Modulation by a sulfonylurea of insulin-dependent glycogenesis, but not of insulin binding, in cultured rat hepatocytes. Evidence for a postreceptor mechanism of action.

To detect potential direct effects of the sulfonylurea glyburide on hepatic carbohydrate metabolism, we tested whether the drug was capable of modulating insulin binding and glycogenesis in primary cultured hepatocytes. After 24-h culture under serum- and hormone-free conditions, cells were incubated with or without 10(-8) M insulin and/or glyburide (0.1-5.0 micrograms/ml) for another 24 h. Then, specific 125I-insulin binding and basal and insulin-stimulated glycogen synthesis were determined. Acute addition of glyburide to previously untreated cells did not modulate any of these parameters. Incubation for 24 h with 2 micrograms/ml of glyburide did not affect the DNA and protein content of the dishes. Cellular glycogen content and basal glycogenesis also remained unchanged by glyburide in hepatocytes incubated in the absence of insulin, but glycogen content was increased and basal glycogen synthesis decreased in insulin-pretreated cells. In contrast, glyburide increased insulin-stimulated glycogenesis in a dose-dependent fashion in both insulin-pretreated and control cells by enhancing responsiveness, but not sensitivity, toward insulin. Pretreating hepatocytes with 10(-8) M insulin caused a 40% reduction in specific insulin binding. Glyburide did not modulate insulin binding or degradation in control cells nor was insulin-induced regulation of insulin receptors affected. These results demonstrate a direct dose-dependent effect of a sulfonylurea on an insulin action toward hepatic carbohydrate metabolism, and suggest that this effect is mediated by a postreceptor mechanism.

Potency of biosynthetic human insulin determined in vitro.

Biosynthetic human insulin (BHI) obtained from separately synthesized A- and B-chains by recombinant DNA technology with Escherichia coli fermentation was compared with human and pork insulin of high purity in vitro. Applying four biologic tests (glucose oxidation and glucose incorporation into the lipids by rat epididymal fat pads, inhibition of lipolysis, and ATP depletion of isolated fat cells) and three receptor assays (binding competition with human fat cells, IM-9 lymphocytes, and rat liver cell plasma membranes), we could not discern significant differences of the half-maximum response by these seven methods. The only variance occurred with the ATP-depletion assay. This method disclosed 10% greater maximum reversion of isoproterenol-induced ATP depletion by BHI when compared with pork insulin.

The human insulin analog insulin lispro improves insulin binding on circulating monocytes of intensively treated insulin-dependent diabetes mellitus patients.

The rapidly absorbed analog of human insulin, insulin lispro (LP), is characterized by a faster onset of action, a higher peak insulin level, and a shorter duration of action compared with regular insulin (RI). The aim of this study was to investigate whether intensified treatment with either LP or RI influences insulin receptor status. Twelve patients with insulin-dependent diabetes mellitus (IDDM) participating in a multicenter randomized cross-over trial were allocated to this study. Four patients began with LP, whereas eight patients started with RI. Each patient was switched to the other insulin after a 3-month treatment period. Competitive [125I]A-14-insulin binding studies were performed with isolated monocytes. Treatment with insulin lispro increased the total number of insulin binding sites from 9,400 +/- 2,200 (RI) to 20,300 +/- 3,000 (LP)/monocyte (P < 0.001). The insulin concentration required for a 50% competition of [125I]insulin binding (IC50) decreased from 0.6 +/- 0.2 (RI) to 0.1 +/- 0.03 (LP) nmol/L, indicating significantly higher affinity of insulin binding sites during LP treatment (P < 0.001). In additional experiments, the time course of insulin binding was determined after an oral meal. In LP-treated IDDM patients, the affinity and capacity of insulin binding showed a nadir 1 h after insulin injection and a regained binding affinity and capacity 5 h later. These changes observed after LP treatment were comparable to the effect of endogenous insulin secretion in healthy control subjects. In contrast, the IDDM patients who injected RI showed a decreasing insulin binding affinity and capacity, most markedly expressed after 5 h. The corresponding serum levels of insulin were inversely correlated with the affinity and capacity of insulin-binding sites. Pretreatment of cultured human IM-9 lymphoblasts with LP or RI yielded no difference in the down-regulation of insulin binding. In summary, intensified conventional insulin therapy with LP increased the number and affinity of insulin receptors on circulating monocytes to a level similar to that observed in healthy subjects. We conclude that the improved insulin receptor status observed during LP treatment is caused by its more physiological pharmacokinetic profile.

A novel method for continuous online glucose monitoring in humans: the comparative microdialysis technique.

The aim of this study was to prove the feasibility of continuous subcutaneous glucose monitoring in humans using the comparative microdialysis technique (CMT). The performance of the CMT was determined by comparing tissue glucose values with venous or capillary blood glucose values in healthy volunteers and type 1 diabetic subjects. The CMT is a microdialysis-based system for continuous online glucose monitoring in humans. This technique does not require calibration by the patient. Physiological saline with glucose (5.5 mM) is pumped in a stop-flow mode through a microdialysis probe inserted into the abdominal s.c. tissue. Tissue glucose concentration is calculated by comparing the dialysate and perfusate glucose concentrations. The time delay due to the measurement process is 9 min. We tested the CMT on six healthy volunteers and six type 1 diabetic patients for 24 h in our clinical setting. Comparisons were made to HemoCue analyzer (Angelholm, Sweden) capillary blood glucose measurements (healthy volunteers) and to venous blood glucose concentration determined with a Hitachi analyzer (diabetic patients). The mean absolute relative error of the CMT glucose values from the blood glucose values was 17.8+/-15.5% (n = 167) for the healthy volunteers and 11.0+/-10.8% (n = 425) for the diabetic patients. The mean difference was 0.42+/-1.06 mM (healthy volunteers) and -0.17+/-1.22 mM (diabetic patients). Error grid analysis for the values obtained in diabetic patients demonstrated that 99% of CMT glucose values were within clinically acceptable regions (regions A and B of the Clarke Error Grid). The study results show that the CMT is an accurate technique for continuous online glucose monitoring.

Lethal exitus of a patient with N-nitrosodimethylamine poisoning, 2.5 years following the first ingestion and signs of intoxication.

The report describes the lethal exitus of a 44-year-old woman after repeated criminal poisoning with N-nitrosodimethylamine. She died 32 months following the initial symptoms and developed a liver cirrhosis with multiple bleeding, a hemolytic syndrome and diabetes mellitus.

High affinity binding sites for proinsulin in human IM-9 lymphoblasts.

Proinsulin and insulin binding in IM-9 lymphoblasts show curvilinear Scatchard plots, which may be explained by two binding sites, negative cooperativity of receptors, or both. Using flow-cytometric analysis of insulin binding, we were able to distinguish and separate two different IM-9 cell fractions. In both fractions, Scatchard plots for specific binding of insulin and proinsulin were linear, suggesting the presence of two distinct populations of receptors. Type 1 cells showed low capacity but high affinity of insulin binding (16,300 +/- 3,000 sites/cell; Kd 0.4 +/- 0.1 nmol/l). Proinsulin and insulin-like growth factor 1 (IGF-1) were significantly less potent in competition. MA-20, a specific antibody against human insulin receptors, inhibited insulin binding by 80%, while the specific antibody against human IGF-1 receptors, alpha IR-3, had no effect. Pretreatment with insulin decreased insulin binding by 90%. 125I-insulin displayed stepwise dissociation with the rate markedly enhanced by cold insulin. Type 2 cells exhibited significantly different binding characteristics with higher capacity but lower affinity of 125I-insulin binding (430,000 +/- 25,000 sites/cell, p < 0.001 vs type 1; Kd 2 +/- 0.4 nmol/l, p < 0.02 vs type 1). Proinsulin competed with similar potency for insulin binding, while IGF-1 was still less potent. 125I-proinsulin showed a significantly higher binding affinity than 125I-insulin (Kd 0.5 +/- 0.3 nmol/l, p < 0.05) with 50,000 +/- 10,000 binding sites/cell. C-peptide was able to compete for 125I-proinsulin, but not for 125I-insulin binding. MA-20 did not influence 125I-proinsulin binding, but inhibited 125I-insulin binding by 50%, whereas alpha IR-3 increased proinsulin binding 1.5-fold with no effect on insulin binding. Preincubation with insulin decreased insulin binding by 50% and proinsulin binding by 10%. The dissociation of 125I-proinsulin showed linear first-order kinetics and was not significantly accelerated by cold proinsulin. Furthermore, the tyrosine phosphorylation of a 65 kDa protein was stimulated to a significantly greater extent by proinsulin than by insulin, indicating activation of different signalling cascades. DNA analysis revealed that type 1 cells were predominantly in the G1 phase, whereas type 2 cells were in the S and G2 + M phases of the cell cycle. We conclude, that IM-9 lymphoblasts were separated by flow-cytometry into one fraction with typical insulin receptors and a second fraction with high affinity binding sites for proinsulin. High affinity proinsulin binding sites were distinguished from typical insulin receptors by: 1) higher affinity for proinsulin than insulin, 2) inhibition of proinsulin binding by C-peptide but not by the insulin receptor antibody MA-20, 3) non-co-operative first order dissociation kinetics of proinsulin binding, 4) resistance to down-regulation by insulin, and 5) differences in signal transduction.

[A new model for the in vitro study of the entero-island of Langerhans axis]. / Un nuovo modello per lo studio in vitro dell'asse entro-insula re.

A new in vitro pancreas-intestine model which makes it possible to study the physiopathology of the intero-insular axis in greater depth, as it eliminates interference from other system, is proposed. The model, which is composed of the rat pancreas, duodenum and the first 25 cm of the small intestine, conserves both the circulation and the in vivo anatomical reltionship intact. Experiments were designed to evaluate the effect of a drug-induced disaccharidase-activity block on insulin secretion. The results obtained confirm the validity of the proposed model in investigating the entero-insular axis.

Glucagon-like peptide-1 improves insulin and proinsulin binding on RINm5F cells and human monocytes.

Glucagon-like peptide-1-(7---36) amide (GLP-1) is a potent incretin hormone secreted from distal gut. It stimulates basal and glucose-induced insulin secretion and proinsulin gene expression. The present study tested the hypothesis that GLP-1 may modulate insulin receptor binding. RINm5F rat insulinoma cells were incubated with GLP-1 (0.01-100 nM) for different periods (1 min-24 h). Insulin receptor binding was assessed by competitive ligand binding studies. In addition, we investigated the effect of GLP-1 on insulin receptor binding on monocytes isolated from type 1 and type 2 diabetes patients and healthy volunteers. In RINm5F cells, GLP-1 increased the capacity and affinity of insulin binding in a time- and concentration-dependent manner. The GLP-1 receptor agonist exendin-4 showed similar effects, whereas the receptor antagonist exendin-(9---39) amide inhibited the GLP-1-induced increase in insulin receptor binding. The GLP-1 effect was potentiated by the adenylyl cyclase activator forskolin and the stable cAMP analog Sp-5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3', 5'-monophosphorothioate but was antagonized by the intracellular Ca(2+) chelator 1,2-bis(0-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. Glucagon, gastric inhibitory peptide (GIP), and GIP-(1---30) did not affect insulin binding. In isolated monocytes, 24 h incubation with 100 nM GLP-1 significantly (P<0.05) increased the diminished number of high-capacity/low-affinity insulin binding sites per cell in type 1 diabetics (9,000+/-3,200 vs. 18,500+/-3,600) and in type 2 diabetics (15,700+/-2,100 vs. 28,900+/-1,800) compared with nondiabetic control subjects (25,100+/-2,700 vs. 26,200+/-4,200). Based on our previous experiments in IEC-6 cells and IM-9 lymphoblasts indicating that the low-affinity/high-capacity insulin binding sites may be more specific for proinsulin (Jehle, PM, Fussgaenger RD, Angelus NK, Jungwirth RJ, Saile B, and Lutz MP. Am J Physiol Endocrinol Metab 276: E262-E268, 1999 and Jehle, PM, Lutz MP, and Fussgaenger RD. Diabetologia 39: 421-432, 1996), we further investigated the effect of GLP-1 on proinsulin binding in RINm5F cells and monocytes. In both cell types, GLP-1 induced a significant increase in proinsulin binding. We conclude that, in RINm5F cells and in isolated human monocytes, GLP-1 specifically increases the number of high-capacity insulin binding sites that may be functional proinsulin receptors.

[Typing of the alpha adrenergic receptors of pancreatic B cells. In vitro studies with the isolated and perfused rat pancreas]. / Tipizzazione dei recettori alpha-adrenergici della cellula b insulare. Studio in vitro con il pancreas isolato e perfuso di ratto.

Two groups of receptors, one which develops a stimulating effect (alpha 1), the other an inhibitory effect hae recently been isolated in the alpha adrenergic group. In order to type the B cell adrenergic receptor of the endocrine pancreas, which mediates the inhibitory action exerted by the catecholamines on insulin secretion, the release of this hormone was evaluated in the presence of five alpha simpathomimetic substances that have a decreasing degree of efficiency on the adrenergic alpha 2 receptor of the presynaptic sympathic nerve terminal. The order of potency with which the alpha agonists tested depressed IRI secretion is superimposable on that of their potency on the sympathetic nerve and alpha 2 receptor. We concluded that adrenergic inhibition of insulin secretion is mediated by an alpha 2 receptor.

Proinsulin stimulates growth of small intestinal crypt-like cells acting via specific receptors.

The mechanisms that regulate cell turnover in the intestinal epithelium are incompletely understood. Here we tested the hypothesis that proinsulin, present in serum and pancreatic juice in picomolar concentrations, stimulates growth of the rat small intestinal crypt-like cell line IEC-6 under serum-free conditions. Proinsulin binding was assessed by competitive ligand binding studies. Proinsulin and insulin-like growth factor I (IGF-I) stimulated cell proliferation up to threefold above controls, with half-maximal action already in the picomolar range and with additive effects. In early confluent cell monolayers, proinsulin bound with higher affinity (IC50 1.3 +/- 0.05 nM) and capacity (87,200 +/- 2,500 receptors/cell) than IGF-I (4.0 +/- 0.6; 23,700 +/- 2,200, P < 0. 05). C-peptide competed with 10-fold lower affinity for binding of 125I-proinsulin but not for 125I-IGF-I or 125I-insulin, suggesting a specific binding epitope of the proinsulin molecule within or close to the C-peptide region. In contrast, insulin showed approximately 100-fold lower binding affinity and growth-promoting potency than proinsulin or IGF-I. We conclude that proinsulin stimulates growth of small intestinal crypt cells through specific binding and may play a physiological role in the regulation of intestinal epithelial cell proliferation.

Differential autocrine regulation of intestine epithelial cell proliferation and differentiation by insulin-like growth factor (IGF) system components.

The mechanisms which regulate cell turnover in the intestinal epithelium are incompletely understood. The present study was performed to characterize the role of autocrine IGF system components in intestine epithelial cell proliferation and differentiation comparing rapidly growing crypt cells (IEC-6) with differentiating enterocytes (CaCo-2). The autocrine release of IGF-I, IGF-II and IGFBP-1 through -3 was determined by specific RIAs and western ligand blotting. In addition, binding and growth-promoting activity of insulin, IGF-I and IGF-II was investigated. Enterocytic differentiation was assessed by measuring the brush-border enzymes alkaline phosphatase and sucrase. During IEC-6 growth, the autocrine release of IGF-I and -II increased, whereas IGFBP-2 levels decreased. Specific receptors for IGF-I and IGF-II but not insulin could be detected. IGF-I was 100-fold more potent than insulin to stimulate IEC-6 cell proliferation. In contrast, CaCo-2 cells revealed higher binding of insulin than IGF-I/-II and no release of IGF-I. At switch from CaCo-2 cell proliferation to differentiation a marked increase in the secretion of IGF-II (10-fold), IGFBP-1 (2.5-fold), IGFBP-2 (3-fold), and IGFBP-3 (6-fold) was measured. Our data indicate that IGF system components differentially modulate enterocytic cell proliferation and differentiation.