakdown of proteins in the cow’s body. A balance model across plasma was solved to integrate entry rates of branched-chain (BCAA) and non-branched-chain EAA (NBAA) with their corresponding rate constants for clearance by mammary glands and the remainder of the body, for selected glucose and fat infusion experiments. Endogenous EAA entry from whole-body proteolysis was reduced by glucose and unchanged or increased by fat, the efficiency of net plasma BCAA clearance by mammary and extra-mammary tissues was elevated by glucose but slightly reduced by fat, and the efficiency of extra-mammary NBAA clearance may have decreased during glucose infusion but it was not affected or slightly increased by fat. These differences between glucose and fat responses can be accounted for by insulin and glucagon. Insulin suppresses endogenous EAA entry through mechanistic target of rapamycin complex 1, integrated stress response, and glycogen synthase kinase 3 signaling networks in skeletal muscle. While these networks can also regulate protein synthesis rates in muscle and the extra-mammary body, they exhibit low sensitivities to insulin in lactating ruminants. However, in the mammary glands, via these same networks, insulin stimulates clearance of EAA from plasma, although the drive to maintain a set point for milk protein yield takes precedence over nutritional signals. The glucose-induced increase in mammary BCAA clearance without an effect on NBAA clearance is due to a pronounced decrease in plasma BCAA concentrations. Because NBAAs do not experience a similar decline in concentration, the BCAA effect must be due to their metabolic transformation as opposed to sequestration in proteins. In adipose, the products of BCAA catabolism are lipogenic precursors. We propose that faster lipogenesis in adipose tissue, stimulated by glucose infusion, also promotes the uptake of precursor BCAA from plasma, causing a drop in their circulating concentrations. In addition, insulin stimulates BCAA oxidation in muscle as an alternative fuel to fatty acids. A lower efficiency of extra-mammary NBAA clearance during glucose infusion may be the consequence of decreased hepatic expression of AA-catabolizing enzymes in response to low glucagon concentration. The proportion of EAA entry partitioned to the mammary glands is a culmination of regulatory shifts at all of the points discussed above according to a regulated or unfair competition between mammary and extra-mammary processes.

Plasma concentrations of glucagon and glucagon-like peptide-1 are reduced in dogs with aminoaciduric canine hypoaminoacidemic hepatopathy syndrome versus healthy dogs: a preliminary study.

To compare plasma concentrations of glucagon and glucagon-like peptide-1 (GLP-1) between healthy dogs and dogs with aminoaciduric canine hypoaminoacidemic hepatopathy syndrome (ACHES) dogs. Privately owned healthy (n = 5) control (CON) and ACHES (8; including 3 with diabetes mellitus) dogs enrolled between October 2, 2019, and March 4, 2020. This was a prospective case-control study. Fasting and 15-minute postprandial plasma glucagon total GLP-1 concentrations were measured with commercial immunoassays. Dogs with ACHES had lower fasting (median, 0.5; mean difference, 3.8; 95% CI, 0.52 to 7.0 pmol/L; P = .021) and postprandial (median, 0.35; mean difference, 5.0; 95% CI, 1.8 to 8.3 pmol/L; P = .002) plasma glucagon concentrations than CON (fasting and postprandial medians, 3.5 and 4.6 pmol/L, respectively). ACHES dogs had significantly (median, 4.15; mean difference, 12.65; 95% CI, 2.0 to 16.3 pg/ml; P = .011) lower postprandial plasma GLP-1 concentrations than CON (median, 16.8 pg/ml). There was no significant difference between fasting GLP-1 levels between the 2 groups. Lower postprandial plasma GLP-1 concentrations may contribute to the propensity of diabetes mellitus in ACHES. Lower glucagon concentrations may reflect an appropriate physiologic response to hypoaminoacidemia.

Incretin-Based Therapies

Incretin mimetics and inhibitors of the protease dipeptidyl peptidase (DPP)-4 are new classes of antidiabetic agents first introduced in the years 2005 (exenatide) and 2007 (sitagliptin), respectively. Both use the antidiabetic properties of the incretin hormone, glucagon-like peptide (GLP)-1 (1). This gut-derived peptide hormone not only augments glucose-induced insulin secretion (required to fulfill the definition of an incretin hormone), but does so in a highly glucose-dependent manner (2), thus preventing GLP-1 alone from provoking hypoglycemia. Additional beneficial effects of GLP-1 on endocrine pancreatic islets are that it 1 ) supports the synthesis of proinsulin to replenish insulin stores in β-cells; 2 ) reduces the rate of β-cell apoptosis when islets are incubated in a toxic environment (glucotoxicity, lipotoxicity, cytotoxic cytokines); and 3 ) promotes differentiation of precursor cells with the ability to develop into β-cells and proliferation of β-cell lines, and in whole animals (rodent studies), this leads to an increased β-cell mass within a few days or weeks (1,3). Furthermore, GLP-1 can lower glucagon concentrations, i.e., induce α-cells to respond again to the inhibitory action of hyperglycemia, while leaving the counterregulatory glucagon responses undisturbed, as in the case of hypoglycemia (2,4). Additional activities of GLP-1 are the deceleration of gastric emptying (5), which slows the entry of nutrients into the circulation after meals, a reduction in appetite, and earlier induction of satiety (6), leading to weight reduction with chronic exposure (7). Renal effects (promotion of sodium and water excretion (8), as well as neuro- (9) and cardioprotective (10) properties of GLP-1, have also been described. While GLP-1 is perfectly suitable for lowering, or even stabilizing, glucose concentrations in short-term experiments, its short half-life (∼1–2 min for the intact, biologically active form) caused by rapid proteolytic degradation and inactivation through the ubiquitous enzyme DPP-4 and renal elimination (Fig. 1 …

Glucagon-like peptide 1 (GLP-1) as a new therapeutic approach for type 2-diabetes.

Glucagon-like peptide 1 (GLP-1) is a physiological incretin hormone in normal humans explaining in part the augmented insulin response after oral versus intravenous glucose administration. In addition, GLP-1 also lowers glucagon concentrations, slows gastric emptying, stimulates (pro)insulin biosynthesis, reduces food intake upon intracerebroventricular administration in animals, and may, in addition, enhance insulin sensitivity. Therefore, GLP-1, in many aspects, opposes the Type 2-diabetic phenotype characterized by disturbed glucose-induced insulin secretory capacity, hyperglucagonaemia, moderate insulin deficiency, accelerated gastric emptying, overeating (obesity) and insulin resistance. The other incretin hormone, gastric inhibitory polypeptide (GIP), has lost almost all its activity in Type 2-diabetic patients. In contrast, GLP-1 glucose-dependently stimulates insulin secretion in diet- and sulfonylurea-treated Type 2-diabetic patients and also in patients under insulin therapy long after sulfonylurea secondary failure. Exogenous administration of GLP-1 ([7-37] or [7-36 amide]) in doses elevating plasma concentrations to approximately 3-4 fold physiological postprandial levels fully normalizes fasting hyperglycaemia in Type 2-diabetic patients. The half life of GLP-1 is too short to maintain therapeutic plasma levels for sufficient periods by subcutaneous injections. Current research activities aim at finding GLP-1 analogues with more suitable pharmacokinetic properties than the original peptide. Another approach could be the augmentation of endogenous release of GLP-1, which is abundant in L cells of the lower small intestine and the colon. Interference with sucrose digestion using alpha-glucosidase inhibition moves nutrients into distal parts of the gastrointestinal tract and, thereby, prolongs and augments GLP-1 release. Enprostil, a prostaglandin E2 analogue, fully suppresses GIP responses, while only marginally affecting insulin secretion and glucose tolerance after oral glucose, suggesting compensatory hypersecretion of additional insulinotropic peptides, possibly including GLP-1. Given the large amount of GLP-1 present in L cells, it appears worthwhile to look for more agents that could 'mobilize' this endogenous pool of the 'antidiabetogenic' gut hormone GLP-1.

Insulin-like stimulation of cardiac fuel metabolism by physiological levels of glucagon: involvement of PI3K but not cAMP

At concentrations around 10(-9) M or higher, glucagon increases cardiac contractility by activating adenylate cyclase/cyclic adenosine monophosphate (AC/cAMP). However, blood levels in vivo, in rats or humans, rarely exceed 10(-10) M. We investigated whether physiological concentrations of glucagon, not sufficient to increase contractility or ventricular cAMP levels, can influence fuel metabolism in perfused working rat hearts. Two distinct glucagon dose-response curves emerged. One was an expected increase in left ventricular pressure (LVP) occurring between 10(-9.5) and 10(-8) M. The elevations in both LVP and ventricular cAMP levels produced by the maximal concentration (10(-8) M) were blocked by the AC inhibitor NKY80 (20 microM). The other curve, generated at much lower glucagon concentrations and overlapping normal blood levels (10(-11) to 10(-10) M), consisted of a dose-dependent and marked stimulation of glycolysis with no change in LVP. In addition to stimulating glycolysis, glucagon (10(-10) M) also increased glucose oxidation and suppressed palmitate oxidation, mimicking known effects of insulin, without altering ventricular cAMP levels. Elevations in glycolytic flux produced by either glucagon (10(-10) M) or insulin (4 x 10(-10) M) were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (10 microM) but not significantly affected by NKY80. Glucagon also, like insulin, enhanced the phosphorylation of Akt/PKB, a downstream target of PI3K, and these effects were also abolished by LY-294002. The results are consistent with the hypothesis that physiological levels of glucagon produce insulin-like increases in cardiac glucose utilization in vivo through activation of PI3K and not AC/cAMP.

Glucagon Amyloid-like Fibril Morphology Is Selected via Morphology-Dependent Growth Inhibition

The 29-residue peptide hormone glucagon readily fibrillates at low pH, but the structure and morphology of the fibrils are very sensitive to the environmental conditions. Here we have investigated the mechanism behind the differences in morphology observed when glucagon fibrils are formed at different peptide concentrations. Electron microscopy shows that fibrils formed at low glucagon concentration (0.25 mg/mL) are twisted, while fibrils formed at high concentration (8 mg/mL) are straight. Monitoring the fibrillation kinetics at different concentrations, we find that the lag time has an unexpected maximum at a concentration of 1 mg/mL, with faster fibrillation at both lower and higher concentrations. Seeding experiments show that small amounts of straight fibril seeds can accelerate fibril growth at both low and high glucagon concentration, while twisted fibril seeds cannot grow at high concentrations. We conclude that there exists a morphology-dependent mechanism for inhibition of glucagon fibril growth. Light scattering experiments indicate that glucagon is mainly monomeric below 1 mg/mL and increasingly trimeric above this concentration. We propose that the glucagon trimer is able to specifically inhibit growth of the twisted fibril morphology. Such inhibitory binding of molecules in an unproductive conformation could also play a role in the selection of morphologies for other fibril-forming peptides and proteins.

Expanded Use of Exenatide in the Management of Type 2 Diabetes

Exenatide is an incretin mimetic agent that possesses multiple mechanisms of glucose lowering. It enhances glucose-dependent insulin secretion by the pancreatic β-cell, leading to insulin release in the presence of elevated glucose concentrations. It also moderates glucagon secretion and lowers serum glucagon concentrations during periods of hyperglycemia. Lower glucagon concentrations lead to decreased hepatic glucose output and decreased insulin demand. Exenatide also slows gastric emptying, thereby reducing the rate at which meal-derived glucose appears in the circulation, thereby further decreasing postprandial blood glucose levels. Finally, administration of exenatide has been shown to reduce subsequent food intake by increasing satiety, often resulting in weight loss.1 Exenatide is indicated as an adjunctive therapy to improve glycemic control in patients with type 2 diabetes who are taking metformin and/or a sulfonylurea but have not achieved adequate glycemic control.1 To decrease gastrointestinal intolerance, exenatide is initiated at 5 μg subcutaneously twice daily and increased after 1 month to a target maintenance dose of 10 μg twice daily. Three 30-week studies as add-on therapy to sulfonylurea, metformin, and sufonylurea/metformin have demonstrated hemoglobin A1c (A1C) reductions of ∼ 1% with an average weight loss of 1.4–1.8 kg.2–4 Therapy was generally well tolerated with the most frequent adverse events classified as mild or moderate and gastrointestinal in nature. Severe hypoglycemia was not observed during these studies.2–4 The concurrent use of exenatide with insulin, meglitinides, or α-glucosidase inhibitors has not been reported. Published information regarding the use of exenatide with thiazolidinediones (TZDs) is limited to two abstracts.5,6 The objective of this study was to determine the effectiveness and safety of off-label exenatide use in patients with type 2 diabetes. A retrospective review of the electronic medical records of one private-practice endocrinologist was conducted. The study was …

Sucrose diets increase glucose-6-phosphatase and glucose release and decrease glucokinase in hepatocytes.

A high-sucrose diet (SU) decreases insulin action in the liver (Pagliassotti MJ, Shahrokhi KA, and Moscarello M. Am J Physiol Regulatory Integrative Comp Physiol 266: R1637-R1644, 1994). The present study was conducted to characterize the effect of SU on glucagon action in isolated periportal (PP) and perivenous (PV) hepatocytes by measuring glucagon-stimulated glycogenolysis and glucose release. Male rats were fed a SU (68% sucrose) or starch diet (ST, 68% starch) for 1 wk, and hepatocytes were isolated from PP or PV regions (n = 4/diet/cell population). Hepatocytes were incubated for 1 h in the presence of varying concentrations of glucagon (0-100 nM). In PP and PV cells, glucagon stimulation of glucose release and glycogenolysis (sum of glucose release and lactate accumulation) was not significantly different between SU and ST cells. However, in the SU PP cells, glucose release was increased compared with ST PP cells, both in the absence of glucagon (76.1 +/- 4 vs. 54.8 +/- 3 nmol x h(-1) x mg cell wet x wt(-1)) and at all glucagon concentrations. In SU-fed PV cells, glucose release was increased compared with ST PV cells in the absence of glucagon (79.3 +/- 5 vs. 56.4 +/- 5 nmol x h(-1) x mg cell wet x wt(-1)) and at low glucagon concentrations. Maximal glucose-6-phosphatase activity (in nmol x min(-1) x mg protein(-1)) was elevated in SU compared with ST cells (61.4 +/- 3 vs. 37.5 +/- 4 in PP and 37.5 +/- 4 vs. 29.5 +/- 3 in PV cells). In contrast, maximal glucokinase activity (in nmol x min(-1) x mg protein(-1)) was elevated in ST compared with SU cells (15.9 +/- 2 vs. 12.1 +/- 1 in PP and 19.4 +/- 2 vs. 14.2 +/- 1 in PV cells). These data demonstrate that SU increases the capacity for glucose release in both PP and PV hepatocytes, in part because of reciprocal changes in glucose-6-phosphatase and glucokinase.

Glucagon-like peptide 1 (GLP-1): a potent gut hormone with a possible therapeutic perspective.

Glucagon-like peptide 1 (GLP-1) is a physiological incretin hormone from the lower gastrointestinal tract, partially explaining the augmented insulin response after oral compared to intravenous glucose administration in normal humans. In addition, GLP-1 also lowers glucagon concentrations, slows gastric emptying, stimulates (pro)insulin biosynthesis, and reduces food intake upon intracerebroventricular administration in animals. Therefore, GLP-1 offers some interesting perspective for the treatment of type 2, and perhaps also for type 1 diabetic patients. The other incretin hormone, gastric inhibitory polypeptide (GIP), has lost almost all its activity in type-2 diabetic patients. In contrast, GLP-1 glucose-dependently stimulates insulin secretion in type-2 diabetic patients and exogenous administration of GLP-1 ([7-37] or [7-36 amide]) in doses elevating plasma concentrations to approximately three to four times physiological postprandial levels fully normalizes fasting hyperglycaemia and reduces postprandial glycaemic increments. Due to rapid proteolytic cleavage, which results in an inactive or even antagonistic fragment. GLP-1 [9-36 amide], and to rapid elimination, the half-life of GLP-1 is too short to maintain therapeutic plasma levels for sufficient periods by subcutaneous injections of the natural peptide hormone. Current research aims to characterize GLP-1 analogues with more suitable pharmacokinetic properties than the original peptide. Given the large amount of GLP-1 present in L cells, it also appears worthwhile to search for more agents that could 'mobilize' this endogenous pool of GLP-1.

Origin of slow and fast oscillations of Ca2+ in mouse pancreatic islets.

1. Pancreatic islets exposed to 11 mM glucose exhibited complex variations of cytoplasmic Ca2+ concentration ([Ca2+]i) with slow (0.3-0.9 min-1) or fast (2-7 min-1) oscillations or with a mixed pattern. 2. Using digital imaging and confocal microscopy we demonstrated that the mixed pattern with slow and superimposed fast oscillations was due to separate cell populations with the respective responses. 3. In islets with mixed [Ca2+]i oscillations, exposure to the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (DTBHQ) resulted in a selective disappearance of the fast pattern and amplification of the slow pattern. 4. In addition, the protein kinase A inhibitor RP-cyclic adenosine 3',5'-monophosphorothioate sodium salt transformed the mixed [Ca2+]i oscillations into slow oscillations with larger amplitude. 5. Islets exhibiting only slow oscillations reacted to low concentrations of glucagon with induction of the fast or the mixed pattern. In this case the fast oscillations were also counteracted by DTBHQ. 6. The spontaneously occurring fast oscillations seemed to require the presence of cAMP-elevating glucagon, since they were more common in large islets and suppressed during culture. 7. Image analysis revealed [Ca2+]i spikes occurring irregularly in time and space within an islet. These spikes were preferentially observed together with fast [Ca2+]i oscillations, and they became more common after exposure to glucagon. 8. Both the slow and fast oscillations of [Ca2+]i in pancreatic islets rely on periodic entry of Ca2+. However, the fast oscillations also depend in some way on paracrine factors promoting mobilization of Ca2+ from intracellular stores. It is proposed that such a mobilization in different cells within a tightly coupled islet syncytium generates spikes which co-ordinate the regular bursts of action potentials underlying the fast oscillations.

EGF- and CPA-induced mitogenic stimuli are differentially down- regulated by TGF-beta1 in cultured rat hepatocytes

Down-regulation of the mitogenic activity of the rodent liver carcinogen cyproterone acetate (CPA) and of epidermal growth factor (EGF) were compared in cultured rat hepatocytes. Both hepatomitogens produce an increase in the expression of proliferating cell nuclear antigen (PCNA) and in [3H]thymidine incorporation in a dose-dependent manner. In combination, the two mitogens induced an additive mitogenic response. Concomitant exposure to the growth inhibitory cytokine transforming growth factor beta1 (TGF-beta1) resulted in a differential dose-dependent down-regulation of PCNA-expressing cells. The corresponding down-regulation of CPA-induced PCNA expression required a 3- to 5-fold higher TGF-beta1 concentration than for EGF-induced expression. In contrast, CPA-exposed hepatocytes become vulnerable to and EGF-exposed cells protected against the apoptosis-inducing activity of TGF-beta1 (>0.1 ng/ml). Under culture conditions that mimicked a pericentral-equivalent microenvironment (low oxygen tension, low glucagon concentration), PCNA expression was 3-fold lower and CPA-specific resistance was no longer detectable. It is concluded that EGF and CPA induce their growth stimuli preferentially in the periportal area of the liver but in different hepatocyte sub-populations, which differ in their down-regulation of premitotic events by TGF-beta1. At low TGF-beta1 concentrations, EGF-stimulated cells shift back into a resting cell cycle phase, whereas CPA-treated hepatocytes are eliminated by apoptosis at higher TGF-beta1 concentrations.

On the mechanisms of the growth‐promoting effect of prostaglandins in hepatocytes: The relationship between stimulation of DNA synthesis and signaling mediated by adenylyl cyclase and phosphoinositide‐specific phospholipase C

While many observations indicate that prostaglandins may act as positive regulators of hepatocyte proliferation, the underlying mechanisms are not known. We have examined some of the signal pathways in the growth response induced by prostaglandins in hepatocytes, with particular focus on adenylyl cyclase and phosphoinositide-specific phospholipase C. Adult rat hepatocytes were cultured as primary monolayers in serum-free medium in the presence of EGF and insulin. PGE2 or PGF2 alpha (added 0-3 h after plating) enhanced the incorporation of [3H]-thymidine into DNA (measured at 50 h); at 100 microM the stimulation was about threefold PGI2 and PGD2 also showed significant but smaller stimulatory effects. No significant increase in the level of cyclic AMP (cAMP) was detected in response to any of the prostaglandins. Low concentrations of glucagon (0.1-10 nM), a potent activator of hepatic adenylyl cyclase, or 8-bromo-cAMP (0.1-10 microM) enhanced the DNA synthesis. When 8-bromo-cAMP was used in maximally effective concentrations, no further stimulation was obtained by combining it with glucagon, whereas the effects of PGE2 and 8-bromo-cAMP were completely additive. All the prostaglandins also showed additivity with the effect of glucagon on the DNA synthesis. PGE2, PGF2 alpha, PGI2, and PGD2 increased intracellular inositol-1,4,5-trisphosphate (InsP3), with a relative order of efficacy roughly corresponding to their activity as stimulators of DNA synthesis. Increases in cytosolic free Ca2+, as measured in single cells, were elicited in a majority of the hepatocytes by all these prostaglandins at 1 microM. Supramaximal concentrations of vasopressin, a strong activator of phospholipase C in hepatocytes, acted additively with PGE2 on the DNA synthesis. Pretreatment of the hepatocytes with a concentration of pertussis toxin that prevented the inhibitory effect of PGE2 on glucagon-induced cAMP accumulation did not abolish the ability of PGE2 to stimulate the DNA synthesis. The results do not support a role for adenylyl cyclase activation in the stimulatory effect of prostaglandins on hepatocyte growth. While the data are compatible with an involvement of phosphoinositide-specific phospholipase C in the growth-promoting effect of prostaglandins in cultured rat hepatocytes, they suggest this may not be the sole mechanism.

Insulin and glucagon regulation of gluconeogenesis in preruminating and ruminating bovine.

Hepatocyte monolayers were established from young preruminating (7 to 14 d of age) or older ruminating (11 to 12 wk of age) calves and used to evaluate the effects of insulin and glucagon on incorporation of carbon from 2.5 mM [2-14C]propionate and 2.0 mM [U-14C]lactate into glucose and glycogen. Developmental state (young preruminating vs older ruminating) of the donor calf did not affect the rate of gluconeogenesis from propionate in the absence of hormones. Insulin decreased (P < .05) gluconeogenesis and increased (P < .05) glycogenesis from propionate and lactate in hepatocytes from preruminating calves but had no effect on hepatocytes from ruminating calves. Lactate was poorly metabolized to glucose and was not responsive to glucagon in hepatocytes from ruminating calves compared with hepatocytes from preruminating calves. Hepatocytes responded to glucagon by increasing (P < .05) gluconeogenesis from propionate. Maximal responsiveness to glucagon did not differ between ruminating and preruminating calves, but hepatocytes from preruminating calves responded at lower glucagon concentrations (P < .05). These data demonstrate a similar capacity of hepatocytes from preruminating and ruminating calves to metabolize propionate to glucose, but there was a seven- to eightfold decrease in gluconeogenesis from lactate in ruminating calves that was accompanied by a decreased response to acute changes in insulin and glucagon.

Characterization of the G protein coupling of a glucagon receptor to the KATP channel in insulin-secreting cells

The G-protein-mediated coupling of a glucagon receptor to ATP-dependent K channels--KATP--has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches, KATP channel activity was inhibited by low concentrations of glucagon (IC50 = 2.4 nM); the inhibitory effect vanished at concentrations greater than 50 nM. In cell-attached patches, inhibition by bath-applied glucagon was seen most often, although stimulation was observed in a few cases. A dual action of the hormone is proposed to resolve these apparently divergent results. In excised inside-out patches, KATP channel activity was inhibited by addition of beta gamma subunits purified from either erythrocyte or retina (IC50 = 50 pM and 1 nM, respectively). Subsequent exposure of the patch to alpha i or alpha o reversed this effect. In excised inside-out patches, increasing Mg2+ in the bath stimulated the channel activity between 0 and 0.5 nM, but blocked it at higher concentrations (IC50 = 2.55 mM). In most cases (70%), GTP had a stimulatory effect at concentrations up to 100 microns. However, in three cases, similar GTP levels had clear inhibitory effects. In excised inside-out patches, cholera toxin (CTX) caused channel inhibition. Although the effect could not be reversed by removal of the toxin, the activity was restored by subsequent addition of purified alpha i or alpha o. These results are compatible with a model whereby channel inhibition by activated Gs-coupled receptors occurs, at least in part, via association of the beta gamma subunits of Gs with alpha i/alpha o subunits and deactivation of the alpha i/alpha o-dependent stimulatory pathway. On the basis of this hypothesis, a model is developed to describe the effects of G proteins on the KATP channel, as well as to account for the concentration-dependent stimulation and inhibition of KATP channel by Mg2+. An interpretation of the ability of glucagon to potentiate, but not initiate, insulin release is also given in terms of this model and the effects of ATP on KATP channels.

Role of calcium fluxes in the action of glucagon on glucose metabolism in rat hepatocytes

The aim of the present study was to assess the role of calcium fluxes in the action of glucagon on glycogenolysis and gluconeogenesis in isolated rat hepatocytes. Calcium influx was blocked by two ways: by use of the compound tetramethrin and by reduction of extracellular calcium to 1 microM. The minimal concentration of tetramethrin that inhibited glucagon-mediated calcium entry was 7.5 x 10(-7) M. In the presence of 7.5 x 10(-7) M tetramethrin, glucagon-induced glycogenolysis was markedly attenuated when glucagon concentration was 10(-9) M or higher. In contrast, tetramethrin had no effect on glucogenolysis evoked by lower concentrations of glucagon. Similarly, tetramethrin greatly reduced gluconeogenesis induced by high concentrations of glucagon without affecting the effect of low concentrations of glucagon. The same results were obtained in the presence of 1 microM extracellular calcium. To abolish glucagon-induced elevation of cytoplasmic free calcium concentration, we heavily loaded quin2 into hepatocytes. In these cells, glycogenolysis evoked by low concentrations of glucagon was completely abolished. Glycogenolysis caused by high concentrations of glucagon was markedly inhibited. These results indicate that glucagon action on hepatic glucose metabolism is mediated by two different mechanisms, which depend on concentrations of glucagon.

Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus.

In type-2 diabetes, the overall incretin effect is reduced. The present investigation was designed to compare insulinotropic actions of exogenous incretin hormones (gastric inhibitory peptide [GIP] and glucagon-like peptide 1 [GLP-1] [7-36 amide]) in nine type-2 diabetic patients (fasting plasma glucose 7.8 mmol/liter; hemoglobin A1c 6.3 +/- 0.6%) and in nine age- and weight-matched normal subjects. Synthetic human GIP (0.8 and 2.4 pmol/kg.min over 1 h each), GLP-1 [7-36 amide] (0.4 and 1.2 pmol/kg.min over 1 h each), and placebo were administered under hyperglycemic clamp conditions (8.75 mmol/liter) in separate experiments. Plasma GIP and GLP-1 [7-36 amide] concentrations (radioimmunoassay) were comparable to those after oral glucose with the low, and clearly supraphysiological with the high infusion rates. Both GIP and GLP-1 [7-36 amide] dose-dependently augmented insulin secretion (insulin, C-peptide) in both groups (P < 0.05). With GIP, the maximum effect in type-2 diabetic patients was significantly lower (by 54%; P < 0.05) than in normal subjects. With GLP-1 [7-36 amide] type-2 diabetic patients reached 71% of the increments in C-peptide of normal subjects (difference not significant). Glucagon was lowered during hyperglycemic clamps in normal subjects, but not in type-2 diabetic patients, and further by GLP-1 [7-36 amide] in both groups (P < 0.05), but not by GIP. In conclusion, in mild type-2 diabetes, GLP-1 [7-36 amide], in contrast to GIP, retains much of its insulinotropic activity. It also lowers glucagon concentrations.

Carbohydrate metabolism in fetuses with Rh alloimmunization.

Glucose, insulin, glucagon and fructosamine were measured in the plasma of control (n = 34) and of Rh fetuses (n = 22) before the first and subsequent transfusion. Prior to treatment, Rh fetuses, compared to controls, had raised concentrations of glucose (p = 0.01), glucagon (p = 0.04) and fructosamine (p = 0.004). Fetal insulin levels were not significantly different. Before subsequent transfusion, despite correction of anemia, Rh fetuses were clearly hyperinsulinemic even after correction for gestation (p = 0.002) and had low glucagon concentrations (p = 0.01) when compared with values at the first transfusion. This metabolic milieu appears similar to that of fetuses of diabetic mothers which have increased plasma glucose and insulin during most of the last third of gestation.

Dual effects of glucagon and cyclic amp on dna synthesis in cultured rat hepatocytes: Stimulatory regulation in early G1 and inhibition shortly before the s phase entry

Although several lines of evidence implicate cyclic AMP in the humoral control of liver growth, its precise role is still not clear. To explore further the role of cyclic AMP in hepatocyte proliferation, we have examined the effects of glucagon and other cyclic AMP-elevating agents on the DNA synthesis in primary cultures of adult rat hepatocytes, with particular focus on the temporal aspects. The cells were cultured in a serum-free, defined medium and treated with epidermal growth factor (EGF), insulin, and dexamethasone. Exposure of the hepatocytes to low concentrations (10 pM-1 nM) of glucagon in the early stages of culturing (usually within 6 h from plating) enhanced the initial rate of S phase entry without affecting the lag time from the plating to the onset of DNA synthesis, whereas higher concentrations inhibited it. In contrast, glucagon addition at later stages (24-45 h after plating) produced only the inhibition. Thus, if glucagon was added at a time when there was a continuous EGF/insulin-induced recruitment of cells to S phase, the rate of G1-S transition was markedly decreased within 1-3 h. This inhibitory effect occurred at low glucagon concentrations (ID50 less than 1 nM) and was mimicked by cholera toxin, forskolin, isobutyl methylxanthine, and 8-bromo cyclic AMP. The results indicate that cyclic AMP has dual effects on hepatocyte proliferation with a stimulatory modulation early in the prereplicative period (G0 or early G1), and a marked inhibition exerted immediately before the transition from G1 to S phase.

Glucagon-(19-29) exerts a biphasic action on the liver plasma membrane Ca2+ pump which is mediated by G proteins.

We have recently shown that nanomolar concentrations of glucagon-(19-29), which can derive from native glucagon by proteolytic cleavage of the dibasic doublet Arg17-Arg18, inhibit the Ca2+ pump in liver plasma membrane vesicles independently of adenylyl cyclase activation (Mallat, A., Pavoine, C., Dufour, M., Lotersztajn, S., Bataille, D., and Pecker, F. (1987) Nature 325, 620-622). We report here that the regulation of the Ca2+ pump by glucagon-(19-29) is dependent on guanine nucleotides. In the presence of 10 microM guanosine 5'-3-O-(thio) triphosphate (GTP gamma S) or 75 microM GTP, glucagon-(19-29) caused a biphasic regulation of the Ca2+ pump. ATP-dependent Ca2+ transport was inhibited in the presence of 10 pM to 1 nM glucagon-(19-29), while higher concentrations of the peptide (1-100 nM) reversed the inhibition caused by lower ones. GTP gamma S alone, at high concentrations (100 microM), reproduced the inhibitory effect of glucagon-(19-29) and induced a 40% inhibition of the basal activity of the Ca2+ pump which was reversed by low concentrations of glucagon-(19-29) (10 pM to 1 nM). Treatment of rats with cholera toxin resulted in a 70% increase in the basal activity of the Ca2+ pump, a loss of sensitivity to GTP gamma S and to the biphasic regulation by glucagon-(19-29). Treatment with pertussis toxin did not affect the response of the Ca2+ pump to GTP gamma S and glucagon-(19-29). We conclude that glucagon-(19-29) can exert a biphasic effect on the Ca2+ pump which is mediated by G protein(s) sensitive to cholera toxin.

Evidence of cyclic AMP-independent action of glucagon on calcium mobilization in rat hepatocytes

Glucagon increases the cytoplasmic free calcium concentration as measured bby aequorin bioluminescence. It has been proposed by Wakelam et al. (Nature 323 (1986) 68–71) that low concentrations of glucagon mobilize calcium from an intracellular pool by causing polyphosphoinositide breakdown. To identify whether cyclic AMP mediates changes in the cytoplasmic free calcium concentration ([Ca 2+] c) induced by glucagon, the effects of forskolin and exogenous cyclic AMP on [Ca 2+] c were compared with that of glucagon in aequorin-loaded hepatocytes. Although the magnitudes of the [Ca 2+] c responses to 250 μM forskolin and 1 mM 8-bromo cyclic AMP were identical to that of 5 nM glucagon, these two agents induced a more prolonged elevation of [Ca 2+] c. Glucagon-induced elevation of [Ca 2+] c was accompanied by a smaller increase in cyclic AMP than that induced by forskolin. When the cyclic AMP response to glucagon was potentiated by an inhibitor of phosphodiesterase, 3-isobutyl-1-methylxanthine, the glucagon-induced increase in [Ca 2+] c was not affected. Conversely, when the cyclic AMP response to glucagon was reduced by pretreatment of the cells with angiotensin II, glucagon-induced changes in [Ca 2+] c were rather enhanced. Furthermore, vasopressin potentiated glucagon-induced changes in [Ca 2+] c despite the reduction of the cyclic AMP response to glucagon. In the presence of 1 μM extracellular calcium, angiotensin II did not enhance glucagon-induced changes in [Ca 2+] c. These results suggest that at least part of the action of 5 nM glucagon on calcium mobilization is independent of cyclic AMP.