Kaluresis independent K homeostasis in dogs: activity after ureter ligation and pancreatectomy

In ureter ligated dogs intravenous administration of KCl stimulates both insulin secretion and activity of a kaluresis independent K homeostatic mechanism (K transfer capacity) that retards the development of hyperkalemia by transferring K to intracellular fluid. If the preparation is K loaded by infusion with 2 mEq KCl/kg/hr until prelethal ECG changes of hyperkalemic cardiotoxicity appear, about 50% of administered K is transferred. An increased proportion--70%--is transferred if the animal is K loaded 70 minutes after pancreatectomy--when serum immunoreactive insulin is fixed at less than 4 uU/ml. That proportion (70%) is unchanged by simultaneous adrenalectomy, but is reduced to less than 40% by propranolol blockade of B receptors. Increased post pancreatectomy K transfer capacity apparently involves K transfer mediated by B receptors that are activated by an extra-adrenomedullary B agonist(s). Findings also indicate that residual post pancreatectomy insulin biological activity mediates K transfer.     

Acetazolamide impairment of the transfer of infused K from extracellular to intracellular fluid.

In intact dogs K loaded by infusion with 2 mEq KC1/kg/hr, treatment with acetazolamide produces both a profound kaluresis and a marked impairment of the animal's ability to transfer the infused K to intracellular fluid. The impairment is unrelated to kaluresis, since it is substantially the same in nephrectomized animals. Neither does the impairment stem fromacetazolamide induced acid- osis - there is a similar fall of blood pH in untreated control animals in which there is brisk transmembrane K transfer of infused K; and the $\text{ability}$ to transfer K is relatively unimpaired in nephrectomized dogs rendered acidotic by HCl administration. Acetazolamide is an effective therapeutic and prophylactic agent in the treatment of hypokalemic periodic paralysis; the results of the present investigation suggest a possible explanation of its therapeutic efficacy.     

Insulin and beta receptor modulation of K homeostasis in nephrectomized dogs with hyperkalemia

In nephrectomized dogs infused with 2 mEq KCl/kg/hr a homeostatic mechanism retards the development of hyperkalemia by transferring about 70% of the K load to intracellular fluid. beta Adrenergic receptor activity is importantly involved in the transfer process; halting it with propranolol reduces the proportion transferred to less than 35%. The addition of pancreatectomy increases the involvement of beta receptor activity; propranolol treatment now reduces the proportion transferred to less than 20%. Insulin treatment, on the other hand, not only improves transfer of a K load, it also alters the response to propranolol. Nephrectomized dogs treated with 2 U insulin/kg/hr deposit some 80% of the infused K in intracellular fluid. After beta receptor blockade, nearly 90% is transferred. The results suggest that in the K homeostatic mechanism of nephrectomized dogs, insulin and beta receptors may be reciprocally related. K transfer mediated by beta receptors improves after pancreatectomy, and insulin mediated K transfer improves after beta receptors are inactivated.     

Heparin mediates transmembrane potassium transfer in hyperkalemic dogs

Unheparinized, ureter-ligated control dogs that are potassium loaded, i.e., infused with 2 mEq of KCl/kg until prelethal electrocardiographic changes of hyperkalemic cardiotoxicity appear (end point), transfer 57 +/- 4% (1.7 +/- 0.1 mEq/kg) of administered potassium to intracellular fluid. Heparinized controls transfer 73 +/- 1% (3.2 +/- 0.2 mEq/kg); with simultaneous alpha-adrenoreceptor blockade, that proportion increases to 81 +/- 2% (4.80 +/- 0.7 mEq/kg) and with simultaneous beta-receptor blockade it is 58 +/- 3% (1.1 +/- 0.1 mEq/kg). In potassium loaded, ureter-ligated dogs, heparin increases transmembrane potassium transfer as effectively as does a dosage of atropine large enough to cross the blood-brain barrier and its influence on potassium transfer, like that of atropine, is suppressed by beta-adrenoreceptor blockade.     

Exenatide can reduce glucose independent of islet hormones or gastric emptying

Exenatide is a long-acting glucagon-like peptide-1 (GLP-1) mimetic used in the treatment of type 2 diabetes. There is increasing evidence that GLP-1 can influence glycemia not only via pancreatic (insulinotropic and glucagon suppression) and gastric-emptying effects, but also via an independent mechanism mediated by portal vein receptors. The aim of our study was to investigate whether exenatide has an islet- and gastric-independent glycemia-reducing effect, similar to GLP-1. First, we administered mixed meals, with or without exenatide (20 microg sc) to dogs. Second, to determine whether exenatide-induced reduction in glycemia is independent of slower gastric emptying, in the same animals we infused glucose intraportally (to simulate meal test glucose appearance) with exenatide, exenatide + the intraportal GLP-1 receptor antagonist exendin-(9-39), or saline. Exenatide markedly decreased postprandial glucose: net 0- to 135-min area under the curve = +526 +/- 315 and -536 +/- 197 mg.dl(-1).min(-1) with saline and exenatide, respectively (P < 0.05). Importantly, the decrease in plasma glucose occurred without a corresponding increase in postprandial insulin but was accompanied by delayed gastric emptying and lower glucagon. Significantly lower glycemia was induced by intraportal glucose infusion with exenatide than with saline (92 +/- 1 vs. 97 +/- 1 mg/dl, P < 0.001) in the absence of hyperinsulinemia or glucagon suppression. The exenatide-induced lower glycemia was partly reversed by intraportal exendin-(9-39): 95 +/- 3 and 92 +/- 3 mg/dl with exenatide + antagonist and exenatide, respectively (P < 0.01). Our results suggest that, similar to GLP-1, exenatide lowers glycemia via a novel mechanism independent of islet hormones and slowing of gastric emptying. We hypothesize that receptors in the portal vein, via a neural mechanism, increase glucose clearance independent of islet hormones.     

Expression of glucagon receptors in tetracycline-inducible HEK293S GnT1- stable cell lines: an approach toward purification of receptor protein for structural studies

Glucagon is a 29-amino acid polypeptide hormone secreted by pancreatic A cells. Together with insulin, it is an important regulator of glucose metabolism. Type 2 diabetes is characterized by reduced insulin secretion from pancreatic B cells and increased glucose output by the liver which has been attributed to abnormally elevated levels of glucagon. The glucagon receptor (GR) is a member of family B G protein-coupled receptors, ligands for which are peptides composed of 30-40 amino acids. The impetus for studying how glucagon interacts with its membrane receptor is to gain insight into the mechanism of glucagon action in normal physiology as well as in diabetes mellitus. The principal approach toward this goal is to design and synthesize antagonists of glucagon that will bind with high affinity to the GR but will not activate it. Site-directed mutagenesis of the GR has provided some insight into the interactions between glucagon and GR. The rational design of potent antagonists has been hampered by the lack of structural information on receptor-bound glucagon. To obtain adequate amounts of receptor protein for structural studies, a tetracycline-inducible HEK293S GnT1(-) cell line that stably expresses human GR at high-levels was developed. The recombinant receptor protein was characterized, solubilized, and isolated by one-step affinity chromatography. This report describes a feasible approach for the preparation of human GR and other family B GPCRs in the quantities required for structural studies.     

Cardiac sensitivity to hyperkalemia in adrenalectomized dogs.

In dogs with bilateral adrenalectomy loaded with K by infusion of 2 mEq KCI/kg/hr there is a marked increase of cardiac sensitivity to hyperkalemia. Typical ECG changes begin at lower serum K levels (5-6 mEq/l) and the prelethal arrhythmias that signal the imminent onset of fatal when mean serum K is 7.6 mEq/l, 2.9 mEq/l above the average pre-infusion level. In control dogs, ECG changes start above 8 mEq K/liter, and prelethal arrhythmias appear between 9.5 and 10.2 mEq/l, a mean increase of 5.6 mEq/l above the average preinfusion level.     

Alterations in hepatic glucagon receptor density and in Gsalpha and Gialpha2 protein content with diet-induced hepatic steatosis: effects of acute exercise

The present study was undertaken to test the hypothesis that a high-fat diet-induced liver lipid infiltration is associated with a reduction of hepatic glucagon receptor density (B(max)) and affinity (K(d)), and with a decrease in stimulatory G protein (G(s)alpha) content while enhancing inhibitory G protein (G(i)alpha(2)) expression. We also hypothesized that, under this dietary condition, a single bout of endurance exercise would restore hepatic glucagon receptor parameters and G protein expression to standard levels. Female Sprague-Dawley rats were fed either a standard (SD) or a high-fat diet (HF; 40% kcal) for 2 wk (n = 20 rats/group). Each dietary group was thereafter subdivided into a nonexercised (Rest) and an acute-exercised group (Ac-Ex). The acute exercise consisted of a single bout of endurance exercise on a treadmill (30 min, 26 m/min, and 0% slope) immediately before being killed. The HF compared with the SD diet was associated with significantly (P < 0.05) higher values in hepatic triglyceride concentrations (123%), fat pad weight, and plasma free fatty acid (FFA) concentrations. The HF diet also resulted in significantly (P < 0.05) lower hepatic glucagon receptor density (45%) and G(s)alpha protein content (75%), as well as higher (P < 0.05) G(i)alpha(2) protein content (27%), with no significant effects on glucagon receptor affinity. Comparisons of all individual liver triglyceride and B(max) values revealed that liver triglycerides were highly (P < 0.003) predictive of the decreased glucagon receptor density (R = -0.512). Although the 30-min exercise bout resulted in some typical exercise effects (P < 0.05), such as an increase in FFA (SD diet), a decrease in insulin levels, and an increase in plasma glucagon concentrations (SD diet), it did not change any of the responses related to liver glucagon receptors and G proteins, with the exception of a significant (P < 0.05) decrease in G(i)alpha(2) protein content under the HF diet. The present results indicate that the feeding of an HF diet is associated with a reduction in plasma membrane hepatic glucagon receptor density and G(s)alpha protein content, which is not attenuated by a 30-min exercise bout. It is suggested that liver lipid infiltration plays a role in reducing glucagon action in the liver through a reduction in glucagon receptor density and glucagon-mediated signal transduction.     

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.     

Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus

The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.     

Chronic propranolol treatment decreases pulmonary artery pressure in conscious dogs

Daily administration of propranolol to 9 chronically instrumented, trained dogs for 2 weeks caused significant (p less than 0.05) decreases in heart rate (70 +/- 8 to 57 +/- 6 beats/min), cardiac output (3.6 +/- 0.3 to 2.9 +/- 0.2 liters/min), pulmonary arterial pressure (15.7 +/- 0.5 to 10.0 +/- 0.5 mm Hg) and total pulmonary vascular resistance (4.6 +/- 0.6 to 3.3 +/- 0.4 units). Nadolol, a structurally dissimilar beta-adrenergic receptor antagonist, caused a similar decrease in total pulmonary resistance. Acute meclofenamate administration did not return to normal pulmonary arterial pressure and resistance in the dogs chronically treated with beta-adrenergic receptor blockers. We therefore conclude that chronic beta-adrenergic receptor blockade lowered pulmonary arterial pressure and resistance by a mechanism independent of cyclooxygenase. In addition, chronic beta-adrenergic receptor blockade did not affect the potential for hypoxic vasoconstriction.     

Leukotriene and purinergic receptors are involved in the hyperpolarizing effect of glucagon in liver cells

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K+ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba2+ (BaCl2, 5 mmol/l) and the blocker of the Na+/K+ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B4 receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 micromol/l). ATPgammaS (10 micromol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 micromol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPgammaS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane.     

Effect of glucagon on glomerulopressin production in diabetic dogs

Glucagon (21.5 +/- 0.23 ng/min/kg) was infused through the portal vein of normal or pancreatectomized dogs. It was observed that a dose of glucagon that produces no significant change in the glycemia of normal dogs has a very small activity in the production of glomerulopressin and does not alter glomerular filtration rate (GRF). In pancreatectomized dogs this same dose of glucagon also does not alter glycemia but it induces a large increase in the production of glomerulopressin and GFR. Our results suggest that in pancreatectomized dogs glomerulopressin production is more sensitive to glucagon infusion than in normal dogs.     

Molecular determinants of glucagon receptor signaling

A 29-amino acid polypeptide hormone, glucagon has been one of the most prolific models in the study of hormone action. The key biologic function of glucagon is to counterbalance the actions of insulin and maintain a normal level of serum glucose. Diabetes mellitus can thus be considered a bihormonal disorder with an excess of glucagon contributing to the hyperglycemic state. The effects of glucagon are mediated by the glucagon receptor, which is itself a prototypical member of a distinct category called family B receptors within the G protein-coupled superfamily of seven-helical transmembrane receptors (GPCRs). At the structural level, the peptide ligands of family B receptors are highly homologous, in particular in the N-terminal region of the molecules. The mechanism by which highly homologous peptide ligands selectively recognize their receptors involves distinct molecular interactions that are gradually being elucidated. This review focuses on structural determinants of the glucagon receptor that are important for its activity with respect to interaction with its ligand and G proteins. Information about the glucagon receptor is presented within the context of what is known about other members of the family B GPCRs.     

The dynamics of protein kinase B regulation during B cell antigen receptor engagement.

This study has used biochemistry and real time confocal imaging of green fluorescent protein (GFP)-tagged molecules in live cells to explore the dynamics of protein kinase B (PKB) regulation during B lymphocyte activation. The data show that triggering of the B cell antigen receptor (BCR) induces a transient membrane localization of PKB but a sustained activation of the enzyme; active PKB is found in the cytosol and nuclei of activated B cells. Hence, PKB has three potential sites of action in B lymphocytes; transiently after BCR triggering PKB can phosphorylate plasma membrane localized targets, whereas during the sustained B cell response to antigen, PKB acts in the nucleus and the cytosol. Membrane translocation of PKB and subsequent PKB activation are dependent on BCR activation of phosphatidylinositol 3-kinase (PI3K). Moreover, PI3K signals are both necessary and sufficient for sustained activation of PKB in B lymphocytes. However, under conditions of continuous PI3K activation or BCR triggering there is only transient recruitment of PKB to the plasma membrane, indicating that there must be a molecular mechanism to dissociate PKB from sites of PI3K activity in B cells. The inhibitory Fc receptor, the FcgammaRIIB, mediates vital homeostatic control of B cell function by recruiting an inositol 5 phosphatase SHIP into the BCR complex. Herein we show that coligation of the BCR with the inhibitory FcgammaRIIB prevents membrane targeting of PKB. The FcgammaRIIB can thus antagonize BCR signals for PKB localization and prevent BCR stimulation of PKB activity which demonstrates the mechanism for the inhibitory action of the FcgammaRIIB on the BCR/PKB response.     

Chloride transport pathways and their bioenergetic implications in the obligate acidophile Bacillus coagulans.

The protonophore-mediated collapse of the large delta pH that acidophiles maintain across their cytoplasmic membranes was augmented by the presence of Cl-, and Cl- influx into the cells occurred evidently in response to the protonophore-induced increase in the inside-positive membrane potential (+ delta psi). In respiring cells, the addition of Cl- but not SO4(2-) salts caused a rapid and precipitous decrease in the + delta psi. A Nernstian relationship between the imposed transmembrane K+ gradient and the valinomycin-induced K+ diffusion potentials was observed when everted membrane vesicles were loaded with K2SO4 or KH2PO4 but not when loaded with KCl or KNO3. Thus, electrogenic Cl- transport occurred in Bacillus coagulans. In addition, a nonelectrogenic temperature-sensitive Cl- transport mechanism, with the net Cl- efflux coefficient (PCl-) ranging from 1.5 x 10(-4) to 6.1 x 10(-6) cm/s, accounted for the massive Cl- efflux from Cl(-)-loaded cells. Thus, B. coagulans, despite its dependence on the + delta psi and therefore the need to exclude anions, apparently possesses specific mechanisms for Cl- permeation. Active cells of B. coagulans prevented Cl- accumulation from attaining an electrochemical equilibrium, maintaining a delta micro Cl- of ca. -63 mV. B. coagulans therefore also possesses an energy-dependent mechanism for Cl- exclusion from the cells.     

Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes

Although many effects of leptin are mediated through the central nervous system, leptin can regulate metabolism through a direct action on peripheral tissues, such as fat and liver. We show here that leptin, at physiological concentrations, acts through an intracellular signaling pathway similar to that activated by insulin in isolated primary rat hepatocytes. This pathway involves stimulation of phosphatidylinositol 3-kinase (PI3K) binding to insulin receptor substrate-1 and insulin receptor substrate-2, activation of PI3K and protein kinase B (AKT), and PI3K-dependent activation of cyclic nucleotide phosphodiesterase 3B, a cAMP-degrading enzyme. One important function of this signaling pathway is to reduce levels of cAMP, because leptin-mediated activation of both protein kinase B and phosphodiesterase 3B is most marked following elevation of cAMP by glucagon, and because leptin suppresses glucagon-induced cAMP elevation in a PI3K-dependent manner. There is little or no expression of the long form leptin receptor in primary rat hepatocytes, and these signaling events are probably mediated through the short forms of the leptin receptor. Thus, leptin, like insulin, induces an intracellular signaling pathway in hepatocytes that culminates in cAMP degradation and an antagonism of the actions of glucagon.