2, 3-Diphosphoglycerate fluctuations in erythrocytes reflecting pronounced blood glucose variation

Summary No significant differences were found in the erythrocyte 2.3-DPG concentration between 14 normals (16.82±0.66 moles 2.3-DPG/g Hb) and 44 diabetic patients (16.22±0.38 moles 2.3-DPG/g Hb). However, in diabetic patients we could demonstrate significant fluctuations determined by the metabolic control of their diabetes. Hyperglycaemic patients (n = 10) developed during treatment, concomitant with declining blood glucose, a significant decrease to 13.97± 0.64 [mioles 2.3-DPG/g Hb. After normalization of blood glucose the 2.3-DPG level rose again. Two patients with islet cell tumors had a fluctuation in the 2.3-DPG concentration of about 20%, when symptomatic hypoglycaemia occurred during an extended fast. This variation in 2.3-DPG dependent upon changes in blood glucose was also demonstrated in-vitro by a dialysis technique where glucose was kept constant at 400 or 80 mg/100 ml. Incubating hyperglycaemic blood (n = 6) of uncontrolled diabetics in a high glucose medium, 2.3-DPG was constant over 7 h, whereas at low glucose concentration 2.3-DPG dropped significantly (p < 0.001). Blood from nondiabetic subjects did not show this phenomenon. In-vitro additions of insulin and tolbutamide failed to produce an effect on 2.3-DPG. Our results suggest that pronounced fluctuations of blood glucose in diabetics influence 2.3-DPG levels in erythrocytes and thus might impair peripheral oxygen supply.Abbreviations 2.3-DPG 2.3-Diphosphoglycerate - Hb Haemoglobin - ATP Adenosine triphosphate - NADH -Diphosphopyridin Nucleotide reduced - Tris Tris (hydroxy methyl) aminomethane - EDTA Ethylendiamine tetraacetic acid     

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance

Summary Plasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca²⁺]_i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca²⁺]_i, lasting 2–3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca²⁺]_i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca²⁺]_i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca²⁺]_i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca²⁺]_i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.Abbreviations [Ca²⁺]_i Cytoplasmic free Ca²⁺ - NIDDM non-insulin-dependent diabetes mellitus - DMSO dimethylsulphoxide - PC pancreatic cancer     

Variations in synalbumin insulin antagonism during tolbutamide tolerance test

Summary Albumin was extracted from plasma and serum taken during intravenous Tolbutamide tolerance tests in 18 healthy volunteers and tested at a concentration of 2.5 g per 100 ml for insulin antagonism. A significant rise in insulin antagonism was observed after 30 and 60 min, falling to the initial level at 90 min following Tolbutamide injection. These changes were inversely related to the corresponding blood sugar levels.

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Schwankungen des Synalbumin-Insulin-Antagonismus während des Tolbutamid Toleranz-Tests

Zusammenfassung Bei 18 stoffwechselgesunden Freiwilligen wurde Albumin aus während eines Tolbutamid-Tests entnommenen Plasma- und Serumproben extrahiert und in einer Konzentration von 2,5 g% auf Insulin-Antagonismus getestet. Nach 30 und 60 min fand sich ein signifikanter Anstieg des Insulin-Antagonismus und 90 min nach der Tolbutamidgabe ein Abfall auf die Ausgangswerte. Die Veränderungen verhielten sich umgekehrt proportional zu den jeweiligen Blutzuckerspiegeln.

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Variations dans l'antagonisme de la synalbumine vis-à-vis de l'insuline au cours de tests de tolérance au tolbutamide

Résumé L'albumine a été extraite du plasma et du sérum prélevé au cours de tests de tolérance au tolbutamide intraveineux chez 18 volontaires en bonne santé, et testée à une concentration de 2.5 g pour 100 ml pour y déceler l'antagonisme à l'insuline. Une augmentation significative de l'antagonisme à l'insuline a été observée après 30 et 60 min, le taux initial était atteint 90 min après l'injection de tolbutamide. Ces modifications étaient inversement proportionnelles aux taux correspondants de la glycémie.

     

Action of some hypoglycaemic sulphonylureas on the oxygen consumption of isolated pancreatic islets of mice

Summary Cartesian divers were used to evaluate the effects in vitro of some hypoglycaemic Sulphonylureas on the oxygen consumption of isolated pancreatic islets. The islet specimens were obtained from obese-hyper-glycaemic mice, and consisted of over 90% B-cells. When incubated with Krebs-Ringer phosphate medium, the islet cells displayed an increased rate of respiration upon addition to the incubation medium of either tolbutamide (D860; 0.1 mg/ml) or glibenclamide (HB 419; 0.1 .g/ ml). The respiratory rate increased with the concentration of HB 419 in the range 0.001–0.1 g/ml, but did not exceed 120% of the respiration in pure phosphate medium. Whereas the physiological excretion product of D 860 did not affect the respiratory rate, the corresponding derivative of HB 419 was still effective in stimulating the oxygen uptake of the islets. When islets were incubated with glucose at a high concentration (3 mg/ml), the oxygen uptake was inhibited by addition of D 860, or its metabolite, or HB 419. The last drug slightly increased the respiration of islets incubated with glucose at a concentration of 1 mg/ml, and a marked stimulation was noted at a still lower glucose concentration, 0.5 mg/ml. Attempts to evaluate the effect of mannoheptulose on the respiratory response of the islets to hypoglycaemic Sulphonylureas produced inconclusive results. It is suggested that Sulphonylureas effect an increased rate of endogenous substrate oxidation in the B-cells of the pancreatic islets.This work was supported by the Swedish Medical Research Council (B68-12X-109-04) and the U.S.Public Health Service (Grant AM-05759-06). The skilful technical assistance of Miss Gunilla Lekselius and Miss Ing-Britt Brolen is greatfully acknowledged.     

The stimulus-secretion coupling of glucose-induced insulin release

Summary Above a threshold of 3.0–4.2 mmol/l, D-glucose provoked a transient increase in ³²P fractional outflow rate from rat pancreatic islets prelabelled with ³²P-orthophosphate. Nutrients which stimulate insulin release in the absence of glucose, -ketoisocaproate and L-leucine, also provoked a phosphate flush. No flush occurred in islets exposed to non-insulinotropic nutrients (L-glutamine and L-lactate) or non-nutrient secretagogues (arginine, tolbutamide, theophylline). A late increase in ³²P fractional outflow rate was observed in Ca²⁺ deprived islets stimulated with BaCl₂ and theophylline. The occurrence of a phosphate flush did not appear to be attributable to changes in insulin release, cyclic AMP content, membrane polarisation, K⁺ conductance, or reduced pyridine nucleotide content. The ³²P response to glucose was slightly decreased in the absence of extracellular Ca²⁺ or HCO₃ ^-, markedly impaired in the absence of K⁺, and virtually abolished in the presence of menadione (10 mol/l). It is proposed that the occurrence of a phosphate flush is linked to the metabolism of nutrient secretagogues, possibly via an increase in O₂ uptake and the production rate of NAD(P)H and ATP.     

Pharmacokinetic interference of doxorubicin with tolbutamide due to reduced metabolic clearance with increased serum unbound fraction in rats

The study examined the effect of doxorubicin (DOX) on the hepatic expression of CYP2C and its activity for metabolizing tolbutamide (TB), a specific CYP2C substrate, in rats and whether the pharmacokinetics of tolbutamide were altered by doxorubicin exposure. The expression level of hepatic CYP2C11 was depressed 1 day after doxorubicin administration (day 1), and this effect on CYP2C11 was augmented on day 4. However, the expression level of hepatic CYP2C6 remained unchanged. The activity of tolbutamide 4‐hydroxylation in hepatic microsomes was decreased with time following doxorubicin administration. Regarding the enzyme kinetic parameters for tolbutamide 4‐hydroxylation on day 4, the maximum velocity (V_max) was significantly lower in the DOX group than that in the control group, while the Michaelis constant (K_m) was unaffected. On pharmacokinetic examination, the total clearance (CL_tot) of tolbutamide on day 4 was increased, despite the decreased metabolic capacity. On the other hand, the serum unbound fraction (f_u) of tolbutamide was elevated with a reduced serum albumin concentration in the DOX group. Contrary to CL_tot, CL_tot/f_u, a parameter approximated to the hepatic intrinsic clearance of unbound tolbutamide, was estimated to be significantly reduced in the DOX group. These findings indicate that the metabolic capacity of CYP2C11 in the liver is depressed time‐dependently by down‐regulation after doxorubicin exposure in rats, and that the decreased enzyme activity of TB 4‐hydroxylation in hepatic microsomes reflects the pharmacokinetic change of unbound tolbutamide, not total tolbutamide, in serum.     

The significance of the blood glucose level for plasma insulin response to intravenously administered tolbutamide in healthy subjects

Summary The effect of intravenous tolbutamide on insulin release in normal human subjects was investigated under various experimental conditions. The blood glucose level was either allowed to fall after i.v. tolbutamide or kept within normal limits by a concomitant glucose infusion. In other experiments, tolbutamide was given during different degrees of hypoglycaemia induced by insulin. It was found that tolbutamide provoked a rapid and short-lasting insulin release as well as a post-initial and extended insulin release, provided the blood glucose concentration was kept within normal limits. The hitherto accepted transiency of tolbutamide action in healthy subjects seems to be due to the hypoglycaemia which follows the administration of the drug. During more marked hypoglycaemia induced by exogenous insulin, the insulin releasing capacity of tolbutamide was almost blunted. Tolbutamide markedly enhanced the insulin release following glucose administration. The findings presented might clarify some of the therapeutic effects of the drug in diabetes mellitus.This study was supported by grants to E.C. and R.L. from the Swedish Medical Research Council (B69-19X-34-05A) and the Knut and Alice Wallenberg Foundation.     

Insulin sensitivity,insulin secretion and glucose effectiveness in diabetic and non-diabetic cirrhotic patients

Summary In cirrhotic patients with normal fasting glucose levels both insulin insensitivity and a blunted early insulin response to oral glucose are important determinants of the degree of intolerance to oral glucose. It is not known whether the ability of hyperglycaemia per se to enhance glucose disposal (glucose effectiveness) is also impaired. It is also unclear whether overt diabetes is due to (1) more marked insulin insensitivity; (2) impaired insulin secretion; (3) reduced glucose effectiveness; or (4) a combination of these mechanisms. We used the minimal model to analyse the results of a 3-h intravenous glucose tolerance test to assess glucose effectiveness, insulin sensitivity and insulin responses in 12 non-diabetic cirrhotic patients, 8 diabetic cirrhotic patients and 10 normal control subjects. Fasting blood glucose levels were 4.8±0.2, 7.5±0.6 and 4.7±0.1 mmol/l, respectively. Fasting insulin and C-peptide levels were higher in both cirrhotic patient groups compared with control subjects. The glucose clearance between 6 and 19 min after i.v. glucose was lower in both cirrhotic groups (non-diabetic, 1.56±0.14, diabetic, 0.76±0.06, control subjects, 2.49±0.16 min^–1%, both p<0.001 vs control subjects). Serum insulin peaked at 3 and 23 min in the non-diabetic cirrhotic patients and control subjects; both peaks were higher in the non-diabetic cirrhotic patients and showed a delayed return to basal levels. In the diabetic cirrhotic patients, the first phase insulin and C-peptide response to i.v. glucose was absent; their early (22–27 min) incremental insulin response to i. v. tolbutamide was however similar to that of control subjects but 43% lower than in the non-diabetic cirrhotic patients (p<0.05). Insulin sensitivity was markedly reduced in both cirrhotic groups (non-diabetic, 1.11±0.24×10^–4, diabetic, 0.33±0.53×10^–4, control subjects, 4.37±0.53×10^–4 min^–1 per mU·l^–1, both p<0.001 vs controls). Glucose effectiveness was normal in the non-diabetic cirrhotic patients but 29% lower in the diabetic group. It would appear that overt diabetes develops in those cirrhotic patients who in addition to insulin insensitivity have a marked impairment of insulin secretion. An associated reduction in glucose effectiveness may be a contributory factor.     

Pharmacological study of a new hypoglycaemic sulphonamide: Glisoxepid (RP 22410)

Summary Glisoxepid or RP 22410 is a new very active hypoglycaemic sulphonylurea. In the normal conscious dog, RP 22410 administered intravenously was 81 or 131 times more active than tolbutamide, depending on whether the dose is expressed in grams or in moles. The hypoglycaemic effect did not occur in the totally pancreatectomized dog. — RP 22410 stimulated insulin secretion.In vivo in the anaesthetized or conscious dog, the action of the drug (whether it be administered intravenously or orally) resulted in a rapid and considerable increase of the amount of insulin secreted by the pancreas. This action lasted several hours.In vitro the direct action of the product on the pancreas was demonstrated on the isolated and perfused rat pancreas, even at very low concentrations. — In the mouse, prolonged oral administration of RP 22410 stimulated neogenesis of the islets of Langerhans and of the beta cells. It therefore possesses betacytotrophic action.

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Pharmakologische Studie über das neue blutzuckersenkende Sulfonamid Glisoxepid RP 22410

Zusammenfassung Glisoxepid oder RP 22410 ist ein neuer, stark aktiver, blutzuckersenkender Sulfonylharnstoff. Je nachdem ob die Dosen in Gewichten oder Mol ausgedrückt werden, ist er am wachen Hund bei intravenöser Applikation 81 bzw. 131mal aktiver als Tolbutamid. Die blutzuckersenkende Wirkung zeigt sich nicht am vollständig pankreatektomierten Hund. -Diese Substanz stimuliert die Insulinsekretion.In vivo zeigt sich ihre Wirkung am anästhesierten oder wachen Hund in einer schnellen und beträchtlichen Erhöhung der vom Pankreas sezernierten Insulinmenge unabhängig davon ob sie intravenös oderper os verabreicht wird. Diese Wirkung bleibt über mehrere Stunden bestehen.In vitro wurde die direkte Wirkung der Substanz auf den isolierten und perfundierten Pankreas der Ratte auch in sehr geringen Konzentrationen nachgewiesen. -Bei der Maus stimuliert RP 22410 bei chronischer Verabreichungper os die Neubildung von Langerhansschen Inseln und B-Zellen. Sie besitzt daher eine betacytotrophe Wirkung.

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Etude pharmacologique d'un nouveau sulfamide hypoglycémiant: le glisoxepid (RP 22410)

Résumé Le glisoxepid ou RP 22410 est un nouveau sulfonylurée hypoglycémiant très actif. Chez le chien normal éveillé, administré par voie intraveineuse, il est 81 ou 131 fois plus actif que le tolbutamide, suivant que les doses sont exprimées en poids ou en moles. Cette action hypoglycémiante ne se manifeste pas chez le chien totalement dépancréaté. — Ce produit stimule la sécrétion d'insuline.In vivo chez le chien anesthésié ou éveillé, l'action du produit (que celui-ci soit administré par voie intraveineuse ou par voie digestive) se traduit par une augmentation rapide et considérable de la quantité d'insuline sécrétée par le pancréas. Cette action se prolonge pendant plusieurs heures.In vitro l'action directe du produit sur le pancréas a été démontrée sur le pancréas isolé et perfusé du rat, même à très faibles concentrations. — Chez la souris le RP 22410 administré chroniquementper os stimule la néogénèse des îlots de Langerhans et des cellules bêta. Il est donc doué de l'action bêtacytotrophe.