Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase

HTC rat hepatoma cells were transfected with human insulin receptor cDNA to a level of 40,000 receptors/cell. In these cells, as well as in nontransfected cells, insulin stimulated the uptake of alpha-aminoisobutyric acid. Two monoclonal antibodies directed against the human insulin receptor alpha subunit, like insulin, stimulated amino acid uptake in transfected HTC cells, but not in nontransfected HTC cells. The antibodies, in contrast to insulin, failed to stimulate insulin receptor tyrosine kinase activity, both in intact transfected cells and in cell free extracts prepared from them. These data suggest, therefore, that activation of insulin receptor tyrosine kinase may not be an obligatory step in all of the transmembrane signaling mechanisms of the insulin receptor.     

Modulation of gastrin-releasing peptide (GRP) receptors in insulin secreting cells

Gastrin-releasing peptide (GRP) receptors are present in pancreatic islets, though their regulation is unknown except for homologous desensitization. The modulation of binding of GRP to mouse pancreatic islets and INS-1 cells was studied. At 60 min (steady-state), total binding of [(125)I-Tyr(15)] GRP was 1.62 per cent of total radioactivity per 50 islets; non-specific binding (presence of 1 mM unlabelled GRP(1-27)) was 0.05 to 0.61 per cent of total radioactivity. A preincubation with 1000 nM cholecystokinin (CCK(8)) or with 1000 nM glucose-dependent insulinotropic peptide (GIP) augmented the number of GRP binding sites but not their affinity. [(125)I-Tyr(15)]GRP binding to INS-1 cells was saturable (90 min) and specific with respect to compounds that are not chemically related to GRP (e.g. calcitonin gene-regulated peptide-CGRP and atrial natriuretic peptide-ANP). Displacement studies showed one binding site with a K(d) of 0.39 nM and a B(max) of 13.2 fmoles mg(-1) protein. When the cells were pretreated for 24 h with 10 nM GIP or CCK(8), only GIP but not CCK(8) increased the B(max) of the GRP binding site. The affinity (K(d)) was not changed by either compound. This effect of GIP pretreatment was not affected by downregulating PKC by TPA (phorbol ester; long-term pretreatment). These data indicate that: (1) specific binding sites for GRP are present in mouse pancreatic islets and INS-1 cells; (2) the GRP binding is upregulated by GIP in both islets and INS-1 cells and additionally by CCK(8 ), albeit only in islets; and (3) PKC does not seem to be involved in the up-regulation process. Thus a positive interplay between both the incretins GIP and CCK(8) and the neurotransmitter GRP is obvious.     

Biological effects of newly synthesized cholecystokinin analogs

Cholecystokinin (CCK) is a gut hormone that regulates pancreatic endocrine functions via CCK(A) receptors. CCK(4) (Trp-Met-Asp-Phe-NH(2)) has an insulinotropic effect, but is 1,000-fold less potent than CCK(8) in rodents. The in vitro potencies with respect to binding, the biological effects and the selectivity of newly synthesized CCK(4) analogs constructed by computer modelling experiments were investigated in vitro in rat pancreas and brain, INS-1 cells, and guinea pig ileum. Exchanging various amino acids, e.g. Met by either Pro or Nle, and modifying Phe by adding various substituents in different positions led to compounds which were more effective as insulin secretagogues than CCK(4) itself and even show insulinotropic effects comparable with those of CCK(8) (e. g. compounds M1 and M2 being substituted at Phe). Some compounds which possess electron withdrawing groups on the C-terminal Phe and possess a Pro instead of a Met were especially effective. The CCK(A) receptor antagonist L-364,718, but not by the CCK(B) receptor antagonist L-365,260, inhibited the insulinotropic effects. The synthetic CCK(4) compounds were not selective for the endocrine pancreas: e.g. M1 and M2 had binding activity with respect to rat brain homogenates but no activity with respect to contraction of the guinea pig ileum. The data indicate that some of the newly synthesized CCK tetrapeptides exhibit a high affinity for the CCK receptor of beta-cells and have an insulinotropic effect much higher than CCK(4).     

Modulation of insulin release by adenosine A1 receptor agonists and antagonists in INS-1 cells: the possible contribution of 86Rb+ efflux and 45Ca2+ uptake

Due to the lack of specific agonists and antagonists the role of adenosine receptor subtypes with respect to their effect on the insulin secretory system is not well investigated. The A1 receptor may be linked to different 2nd messenger systems, i.e. cAMP, K+- and 45Ca2+ channel activity. Partial A1 receptor agonists are going to be developed in order to improve diabetes (increase in insulin sensitivity, lowering of FFA and triglycerides). In this study newly synthesized selective A1 receptor agonists and antagonists were investigated thereby integrating three parameters, insulin release (RIA), 45Ca2+ uptake and 86Rb+ efflux (surrogate for K+ efflux) of INS-1 cells, an insulin secretory cell line. The presence of A1-receptors was demonstrated by Western blotting. The receptor nonselective adenosine analogue NECA (5-N-ethylcarboxyamidoadenosine) at high concentration (10 microM) had no effect on insulin release and 45Ca2+ uptake which could be interpreted as the sum of effects mediated by mutual antagonistic adenosine receptor subtypes. However, an inhibitory effect mediated by A1 receptor agonism was detected at 10 nM NECA and could be confirmed by adding the A1 receptor antagonist PSB-36 (1-butyl-8-(3-noradamantyl)-3-(3-hydroxy-propyl)xanthine). NECA inhibited 86Rb+ efflux which, however, did not fit with the simultaneous inhibition of insulin secretion. The selective A1 receptor agonist CHA (N6-cyclohexyladenosine) inhibited insulin release; the simultaneously increased Ca2+ uptake (nifedipine dependent) and inhibition of 86Rb+ efflux did not fit the insulin release data. The CHA effect (even the maximum effect at 50 microM) can be increased by 10 microM NECA indicating that CHA and NECA have nonspecific and physiologically non-relevant effects on 86Rb+ efflux in addition to their A1-receptor interaction. Since PSB-36 did not influence the NECA-induced inhibition of 86Rb+ efflux, the NECA effect is not mediated by potassium channel-linked A1 receptors. The nonselective adenosine receptor antagonist caffeine increased insulin release which was reversed by CHA as expected when hypothesizing that both act via A1 receptors in this case. In conclusion, stimulation of A1 receptors by receptor selective and nonselective compounds reduced insulin release which is not coupled to opening of potassium channels (86Rb+ efflux experiments) or inhibition of calcium channels (45Ca2+ uptake experiments). It may be expected that of all pleiotropic 2nd messengers, the cAMP system (not tested here) is predominant for A1 receptor effects and the channel systems (K+ and Ca2+) are of minor importance and do not contribute to insulin release though being coupled to the receptor in other tissues.     

Antispasmodic activity of an extract from Plantago lanceolata L. and some isolated compounds

An ethanolic spissum extract of the aerial parts of Plantago lanceolata L. was examined for antispasmodic activity on isolated ileum and trachea of the guinea-pig. Isolated constituents were investigated as well. The P. lanceolata extract inhibited the contractions of the guinea-pig ileum that were induced by various agonists such as acetylcholine (ACh), histamine, potassium and barium ions. Additionally the trachea contractions induced by barium ions were inhibited. The compounds luteolin, acteoside, plantamajoside an catalpol peracetate but not catalpol, isoacteoside, lavandulifolioside and aucubin inhibited the ACh-induced contractions of the guinea-pig ileum. Luteolin and acteoside reduced the barium-induced contractions of the guinea-pig trachea. Two recently isolated compounds did not show antispasmodic activity: luteolin-3',7-diglucuronide and beta-hydroxy-acteoside.     

Local formation of angiotensin peptides with paracrine activity by adipocytes

A local paracrine angiotensin (ANG) system influences the insulin sensitivity and cell differentiation of adipose tissue. The limited view of a merely systemic renin‐angiotensin‐aldosterone‐system with ANG II (1–8) as the main mediator of ANG‐related effects may oversimplify the situation. The aim was to analyze the degradation of ANG by using capillary electrophoresis (CE) techniques. The supernatant of cultured 3T3‐L1 adipocytes was used directly, and some data on degraded peptides were combined with a biological effect. The formation of several peptides such as ANG II (1–8), —III (2–8), —IV (3–8), and ANG (1–7) as degradation products is demonstrated; in addition low levels of ANG (3–7) are identified. The concentrations of the peptides ANG III (2–8) and ANG IV (3–8) (both are AT₄ receptor agonists) are modified in the vicinity of adipose tissue cells by amino‐terminal degradation which resulted in ANG (3–8), —(4–8) and —(5–8). ANG IV (3–8) and ANG II (1–8) were biologically highly effective in inhibiting IRAP (insulin regulated aminopeptidase, part of the AT₄ receptor). It is observed that ANG (1–7) is the main degradation product derived from ANG I via ANG (1–9) and that ANG III (2–8) is one important regulated peptide for IRAP. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Significance of Ca2+, Rb+ fluxes, of cAMP and cGMP for the CCK8-modulated insulin release

In rat pancreatic islets the effects of cholecystokinin-8 (CCK8) on glucose-mediated insulin release, 45Ca2+ net uptake, 45Ca2+ efflux, 86Rb+ efflux, cAMP- and cGMP levels were studied. In the presence of a substimulatory glucose concentration (3 mM) CCK8 concentrations of up to 1 microM had no effect on insulin release, but CCK8 at 10 nM potentiated the stimulatory effect of glucose (11.1 mM). 10 nM CCK8 enhanced glucose-stimulated 45Ca2+ net uptake but was ineffective at substimulatory glucose levels. CCK8 had no effect on cAMP and cGMP levels in the presence of 11.1 mM glucose, CCK8 increased 86Rb+ (a measure of K+) in the presence of both 3 and 11.1 mM glucose. This effect was abolished when Ca2+ was omitted from the perifusion medium. CCK8 did not alter glucose (11.1 mM)-stimulated 45Ca2+ efflux rate. These data indicate that (1) CCK8 potentiates glucose-stimulated insulin secretion possibly via an effect on Ca2+ uptake, 2) by affecting Ca2+ uptake, CCK8 enhances K+ efflux, and 3) CCK8 does not mediate its effect via cAMP or cGMP. With respect to 86Rb+ efflux the mechanism of CCK8 action appears to be different from that of glucose. When the mechanism of CCK action on islets is compared with that on exocrine pancreas (data from others) there are similarities (importance of Ca2+ uptake and non-importance of cAMP and cGMP).     

The role of old age in the effects of glucose on insulin secretion, pentosephosphate shunt activity, pyridine nucleotides and glutathione of rat pancreatic islets

In pancreatic islets of adult (three month) and old (24 month) rats the effect of glucose on glucose oxidation, pyridine nucleotides, glutathione and insulin secretion was studied. DNA content was similar in both groups of animals; however, islets of old rats exhibited 30% less insulin content. While glucose-induced (16.7 mM) insulin secretion in islets of old rats was approximately 50% less than in islets of adults, no significant difference was observed in the insulin releasing effect of theophylline (1 mM). Although islet production of 14CO2 in the presence of 16.7 mM glucose increased equally in both groups, elevation of glucose failed to increase the percentage of total glucose oxidation via the pentose phosphate shunt in islets of old rats. Elevation of glucose increased the NADPH/NADP and the NADH/NAD ratio in both groups of islets in a similar manner. The effect of glucose on the GSH/GSSG ratio revealed a dose-related increase in the islets of adult rats, whereas islets of old rats did not respond to elevation of glucose. Our data seem to indicate that the lower secretory response of islets of old rats is related to the failure of glucose to increase the GSH/GSSG ratio. In contrast the insulin release induced by theophylline does not appear to depend on islet thiols.