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P Ronner

Publications and source records attributed to P Ronner.

18 recordsLinked to original sources
PubMed2001

Effects of glucose and amino acids on free ADP in betaHC9 insulin-secreting cells.

Stimulation of insulin release by glucose is widely thought to be coupled to a decrease in the activity of ATP-sensitive K+ channels (KATP channels) that is caused by a decreased concentration of free ADP. To date, most other investigators have reported only on total cellular ADP concentrations, even though only a small fraction of all ADP is free and only the free ADP affects KATP channels. We tested the hypothesis that amino acids elicit insulin release via a decrease in the activity of KATP channels owing to a decrease in the level of free ADP. We estimated the concentration of free ADP in betaHC9 hyperplastic insulin-secreting cells based on the cell diameter and on luminometric measurements of ATP, phosphocreatine, and total creatine. The concentration of free ADP fell exponentially as the concentration of glucose increased. A physiological mixture of amino acids greatly stimulated insulin release at 0-30 mmol/l glucose but affected the concentration of free ADP only to a minor degree and significantly so only at < or = 2 mmol/l glucose. In the presence of 2-deoxyglucose and NaN3, amino acids were unable to stimulate insulin release. When KATP channels were held open with diazoxide (and the plasma membrane partially depolarized with high extracellular KCl), amino acids still stimulated insulin release. We conclude that amino acid-induced insulin release depends on two components: a yet-unknown amino acid sensor and KATP channels, which serve to attenuate hormone release when cellular energy stores are low. We propose that glucose-induced insulin release may be regulated similarly by two components: glucokinase and KATP channels.

Adenosine Diphosphate↗
PubMed1999

Luminometric assays of ATP, phosphocreatine, and creatine for estimation of free ADP and free AMP.

We present methods to measure ATP, phosphocreatine, and total creatine (the sum of creatine and phosphocreatine) in alkaline cell extracts. Knowledge of these parameters, together with the known equilibrium constants for the creatine kinase and adenylate kinase-catalyzed reactions, allows one to estimate the levels of free ADP and free AMP inside cells. The enzymatic assays for the above-mentioned metabolites all lead up to the production of ATP, which is measured luminometrically with the ATP-dependent oxidation of luciferin catalyzed by firefly luciferase. To determine phosphocreatine, endogenous ATP is first destroyed, and phosphocreatine is then quantitatively reacted with exogenous ADP to form ATP. Total creatine is measured after quantitative conversion of creatine to phosphocreatine with a large excess of exogenous ATP, conversion of all ATP to ADP, and final reaction of phosphocreatine with ADP to form ATP. We used 5-microl samples in 0.5-ml microcentrifuge tubes and subsequent 5-microl additions of analytical reagents. We expect that the volumes can be changed easily. We tested the methods with glucagon- and insulin-secreting cells. Estimates of free ADP and AMP are expected to be useful in many different areas of research, such as cellular energy metabolism, purine nucleotide metabolism, adenine nucleotide gating of ion channels, and release of vasoactive or angiogenic factors.

Adenosine Diphosphate↗
PubMed1993

Sulfonylurea-binding sites and ATP-sensitive K+ channels in alpha-TC glucagonoma and beta-TC insulinoma cells.

alpha-Cells secrete glucagon in a fuel-dependent fashion. We tested the hypothesis that alpha-cells contain sulfonylurea- and ATP-sensitive K+ channels. We studied two clonal lines of alpha-TC cells (simian virus 40 T-antigen induced glucagonoma cells) and for reference purposes, similarly transformed beta-TC insulinoma cells. alpha-TC cells each contained approximately 3000 high-affinity binding sites for the sulfonylurea [3H]glyburide. Whole-cell ATP- and tolbutamide-sensitive K+ currents of alpha-TC and beta-TC cells, relative to cell surface area, were comparable. In cell-attached membrane patches of alpha-TC cells, two types of K+ channels were observed. They had slope conductances of approximately 63 and 33 pS when the electrode contained 151 mM K+. Tolbutamide and diazoxide decreased and enhanced, respectively, the open probability of these channels. The membrane of alpha-TC cells depolarized periodically. This electrical activity was inhibited by diazoxide. A physiological mixture of amino acids enhanced glucagon release, and high glucose partially inhibited this release. Tolbutamide also enhanced glucagon release, whereas diazoxide inhibited it. Thus, alpha-TC glucagonoma cells contain ATP-sensitive K+ channels that regulate glucagon release, yet allow inhibition of hormone release by glucose.

Adenosine Triphosphate↗
PubMed1992

Effect of the hypoglycaemic drug (-)-AZ-DF-265 on ATP-sensitive potassium channels in rat pancreatic beta-cells.

1. It has previously been shown that the hypoglycaemic drug (-)-AZ-DF-265 stimulates insulin release from mouse islets; in the presence of 3 mM glucose it also inhibits 86Rb-efflux from 86Rb-loaded islets. Based on these data we tested the hypothesis that (-)-AZ-DF-265 inhibits ATP-sensitive potassium channels. 2. We voltage-clamped the plasma membrane of single rat pancreatic beta-cells in the whole-cell configuration and measured the current flowing through ATP-sensitive potassium channels. (-)-AZ-DF-265 was applied to the outside of the cell; it inhibited the current half-maximally at a concentration of 1.2 +/- 0.2 nM with a Hill coefficient of 0.7 +/- 0.1. The inhibition was reversible on washing. 3. In intact RINm5F cells, the sulphonylurea [3H]-glibenclamide bound with an affinity of 0.24 +/- 0.01 nM and a Hill coefficient of 2.1 +/- 0.4. Treatment with (-)-AZ-DF-265 led to the displacement of [3H]-glibenclamide; this effect was half-maximal at 8.6 +/- 1.7 nM and displayed a Hill coefficient of 0.53 +/- 0.01. Meglitinide and tolbutamide, which represent, respectively, the benzamido and sulphonylurea moieties of glibenclamide, showed Hill coefficients of 0.8 +/- 0.1 and 0.9 +/- 0.1, respectively. 4. At pH 7.4 and with phosphate/borate buffer, (-)-AZ-DF-265 had an apparent octanol/water partition coefficient of about 53, which is intermediate between the partition coefficients of glibenclamide and glipizide. Nevertheless, (-)-AZ-DF-265 bound only to a minor degree to glass and plastic.5. In conclusion, (-)-AZ-DF-265 inhibits ATP-sensitive potassium channels and displaces [3H]-glibenclamide from the sulphonylurea receptor.

Adenosine Triphosphate↗
PubMed1991

The role of ATP and free ADP in metabolic coupling during fuel-stimulated insulin release from islet beta-cells in the isolated perfused rat pancreas.

The isolated perfused rat pancreas was used to test the hypothesis that total cellular ATP or the ratio of ATP/free ADP plays the primary role in coupling intermediary metabolism to the biophysical events that are the basis of glucose-stimulated insulin release. The pancreas was preperfused for 20 min with 4.0 mM of a physiological mixture of 20 amino acids plus 4.2 mM glucose, and insulin release was then stimulated for 150 s by suddenly increasing the glucose to 8.3 mM. The pancreas was sampled at 24, 48, 72, and 150 s after the switch. The content of total ATP, ADP, AMP, Pi, phosphocreatine, and creatine were measured in beta-cell enriched cores of pancreatic islets microdissected from freeze-dried pancreas cryostat sections. Metabolites were measured by quantitative histochemical enzymatic cycling techniques. Modeling studies were carried out to assess the impact of biochemical analytical results on the membrane potential of the beta-cells. The level of free ADP was calculated using the creatine kinase equilibrium reaction and an intracellular pH of 7.2. First phase insulin release was stimulated at least 10-fold with the maximum reached 45 s after adding high glucose. The biochemical analytical data demonstrate that the total cellular level of the putative coupling factor ATP and of the ratios ATP/free ADP and ATP/free ADP x Pi are not significantly influenced by a glucose level change that causes a more than 10-fold surge of insulin release. The strength and limitations of the present experimental strategy and the implications of the results for our understanding of metabolic coupling in glucose-stimulated insulin release are discussed.

Adenosine Diphosphate↗
PubMed1991

2-Deoxyglucose stimulates the release of insulin and somatostatin from the perfused catfish pancreas.

Glucokinase was proposed to function as a glucose sensor in pancreatic B-cells, acting possibly as a pacemaker of the rate of glycolysis. Glucose, mannose, and 2-deoxyglucose are good substrates of glucokinase which are easily taken up into B-cells. Glucose and mannose are well-known stimuli of insulin release in mammals and fish. I report here that 2-deoxyglucose is also a strong stimulus of insulin and somatostatin release from the in vitro perfused pancreas (i.e., splenic Brockmann body) of channel catfish (Ictalurus punctatus). This is surprising because the product of the glucokinase-catalyzed phosphorylation of 2-deoxyglucose. 2-deoxyglucose-6-phosphate, cannot be metabolized further at an appreciable rate. 3-O-Methylglucose, which does not bind appreciably to mammalian glucokinase, stimulated neither insulin nor somatostatin release. Glucosamine, which binds tightly to glucokinase but is phosphorylated only at a very low rate, did not stimulate insulin release either, but did cause a small amount of somatostatin to be released. The results suggest that glucose-activated glucokinase itself may serve as a signal molecule in glucose recognition by B- and D-cells.

Animals↗
PubMed1991

Effect of linogliride on hormone release from perfused rat pancreas. Fuel dependence and desensitization by tolbutamide.

We examined the effect of the hypoglycemic drug linogliride on hormone release from the in vitro perfused rat pancreas. Linogliride stimulated insulin release in the absence of glucose either in the presence or absence of a physiological mixture of amino acids. In addition, linogliride inhibited amino acid-induced glucagon release. Half-maximal effects of linogliride on insulin and glucagon release were achieved at concentrations as low as 26 and 3 microM, respectively. The effects of linogliride on hormone release largely resembled those of tolbutamide. In the absence of amino acids, the stimulation of insulin release by linogliride or tolbutamide was transient. When the pancreas had been preperfused for 20 min with tolbutamide, linogliride no longer had an effect on hormone release. Likewise, tolbutamide remained without effect in pancreases preperfused with linogliride. These data suggest that linogliride and tolbutamide may have a similar mechanism of action.

Amino Acids↗
PubMed1991

Inhibition of ATP-sensitive K+ channels in pancreatic beta-cells by nonsulfonylurea drug linogliride.

Linogliride is a nonsulfonylurea drug that lowers blood glucose levels in nondiabetic and diabetic humans and animals. Linogliride also stimulates insulin release in vitro. In the perfused pancreas, pretreatment with tolbutamide desensitizes beta-cells to the action of linogliride. We tested the hypothesis that linogliride, like tolbutamide, affects the activity of ATP-sensitive K+ channels, which are thought to control insulin release. We used the whole-cell voltage-clamping technique to measure the K+ current through ATP-sensitive K+ channels in the plasma membrane of single rat beta-cells, which were dialyzed with 30 microM ATP. Linogliride (10-300 microM) inhibited the K+ current; half-maximal inhibition was observed at 6-25 microM, depending on how much time was allowed for equilibration of the drug. Reversal of the inhibition was slow (t1/2 approximately 4 min). In summary, linogliride leads to a decrease in the activity of ATP-sensitive K+ channels.

Adenosine Triphosphate↗
PubMed1987

Secretagogues for pancreatic hormone release in the channel catfish (Ictalurus punctatus).

We investigated the effect of several potential carbohydrate secretagogues, amino acids, a ketoacid, and potassium chloride on insulin, glucagon, and somatostatin release from the in vitro perfused Brockmann body of channel catfish (Ictalurus punctatus). Mannose (15 mM) stimulated the release of insulin and somatostatin. Fructose (30 mM) induced only a small and transient release of somatostatin. Galactose (15 mM) was not a secretagogue. Likewise, glyceraldehyde failed to stimulate hormone release. Among the amino acids newly tested, alanine and leucine, and also alpha-ketoisocaproic acid were without effect. A high concentration of potassium (25 mEq/liter) induced a pronounced release of insulin and glucagon and a moderate release of somatostatin. In conclusion, a striking similarity exists between catfish and higher vertebrates in their pancreatic endocrine response to hexoses; on the other hand, the catfish Brockmann body appears to respond only to a few of the common stimuli of pancreatic hormone release in mammals.

Amino Acids↗
PubMed1986

Transmembrane ion distribution and insulin secretion.

The sodium dependence of insulin release was studied with the in vitro perfused principal islet of channel catfish. When extracellular sodium was replaced with choline, glucose-induced insulin release and glucose enhancement of arginine-induced insulin release were selectively abolished. Lowering the extracellular pH from 7.6 to 7.1 had a similar selective effect on glucose-induced insulin release. It is suggested that the Na:H antiport is responsible for the sodium dependence of insulin release and that a lowered cytosolic pH preferentially inhibits the glucose sensing mechanism.

Animals↗
PubMed1985

Isolation and structures of glucagon and glucagon-like peptide from catfish pancreas.

Both glucagon and the structurally similar glucagon-like peptide proteolytically derived from preproglucagon were purified from the endocrine pancreas of the channel catfish (Ictalurus punctata). This study represents the first report of the isolation of glucagon-like peptide from any source. Peptide sequences of glucagon-like peptide from other species have only been deduced from the cDNA sequences for preproglucagon. The sequence of the 34-residue glucagon-like peptide was found to be HADGTYTSDVSSYLQDQAAKDFITWLKSGQPKPE. Catfish glucagon-like peptide shares sequence identity at 26 of 31 residues with the putative glucagon-like peptide from anglerfish preproglucagon II. The mass of catfish glucagon-like peptide was found by fast atom bombardment-mass spectrometry to be 3785, identical with the value predicted by sequence analysis. This suggests that no post-translational modification occurs beyond proteolytic processing. The sequence of catfish glucagon was determined to be HSEGTFSNDYSKYLETRRAQDFVQWLM(N,S). Catfish glucagon exhibits a high degree of immunologic similarity with porcine glucagon by radioimmunoassay, whereas catfish glucagon-like peptide does not.

Adenylyl Cyclases↗
PubMed1984

Difference in glucose dependency of insulin and somatostatin release.

The splenic Brockmann body of channel catfish (Ictalurus punctatus) was isolated and perfused in vitro. It has been shown previously that the hormone release from the catfish pancreas in response to various stimuli resembled that of a mammalian pancreas. The release of insulin and somatostatin was measured at various concentrations of glucose in the perfusate, and the corresponding dose-response curves were derived. As in a variety of mammalian pancreases, insulin release was stimulated half-maximally at ca. 9 mM glucose. In contrast, half-maximal somatostatin release occurred already at ca. 5 mM glucose. Although a comparable finding with mammalian D cells has not yet been reported, histological and physiological resemblances of the mammalian and the fish pancreas suggest that mammalian D cells may also have a glucose sensitivity in the 5 mM range.

Animals↗
PubMed1982

Isolated perfused Brockmann body as a model for studying pancreatic endocrine secretion.

The release of insulin, glucagon, and somatostatin was investigated in a newly developed in vitro perfusion preparation of the splenic Brockmann body (principal pancreatic islet) of the channel catfish (Ictalurus punctatus). The preparation was viable for up to 7 h by functional and morphological criteria. Glucose evoked a biphasic release of insulin and somatostatin, but had no effect on the release of glucagon. Arginine at basal glucose concentration caused a marked release of insulin and glucagon, but only a minor release of somatostatin.

Animals↗
PubMed1975

A hydrophobic form of the small-intestinal sucrase-isomaltase complex.

A large scale preparation of brush border membranes is described. Solubilized by either papain or Triton X-100, the sucrase-isomaltase complex is purified in a three-step procedure, including differential centrifugation, Sephadex G-200 and DEAE-cellulose chromatography. Detergent solubilized and protease solubilized sucrase-isomaltase differ in tendency to aggregate but not in enzymatic characteristics. The chemical composition and the molecular weight of the two enzyme complexes are almost identical. Limited digestion of the Triton-solubilized sucrase-isomaltase complex by papain produces a protein electrophoretically indistinguishable from papain-solubilized sucrase-isomaltase together with low molecular proteolytic fragments.

Amino Acids↗