Kaluresis independent K-homeostasis: glucagon and B receptor blockade in pancreatectomized dogs

Seventy minutes post pancreatectomy, in dogs that are K loaded - made abruptly hyperkalemic and "life threatened" - by infusion with 2 mEgKC1/kg-/hr until prelethal ECG changes of hyperkalemic cardiotoxicity appear, a kaluresis independent K homeostatic mechanism transfers about 2/3 of administered K to intracellular fluid. Treatment of K loaded pancreatectomized dogs with glucagon or a B receptor blockading dosage of propranolol does not alter the proportion transferred, but treatment with glucagon and propranolol reduces it. It appears that in pancx dogs there is a reciprocal relation between insulin and B receptor mediated K transfer and that glucagon is involved in activity of the kaluresis independent K homeostatic mechanism.     

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.     

Effect of isoproterenol on the plasma C-peptide and insulin levels in humans

There are conflicting reports on the effect of stimulation of the beta-adrenergic receptors on insulin removal by the liver. It was, therefore, the aim of the present study to clarify that problem. Four experiments have been carried out on a group of 8 healthy female volunteers: (1) isoproterenol was infused intravenously, (2) glucose was infused intravenously, (3) isoproterenol was infused with glucose, and (4) infusion of isoproterenol and glucose was preceded by administration of propranolol (the beta-adrenergic blocking agent). The concentration of C-peptide and insulin was determined in plasma from the antecubital vein. It has been found that stimulation of the beta-adrenergic receptors with isoproterenol reduces insulin removal by the human liver. This effect of isoproterenol is prevented by blockade of the beta-adrenergic receptors with propranolol.     

A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis

The cDNAs encoding the normal human insulin receptor (HIRc) and a receptor that had lysine residue 1018 replaced by alanine (A/K1018) were used to transfect Rat 1 fibroblasts. Lysine 1018 is a critical residue in the ATP binding site of the tyrosine kinase domain in the receptor beta-subunit. Untransfected Rat 1 cells express 1700 endogenous insulin receptors. Expressed HIRc receptors had levels of insulin-stimulable autophosphorylation in vitro comparable to normal receptors, whereas A/K1018 receptors had less than 1% of that activity. Stimulation by insulin of HIRc receptors in situ in intact cells led to phosphorylation of beta-subunit tyrosine residues and activation of tyrosine kinase activity that could be preserved and assayed in vitro after receptor purification. In contrast, A/K1018 receptors showed no such activation, either of autophosphorylation or of kinase activity toward histone. Cells expressing HIRc receptors display enhanced sensitivity to insulin of 2-deoxyglucose transport and glycogen synthase activity. This increased sensitivity was proportional to insulin receptor number at low but not at high levels of receptor expression. A/K1018 receptors were unable to mediate these biologic effects and actually inhibited insulin's ability to stimulate glucose transport and glycogen synthase through the endogenous Rat 1 receptors. Expressed HIRc receptors mediated insulin internalization and degradation, whereas A/K1018 receptors mediated little, if any. Endocytotic uptake of the expressed A/K1018 insulin receptors was also markedly depressed compared to normal receptors. Unlike HIRc receptors, A/K1018 receptors also fail to undergo down-regulation after long (24 h) exposures to high (170 nM) concentrations of insulin. We conclude the following. 1) Normal human insulin receptors expressed in Rat 1 fibroblasts display active tyrosine-specific kinase, normal intracellular itinerary after endocytosis, and normal coupling to insulin's biologic effects. 2) A receptor mutated to alter the ATP binding site in the tyrosine kinase domain had little if any tyrosine kinase activity. 3) This loss of kinase activity was accompanied by a nearly complete lack of both endocytosis and biologic activity.     

Insulin-stimulated insulin secretion in single pancreatic beta cells

Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca2+ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assembly of insulin/insulin-like growth factor-1 hybrid receptors in vitro

Insulin and Mn/MgATP treatment of immunoaffinity-purified alpha beta heterodimeric insulin receptors induced the formation of an alpha 2 beta 2 heterotetrameric insulin receptor complex. In contrast, insulin-like growth factor-1 (IGF-1) treatment was completely ineffective in inducing the association of alpha beta heterodimeric insulin receptors. Similarly, IGF-1 or Mn/MgATP, but not insulin, treatment of immunoaffinity-purified alpha beta heterodimeric IGF-1 receptors induced the formation of an alpha 2 beta 2 heterotetrameric IGF-1 receptor complex. A monoclonal antibody specific for the insulin receptor (MA5) completely immunoprecipitated all the insulin binding activity from both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes but did not immunoprecipitate IGF-1 receptors. Conversely, the IGF-1 receptor-specific monoclonal antibody (alpha IR-3) immunoprecipitated all the IGF-1 binding activity, but not insulin receptors. The simultaneous treatment of pooled equal amounts of alpha beta heterodimeric insulin and IGF-1 receptors with a combination of insulin and IGF-1 resulted in the formation of alpha 2 beta 2 heterotetrameric insulin and IGF-1 receptor complexes. However, in the mixed alpha 2 beta 2 heterotetrameric receptor fraction MA5 immunoprecipitated 94% of the insulin binding in addition to 27% of the IGF-1 binding activity whereas alpha IR-3 immunoprecipitated 97% of the IGF-1 binding in addition to 38% of the insulin binding activity. Treatment of the mixed alpha beta heterodimeric insulin and IGF-1 receptors with Mn/MgATP also resulted in the formation of cross-immunoreactive (42-46%) alpha 2 beta 2 heterotetrameric receptors. These data directly demonstrate the formation of insulin/IGF-1 hybrid receptors by both a combination of insulin plus IGF-1 or Mn/MgATP treatment of purified human placenta alpha beta heterodimeric insulin and IGF-1 half-receptors in vitro.     

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.     

Insulin receptors with defective tyrosine kinase inhibit normal receptor function at the level of substrate phosphorylation

Rat 1 fibroblasts have been transfected with the cDNA encoding a kinase-defective mutant human insulin receptor (A/K1018). Expression of this cDNA results in a receptor that is not only biologically inactive but also inhibits normal insulin action through the normal endogenous rat receptors in this fibroblast line (McClain, D. A., Maegawa, H., Lee, J., Dull, T. J., Ullrich, A., and Olefsky, J. M. (1987) J. Biol. Chem. 262, 14663-14671). We have investigated the mechanism of this inhibition and show that: 1) rat receptors are expressed at normal to increased levels in two cell lines which also express A/K1018 receptors at low (A/K1018-A, 5700 total receptors) or high (A/K1018-B, 2.2 x 10(5) total receptors) levels. 2) The rat receptors in the A/K1018 lines can be normally autophosphorylated under the control of insulin in vitro. 3) A/K1018 receptors do not inhibit the kinase activity of normal receptors when mixed together in vitro. 4) In intact A/K1018-B cells, the ability of insulin to stimulate autophosphorylation of the rat receptor is unimpaired; furthermore, the autophosphorylated rat receptor becomes normally activated as a tyrosine kinase. 5) The expression of receptors for insulin-like growth factor I and stimulation of hexose uptake mediated by this receptor are unaffected in cells expressing inhibitory A/K1018 receptors. 6) Expression of the A/K1018 receptor inhibits insulin-stimulated phosphorylation of two endogenous protein substrates (pp220 and pp170) by the native rat receptors. We conclude that the inhibition of insulin action seen in the A/K1018 cells is not mediated at the levels of native receptor expression or activation, nor is the effector (hexose uptake) mechanism affected by the A/K1018 receptors. The expression of this kinase-defective receptor does, however, inhibit the phosphorylation of substrate molecules by the normally activated endogenous rat receptors.     

Stimulation of gastric glucagon secretion by epinephrine administration in dogs

To determine the response of gastric A-cells to adrenergic substances, immunoreactive glucagon was determined simultaneously in the jugular vein and in the left gastroepiploic vein of totally depancreatized dogs. Under basal conditions a significant gradient of glucagon concentrations between the jugular and gastric veins was observed, whereas plasma insulin values were almost undetectable. Intravenous administration of epinephrine elicits a prompt and significant increase in glucagon concentrations in the gastric vein which persist during the time of hormone infusion. To ensure adequate adrenergic blockade, blockers were infused before epinephrine administration. Accordingly, after phentolamine, the infusion of epinephrine failed to increase gastric glucagon concentrations, while after propranolol, epinephrine induced a significant release of gastric glucagon. These results indicate that epinephrine stimulates gastric glucagon secretion and that this effect is mediated through alpha-adrenergic receptors.     

Insulin modulation of chronotropic response to Adrenaline in diabetic dogs.

In 8 unanesthetized dogs, 10-21 days post pancreatectomy, the cardiac chronotropic response to rapid infusion of a pharmacological dosage of Adrenaline was begun. During the subsequent month, the response was recorded electrocardiographically on 19 occasions. On 8 occasions, animals were treated with continuous intravenous administration of fluids and insulin up to the time of the test; on 11, insulin was omitted for 18 hours before Adrenaline injection. Insulin treated animals responded with the typical brief initial bradycardia, followed by some 2 minutes of ventricular tachycardia, and restoration of preinjection heart rate and electrocardiograph pattern within 5 minutes. On those occasions when insulin was omitted, the tachycardia was replaced by ventricular bradycardia. The altered chronotropic response of non-insulin treated dogs indicates impairment of their cardiac beta receptors.     

Suppression of renin secretion by propranolol in salt-depleted dogs

d, 1-propranolol was infused into salt-depleted, conscious dogs at two dosages: 1 mg/kg followed by 0.60 ? 0.67 mg/kg/hr, and 5 mg/kg followed by 1.57 ? 1.76 mg/kg/hr. At both dosages, propranolol decreased plasma renin activity (PRA), plasma aldosterone concentration, and heart rate significantly. Renin substrate concentration remained unchanged. PRA was suppressed with the higher dosage but not with the lower dosage, to values found with dietary salt loading. Mean arterial blood pressure (MABP) remained unchanged with the low-dose infusions, but decreased significantly with the high-dose infusions. The data suggest that the mechanism(s) for the increase in PRA with low-salt diets is sensitive to propranolol and that the effect of propranolol on MABP is dependent on the salt intake and on the dose administered.     

Intramolecular subunit interactions between insulin and insulin-like growth factor 1 alpha beta half-receptors induced by ligand and Mn/MgATP binding.

We have previously demonstrated that isolated insulin and IGF-1 alpha beta half-receptors can be reconstituted into a functional alpha 2 beta 2 hybrid receptor complex [Treadway et al. (1989) J. Biol. Chem. 264, 21450-21453]. In the present study, we have examined this assembly process by determining the effect of ligand occupancy and Mn/MgATP binding on the dimerization of mutant and wild-type insulin and IGF-1 alpha beta half-receptors. IGF-1 or Mn/MgAMPPCP binding to wild-type IGF-1 alpha beta half-receptors resulted in the specific assembly of the alpha beta half-receptors into an alpha 2 beta 2 heterotetrameric IGF-1 holoreceptor complex. Similarly, insulin binding to the kinase-deficient mutant (A/K1018) insulin alpha beta half-receptor also resulted in the specific assembly into an alpha 2 beta 2 holoreceptor complex. In contrast, Mn/MgAMPPCP treatment of A/K1018 mutant insulin alpha beta half-receptors did not induce heterotetramer assembly, consistent with the inability of this mutant receptor to bind ATP. The ability of the insulin alpha beta receptors to assemble with the IGF-1 alpha beta half-receptors was used to examine the intermolecular subunit interactions responsible for dimerization. In the presence of Mn/MgAMPPCP, the wild-type insulin and wild-type IGF-1 alpha beta half-receptors were observed to assemble into an insulin/IGF-1 alpha 2 beta 2 hybrid receptor complex. Similarly, a combination of insulin and IGF-1 induced hybrid receptor formation between wild-type IGF-1 and A/K1018 mutant insulin alpha beta half-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased alpha2-adrenergic receptor in the brain stem and pancreatic islets during pancreatic regeneration in weanling rats

Sympathetic stimulation inhibits insulin secretion. alpha(2)-Adrenergic receptor is known to have a regulatory role in the sympathetic function. We investigated the changes in the alpha(2)-adrenergic receptors in the brain stem and pancreatic islets using [(3)H]Yohimbine during pancreatic regeneration in weanling rats. Brain stem and pancreatic islets of experimental rats showed a significant decrease (p<0.001) in norepinephrine (NE) content at 72 h after partial pancreatectomy. The epinephrine (EPI) content showed a significant decrease (p<0.001) in pancreatic islets while it was not detected in brain stem at 72 h after partial pancreatectomy. Scatchard analysis of [(3)H]Yohimbine showed a significant decrease (p<0.05) in B(max) and K(d) at 72 h after partial pancreatectomy in the brain stem. In the pancreatic islets, Scatchard analysis of [(3)H]Yohimbine showed a significant decrease (p<0.001) in B(max) and K(d) (p<0.05) at 72 h after partial pancreatectomy. The binding parameters reversed to near sham by 7 days after pancreatectomy both in brain stem and pancreatic islets. This shows that pancreatic insulin secretion is influenced by central nervous system inputs from the brain stem. In vitro studies with yohimbine showed that the alpha(2)-adrenergic receptors are inhibitory to islet DNA synthesis and insulin secretion. Thus our results suggest that decreased alpha(2)-adrenergic receptors during pancreatic regeneration functionally regulate insulin secretion and pancreatic beta-cell proliferation in weanling rats.     

Enhanced β-adrenergic receptors in the brain and pancreas during pancreatic regeneration in weanling rats

Adrenergic stimulation has an important role in the pancreatic β-cell proliferation and insulin secretion. In the present study, we have investigated how sympathetic system regulates the pancreatic regeneration by analyzing Epinephrine (EPI), Norepinephrine (NE) and β-adrenergic receptor changes in the brain as well as in the pancreas. EPI and NE showed a significant decrease in the brain regions, pancreas and plasma at 72 hrs after partial pancreatectomy. We observed an increase in the circulating insulin levels at 72 hrs. Scatchard analysis using [³H] propranolol showed a significant increase in the number of both the low affinity and high affinity β-adrenergic receptors in cerebral cortex and hypothalamus of partially pancreatectomised rats during peak DNA synthesis. The affinity of the receptors decreased significantly in the low and high affinity receptors of cerebral cortex and the high affinity hypothalamic receptors. In the brain stem, low affinity receptors were increased significantly during regeneration whereas there was no change in the high affinity receptors. The pancreatic β-adrenergic receptors were also up regulated at 72 hrs after partial pancreatectomy. In vitro studies showed that β-adrenergic receptors are positive regulators of islet cell proliferation and insulin secretion. Thus our results suggest that the β-adrenergic receptors are functionally enhanced during pancreatic regeneration, which in turn increases pancreatic β-cell proliferation and insulin secretion in weanling rats.     

The pancreatic glucagon and C-peptide secretion during hyperinsulinemia in euglycemic glucose clamp with or without somatostatin infusion in normal man

6 normal subjects received two times of 2 hr euglycemic glucose clamp studies (insulin infusion rate 40 mU/M2/min) one with and the other without somatostatin (SRIF) infusion (500 microgram/hr). Serum C-peptide and glucagon levels were measured during clamp to study the sensitivity of pancreatic alpha and beta cells to the suppressive effects of exogenous hyperinsulinemia during normoglycemia in normal subjects and to find whether SRIF had any modulative effects on endocrine pancreas secretion at the status of hyperinsulinemia. The results showed that in normal man the degree of suppression of pancreatic glucagon secretion by hyperinsulinemia (approximately 100 uU/ml) during euglycemic glucose clamp without SRIF infusion was less than that of C-peptide with mean value of 62 +/- 4% of basal glucagon remained at the end of clamp study; while only about 30 +/- 2% of basal C-peptide concentrations remained. But during SRIF infused glucose clamp studies (SRIF was infused from 60 to 120 min), 32 +/- 2% of mean basal C-peptide concentrations and 38 +/- 6% of mean basal glucagon concentrations left at the end of 2 hr clamp studies when serum insulin level was about 100 uU/ml. For the glucose infusion rate (M value), it was significantly greater in our normal subjects in response to insulin + SRIF as compared to insulin alone (12.0 + 0.9 vs 8.8 +/- 1.4; P less than 0.01). We concluded: during hyperinsulinemia (100 uU/ml), the sensitivity of pancreatic alpha cells to insulin seems less than that of beta cells in normal man at normoglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin activation of mitogen-activated protein kinases Erk1,2 is amplified via beta-adrenergic receptor expression and requires the integrity of the Tyr350 of the receptor

Insulin activates a complex set of intracellular responses, including the activation of mitogen-activated protein kinases Erk1,2. The counterregulatory actions of insulin on catecholamine action are well known and include phosphorylation of the beta(2)-adrenergic receptor on Tyr(350), Tyr(354), and Tyr(364) in the C-terminal cytoplasmic domain, as well as enhanced sequestration of the beta(2)-adrenergic receptor. Both beta-adrenergic agonists and insulin provoke sequestration of beta(2)-adrenergic receptors in a synergistic manner. In the current work, cross-talk between insulin action and beta(2)-adrenergic receptors revealed that insulin activation of Erk1,2 was amplified via beta(2)-adrenergic receptors. In Chinese hamster ovary cells, expression of beta(2)-adrenergic receptors enhanced 5-10-fold the activation of Erk1,2 by insulin and prolonged the activation, the greatest enhancement occurring at 5 min post-insulin. The potentiation of insulin signaling on Erk1,2 was proportional to the level of expression of beta(2)-adrenergic receptor. The potentiation of insulin signaling requires the integrity of Tyr(350) of the beta(2)-adrenergic receptor, a residue phosphorylated in response to insulin. beta(2)-adrenergic receptors with a Y350F mutation failed to potentiate insulin activation of Erk1,2. Expression of the C-terminal domain of the beta(2)-adrenergic receptor (Pro(323)-Leu(418)) in cells expressing the intact beta(2)-adrenergic receptor acts as a dominant negative, blocking the potentiation of insulin activation of Erk1,2 via the beta(2)-adrenergic receptor. Blockade of beta(2)-adrenergic receptor sequestration does not alter the ability of the beta(2)-adrenergic receptor to potentiate insulin action on Erk1,2. We propose a new paradigm in which a G-protein-linked receptor, such as the beta(2)-adrenergic receptor, itself acts as a receptor-based scaffold via its binding site for Src homology 2 domains, facilitating signaling of the mitogen-activated protein kinase pathway by insulin.     

Differences in signaling properties of the cytoplasmic domains of the insulin receptor and insulin-like growth factor receptor in 3T3-L1 adipocytes.

Insulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase.     

Comparative regulation of potassium and amphetamine induced release of 3H-norepinephrine from rat brain via presynaptic mechanisms.

This study examined the ability of various drugs to modify the potassium (K) or d-amphetamine (d-A) induced release of ³H-norepinephrine ³HNE) from chopped rat cortical tissue. The K induced release of the transmitter, which occurs from reserpine sensitive sites of cortical tissue, was significantly reduced by the beta receptor antagonist propranolol, the alpha receptor agonist clonidine and also by PGE₂. Pretreatment with eicosatetrynoic acid, an inhibitor of prostaglandin synthesis, did not influence the effect of clonidine on ³HNE release; thus this latter effect appears to be independent of enhanced prostaglandin formation. The proposed alpha receptor mediated negative feedback exhibits stereospecificity since addition of exogenous 1-, but not d-, NE decreased release of the transmitter. Blockade of alpha receptors by phentolamine or stimulation of beta receptors by isoproterenol significantly enhanced the K induced release of ³HNE from cortical tissue. By contrast, the d-A induced release of ³HNE which occurs from reserpine-insensitive sites, was reduced by propranolol and clonidine; and was not altered by phentolamine, isoproterenol or PGE₂. These data indicate that the K, but no d-A, induced release of ³HNE from cortical tissue is modified in accordance with postulated presynaptic negative and positive feedback mechanisms.