Inhibition of intestinal amino acid transport by blood sugar lowering biguanides

Summary The effect of blood-glucose lowering biguanides (phenethyl- and butylbiguanide) on active intestinal transport of different amino acids has been tested in hamster small intestinein vitro. —.Biguanides inhibited active transport of all amino acids tested. The inhibitory effect of biguanides increased with incubation time, was more pronounced after preincubation of intestinal tissue and was found to be non-competitive. The minimal inhibitory concentration of phenethylbiguanide on amino acid transport was 5×10^–4 M. —¹⁴C-butylbiguanide was found to be transported into hamster small intestine by a concentration-independent, energy-independent uptake mechanism and was accumulated in intestinal tissue against a concentration gradient. — In accord with earlier results on the inhibitory effect of biguanides on active intestinal hexose transport it is concluded that biguanides do not act as specific inhibitors for glucose transport, but rather affect active, energy-requiring intestinal transport mechanisms in general (hexose-, amino acid-, calcium- and myo-inositol transport), most likely due to their known inhibitory effect on mitochondrial respiration, thus depriving mucosal cells of ATP required to translocate substrates against a concentration gradient.Part of this work has been presented at the International Diabetes Congress, Buenos Aires, Argentina, August 1970 and at the Meeting of German and Italian Pharmacology Society, Heidelberg, September 1970.     

Inhibition of bile salt absorption by blood-sugar lowering biguanides

Summary The effect of blood sugar lowering biguanides (phenethyl-, butyl- and dimethylbiguanide) upon jejunal and ileal transport of bile salts (tauro- and glycocholate) was tested in rat small intestine by an in vitro technique. Biguanides inhibited active transport of bile salts in the ileum, but did not affect diffusional absorption of bile salts in the jejunum. The inhibitory effect was time-dependent and not reversible under in vitro incubation conditions, suggesting that biguanides must enter intestinal mucosal cells in order to exert their inhibitory action on active transport of glucose analogues, amino acids, calcium and bile salts. Since biguanides achieve high tissue concentrations in the small intestine even after parenteral administration, inhibition of ileal bile salt reabsorption by biguanides could possibly explain the lipid- and cholesterol-lowering effect of these oral antidiabetic drugs.     

Die Wirkung von N1,n-Butylbiguanid auf den Stoffwechsel der isolierten perfundierten Leber normaler und alloxandiabetischer ketotischer Ratten

Zusammenfassung An isolierten perfundierten Lebern von normalen und von alloxandiabetischen ketotischen Ratten wurde der Effekt von N₁,n-Butylbiguanid auf den Leberstoffwechsel untersucht. — N₁,n-Butylbiguanid in einer Konzentration von 2.55 · 10^–5 M im Perfusionsmedium hatte folgende Effekte. — 1. Die Aminosäurebilanz normaler und diabetischer Lebern wurde in positiver Richtung verändert. Die in Kontrollversuchen feststellbare kontinuierliche Nettoabgabe von -Aminosäuren nahm unter Butylbiguanid signifikant ab oder es kam sogar zu einer Nettoaufnahme von -Aminosäuren. — 2. Die Nettoabgabe von anorganischem Phosphat durch normale und diabetische Lebern wurde durch Butylbiguanid signifikant vermindert. — 3. Die erhöhte Nettoharnstoffabgabe diabetischer Lebern wurde durch Butylbiguanid signifikant vermindert. Die Harnstoffbilanz normaler Lebern blieb unbeeinflußt. — 4. Die Nettoabgabe von Kalium durch diabetische Lebern wurde durch Butylbiguanid signifikant vermindert. — 5. Der in Experimenten mit diabetischen Lebern erhöhte Lactat/Pyruvat-Quotient im Perfusionsmedium normalisierte sich bei Butylbiguanidzusatz rascher. — 6. Die Gallebildung normaler und diabetischer Lebern ist unter Butylbiguanid gesteigert. Die Steigerung ist in den Experimenten mit diabetischen Lebern signifikant. — N₁,n-Butylbiguanid in einer Konzentration von 2.55 · 10^–5 M hatte keinen signifikanten Effekt auf die Nettoglucosebilanz, die Aufnahme unveresterter Fettsäuren, die Ketonkörperbildung, die Leberdurchblutung und die Änderungen des Leberglykogengehaltes in Experimenten mit normalen und diabetischen Lebern. — N₁,n-Butylbiguanid in einer 4-fach höheren Konzentration (1.02 · 10^–4 M) hatte in Experimenten mit normalen Lebern folgende Wirkung: 1. Der positive Effekt der kleineren Biguanidkonzentration (2.55 · 10^–5 M) auf die Aminosäurebilanz war nicht mehr nachweisbar. — 2. Die Lactatkonzentrationen im Medium waren deutlich erhöht. — 3. Der Lactat/Pyruvat-Quotient im Medium lag deutlich höher als in den Kontrollexperimenten. — Bei einer Butylbiguanidkonzentration von 2.55 · 10^–5 M befand sich das zugegebene Biguanid ausschließlich im Plasmaanteil des Perfusionsmediums. Bei einer Butylbiguanidkonzentration von 1.02 · 10^–4 M war der Verteilungsraum des Butylbiguanids größer, als dem Plasmavolumen entsprach. Weder bei Verwendung der niedrigen, noch bei Verwendung der hohen Biguanidkonzentration trat ein meßbarer Konzentrationsabfall im Medium während der dreistündigen Perfusion ein. Die Biguanidausscheidung mit der Galle war minimal.

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The effect of N₁,n-butylbiguanide on the metabolism of isolated perfused livers of normal and alloxan-diabetic ketotic rats

Summary The effect of N₁, n-butylbiguanide on the metabolism of isolated perfused livers of normal and alloxan-diabetic ketotic rats was studied. — N₁, n-butylbiguanide in a concentration of 2.55 · 10^–5 M in the perfusion medium had the following effects: 1. The amino acid balance of normal and diabetic livers was altered in a positive direction. The continuous net output of -amino acids seen in control experiments decreased significantly under butylbiguanide and a net uptake of -amino acids took place. 2. The net output of inorganic phosphate by normal and diabetic livers was significantly diminished by butylbiguanide. 3. The elevated net output of urea by diabetic livers decreased significantly under butylbiguanide. The urea balance of normal livers remained unchanged. 4. The net output of potassium by diabetic livers was significantly diminished by butylbiguanide. 5. The lactate/ pyruvate-ratio, which was elevated in experiments with diabetic livers became normalized more rapidly in presence of butylbiguanide. 6. Bile production of normal and diabetic livers was stimulated by butylbiguanide. This stimulation was significant in experiments with diabetic livers. — In experiments with normal and diabetic livers N₁,n-butylbiguanide in a concentration of 2.55 · 10^–5 M did not have any significant effect on the glucose balance, the uptake of non-esterified fatty acids, the production of ketone bodies, the liver blood flow, or liver glycogen content. — N₁,n-butylbiguanide at a concentration four times greater (1.02 · 10^–4 M) had the following effects in experiments with normal livers: 1. The positive effect of the smaller biguanide concentration (2.55 · 10^–5 M) on the amino acid balance could not be demonstrated with the higher concentration. 2. The concentration of lactate in the medium showed a significant increase. 3. The lactate/pyruvate-ratio was significantly higher than in control experiments. — At a concentration of 1.02 · 10^–4 M the equilibration volume for butylbiguanide was greater than the plasma volume. A measurable decrease in the medium concentration of butylbiguanide did not occur during the 3 hours of perfusion with either a low or a high concentration. The biguanide excretion with bile was minimal.

Teilergebnisse wurden auf dem II. Internat. Symposion über Diabetesfragen (1963) vorgetragen (s. Mohnike, G., ed., II. Internat. Sympos. über Diabetesfragen, S. 221ff., Karlsburg b. Greifswald).     

Paradoxical effects of endogenous and exogenous insulin on amino acid transport activity in the isolated rat pancreas: somatostatin-14 inhibits insulin action

Summary Regulatory effects of insulin, somatostatin and cholecystokinin on amino acid transport in the isolated perfused rat pancreas have been studied using a rapid dual isotope dilution technique. Uni-directional L-serine transport (15 s) was quantified relative to an extracellular tracer D-mannitol over a wide range of substrate concentrations. In pancreata perfused with 2.5 mmol/l D-glucose, a weighted nonlinear regression analysis of overall transport indicated an apparent K_m=14.4±1.6 mmol/l and V_max=25.9±1.4 mol ·min^–1·g^–1 (n=6). Although L-serine transport was stimulated during perfusion with 100 U/ml bovine insulin, endogenous insulin (7–25 ng·min^–1·g^–1) released during continuous perfusion with either 8.8 mmol/l or 16.8 mmol/l D-glucose had no such effect. Exogenous somatostatin-14 (250 pg/ml) or cholecystokinin octapeptide (CCK-8, 3 × 10^–11mol/l) appeared to increase only the K_m for transport. Only CCK-8 evoked a notable protein output (2.9±0.3 mg·30min^–1·g^–1) and juice flow (68±10l·30min^–1·g ^–1, n=3) from the exocrine pancreas. When pancreata were perfused with bovine insulin (100 U/ml) and somatostatin-14 (250 pg/ml), the stimulatory action of exogenous insulin on L-serine transport was abolished. If endogenous insulin and somatostatin, released concurrently in response to 16.8 mmol/l D-glucose, were conveyed to the exocrine epithelium via an islet-acinar portalaxis, it is conceivable that somatostatin modulates the stimulatory action of insulin on basolateral amino acid transport in the exocrine pancreas.     

Inhibition of intestinal absorption and improvement of oral glucose tolerance by biguanides in the normal and in the streptozotocin-diabetic rat

Summary In the rat the assumption of a primary effect of moderate doses of biguanides on intestinal absorption is supported by the following results: 1. The tolerance to parenteral glucose administration was not improved. — 2. Afteroral pretreatment the hyperglycaemia induced byoral glucose was decreased. — 3. After pretreatmentin vivo the transport of glucose through the intestinal wall was inhibited. — 4. The intestinal glucose transport was inhibited alsoin vitro in the presence of biguanides at concentrations to be expected in intestinal tissuein vivo after efficient doses. — 5. In the streptozoocin-diabetic rat there was a strong correlation between the feeding state and the hypoglycaemie effect of biguanides. In streptozotocin-diabetic rats previously starved and then refed, the hypoglycaemie effect decreased with decreasing doses of the diabetogenic agent. — 6. The relative potency of various biguanides was similar with regard to their effects on hyperglycaemia and on the intestinal glucose transport. — The results suggest, however, that in the rat further events, probably triggered by the inhibition of intestinal absorption, are involved in the mechanism of action of biguanides.

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Hemmung der intestinalen Glucoseresorption und Verbesserung der oralen Glucosetoleranz durch Biguanide bei normalen und Streptozotocin-diabetischen Ratten

Zusammenfassung Folgende Befunde lassen darauf schließen, daß Biguanide in mäßiger Dosierung bei der Ratte primär die intestinale Absorption beeinflussen: 1. Die parenterale Glucosetoleranz wurde nicht verbessert. — 2. Nachoraler Vorbehandlung wurde die durchorale Glucosegabe induzierte Hyperglykämie vermindert. — 3. NachIn-vivo-Vorbehandlung war der Glucosetransport durch die Darmwand gehemmt. — 4. Der intestinale Glucosetransport wurde auchin vitro in Gegenwart von Biguanid-Konzentrationen gehemmt, die man im intestinalen Gewebe nach Applikation wirksamer Dosen erwarten kann. — 5. In der Streptozotocin-diabetischen Ratte bestand eine strenge Korrelation zwischen Fütterungszust and und hypoglykämischem Effekt. In wiedergefütterten Streptozotocin-Ratten nahm die hypoglykämische Wirkung mit abnehmenden Dosen des diabetogenen Agens ab. — 6. Die relative Wirkungsstärke verschiedener Biguanide bezüglich Verminderung der Hyperglykämie und Hemmung der intestinalen Glucoseaufnahme war vergleichbar. — Die erhobenen Befunde deuten jedoch darauf hin, daß in der Ratte zusätzliche Vorgänge, die möglicherweise durch die Absorptionshemmung ausgelöst werden, am Wirkungsmechanismus der Biguanide wesentlich beteiligt sind.

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Inhibition de l'absorption intestinale et amélioration de la tolérance orale au glucose avec des biguanides chez le rat normal et chez le rat rendu diabétique par le streptozotocine

Résumé Les constatations suivantes permettent la conclusion que l'application de faibles doses de biguanides influence chez le rat premièrement l'absorption intestinale: 1. Aucune amélioration de la tolérance parentérale au glucose. 2. Après un prétraitementoral l'hyperglycémie induite par l'administrationorale de glucose est diminuée. — 3. Après un prétraitementin vivo le transport de glucose par la paroi intestinale est freiné. — 4. Des concentrations de biguanides que l'on pourrait rencontrerin vivo dans le tissu intestinal (après traitement avec des dosages efficaces) provoque également une inhibition du transport de glucose dans l'intestinin vitro. — 5. Chez le rat rendu diabétique par streptozotocine il existe une forte corrélation entre l'alimentation et l'effet hypoglycémique. Chez le rat réalimenté l'effet hypoglycémique diminue après l'administration de doses décroissantes de l'agent diabétogène. — 6. La puissance relative de divers biguanides par rapport à la diminution de l'hypoglycémie et l'inhibition du transport intestinal de glucose peut être comparée. — Les résultats trouvés chez le rat font cependant allusion au fait que l'inhibition de l'absorption intestinale n'est pas la seule action des biguanides. Il semble qu'il y a encore d'autres facteurs reliés à cette inhibition.

     

Glucose regulates both glucose transport and the glucose transporter gene expression in a hamster-derived pancreatic Beta-cell line (HIT)

Summary We studied the effect of chronic exposure to high glucose on the glucose transport regulation in hamster pancreatic Beta cells in permanent culture (HIT). Cells were exposed to either 5.5 mmol/l or 16.7 mmol/l glucose for 48 h and then glucose transport was studied by measuring the (³H)-2-deoxyglucose uptake for 5 and 10 min at 37 °C. The 2- deoxyglucose uptake was lower in cells pre-exposed to glucose 16.7 mmol/l for 48 h compared to cells pre-exposed to glucose 5.5 (12.0±1.6 vs 19.1±1.2 nmol/0.1 mg after 5 min, and 22.2±2.6 vs 39.0±2.9 after 10 min respectively, mean ±SEM, n=5, p < 0.01). In order to investigate the mechanism(s) for glucose impairment of glucose transport, we studied the glucose carrier gene expression in the same cells by Northern and slot-blot analysis. When total RNA was extracted from HIT cells cultured at either 5.5 or 16.7 mmol/l glucose and then hybridized to ³²P-labelled cDNA probes for the glucose transporter 1, the glucose transporter 2 and -actin, a significant reduction of both glucose transporter 1 (–63.9±4.1%, mean±SEM, n=3) and glucose transporter 2 (–48.9±3.2%) mRNA was observed in HIT cells cultured with high glucose. In the same experiments no change of -actin mRNA was observed, suggesting that the effect of high glucose was specific on the glucose-transporter mRNAs. In HIT cells cultured at glucose 16.7 mmol/l the glucose-induced insulin release was also reduced compared to cells cultured at glucose 5.5 (715±19 U · h^–1 · mg^–1 vs 1301±28 U · h^–1 · mg^–1, respectively, mean ±SEM, n=3, p < 0.05). In conclusion, in hamster pancreatic Beta-cells, chronic exposure to high glucose concentrations impairs glucose transporter mRNA levels, glucose transport, and glucose-induced insulin secretion in a co-ordinate manner. Note: After this paper was submitted we became aware of two recent publications [9, 10] showing in animal models of non-insulin-dependent diabetes a decreased expression of the Beta-cell glucose transporter in pancreatic islets unresponsive to glucose, further supporting the view that this transporter may play a role in the glucosesensing mechanism.     

Enhancement of glucose transport by insulin at 37 °C in rat adipocytes is accounted for by increased Vmax

Summary The kinetics of 3-O-methyl-D-glucose and D-glucose transport was reinvestigated at 37 °C in rat adipocytes exhibiting very slow transport rates in the absence of insulin and approximately 30-fold insulin stimulation. The apparent maximal velocity for 3-O-methylglucose was increased at least 15-fold from 0.04±0.01 to 0.73±0.17 mmol ·1^–1·s^–1 (SD, n=7). The apparent half saturation constant was decreased by insulin from 8.1±1.6 mmol/l to 3.3±0.8 mmol/l. However, approximately 10% of the 3-O-methylglucose at a very low concentration was transferred by routes other than the glucose transporter, e. g. via nonmediated diffusion or via the fructose transporter, and the estimated value for the half saturation constant was therefore too high in the nonstimulated cells. After appropriate correction this value was estimated as 5.0±1.8 mmol/l. The inhibition constant of 3-O-methylglucose on the conversion of 12 mol/l ¹⁴C-labelled glucose to cell-associated products was about 4 mmol/l and was not changed significantly by insulin. Transport experiments with labelled glucose gave results similar to those obtained for transport of 3-O-methylglucose. It is concluded that insulin can cause a 15- to 30-fold increase in the maximal velocity for transport of 3-O-methylglucose and glucose, and that a concomitant decrease in the half saturation constant, if present, is insignificant.     

Sulphonylurea stimulates glucose uptake in rats through an ATP-sensitive K+ channel dependent mechanism

Summary We studied the effect of gliclazide, a second-generation sulphonylurea, on rat skeletal muscle glucose uptake using perfused hindquarter muscle preparations. Gliclazide at concentrations of 10 to 1000 g/ml increased (p<0.05) the basal glucose uptake. The effect of gliclazide on glucose uptake was immediate and dose-dependent, reaching a plateau at a concentration of 300 g/ml; the half-maximal effect was obtained between 25 and 50 g/ml. The glucose uptake stimulated by gliclazide (300–1000 g/ ml) did not differ from that achieved by 10^–9 mol/l insulin, and was lower (p<0.05) than that obtained with 10^–7 mol/l insulin. The combination of gliclazide (300 g/ml) and 10^–9 mol/l insulin produced an increase in glucose uptake (7.7±0.6 mol · g^–1 · h^–1, n=8, mean±SEM) which was higher (p<0.05) than that achieved with 10^–9 mol/l insulin (5.6±0.7 mol · g^–1 · h^–1, n=11) and not different from that obtained with 10^–7 mol/l insulin (9.8±1.0 mol · g^–1 · h^–1, n=11). Diazoxide (100 mol/l), an ATP-sensitive K⁺ channel opener, reversed the stimulatory effect of gliclazide (100 g/ml) on muscle glucose uptake from 3.1±0.4 to 0.5±0.2 mol · g^–1 · h^–1, (n=7, p<0.001). The addition of diazoxide prior to gliclazide into the perfusion medium blocked the gliclazide-induced glucose uptake by the hindquarter muscle preparations. In conclusion, gliclazide alone has an immediate stimulatory effect on glucose uptake by skeletal muscle and together with insulin has an additive effect on muscle glucose uptake. The effect of gliclazide on muscle glucose uptake seems to be due to the inhibition of ATP-sensitive K⁺ channels.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - GLUT glucose transporter     

Effect of human recombinant Endostatin protein on human angiogenesis

Tumor growth and metastasis are dependent on the development of new blood vessels. Inhibitors of new vessel growth have been widely investigated as anti-tumor agents. Endostatin, a 20 kDa C-terminal fragment of collagen XVIII inhibits endothelial cell proliferation, induces endothelial cell apoptosis, and can both inhibit and reverse tumor growth in mice. However, human recombinant endostatin has had limited testing against human tissue targets. To investigate the effect of human endostatin on a human vessel target over a broad range of concentrations (10^–12–10^–4 M), human placental vein disks were grown for a period of 2 weeks in a 0.3% fibrin clot overlayed with growth medium. Disks from five individual placentas were tested. For each placenta utilized, a control (medium and 20% fetal bovine serum [FBS]) group and a group treated with heparin (300 g/ml) and hydrocortisone 21-phosphate (350 g/ml) (heparin-steroid) at a dose known to inhibit angiogenesis were included. Endostatin was tested at concentrations of 10^–12–10^–4 M in medium containing 20% FBS. The rate of initiation and the angiogenic growth index (on a visually graded semi-quantitative scale of 0–16) were determined for all experimental conditions. Endostatin inhibited angiogenesis in our model only in high concentrations. At 10^–5 M, endostatin did not alter the percent of wells that initiated an angiogenic response, but significantly inhibited subsequent vessel growth. At 10^–4 M, endostatin was able to inhibit both initiation and subsequent new vessel growth. Human endostatin can inhibit the initiation of a human angiogenic response and inhibit the subsequent proliferation of human neovessels when used at high doses in a continuous exposure model.     

Regulation of energy metabolism in pancreatic islets by glucose and tolbutamide

Summary The kinetics of insulin secretion and oxygen uptake in response to D-glucose and tolbutamide were compared in mouse pancreatic islets. In addition, the role of decreased ATP as a driving force for secretagogue-induced oxygen consumption was examined. D-glucose (10–30 mmol/1) triggered a biphasic insulin release which always coincided with a monophasic increase in islet oxygen uptake. In the presence of D-glucose (5–30 mmol/1), tolbutamide (3–500 g,mol/1) consistently elicited an initial peak of insulin secretion which was followed by a continued decline. Tolbutamide-induced secretory profiles were accompanied by similar respiratory profiles. Oxygen consumption per ng of insulin released during the test phase was higher after elevation of the glucose concentration than after addition of tolbutamide. In conjunction with 5 or 10 mmol/l D-glucose, but not with 15 or 30 mmol/1 D-glucose, tolbutamide (30–100 mol/1) lowered islet ATP content significantly (p < 0.02). Phosphocreatine was not found in isolated islets, although they contained substantial creatine kinase activity. It is concluded that the driving force for Tobutamide-induced oxygen uptake is a decrease in the phosphorylation potential caused by the work load imposed by stimulation of the secretion process. However, a major proportion of the respiratory response to glucose also results from enhancement of biosynthesis.     

Hepatic bile formation in the rat

While changes in gastric, pancreatic, and intestinal secretion in response to more recently identified gastrointestinal peptides have been characterized, there has been less investigation into effects of these hormones on hepatic bile production. The isolated perfused rat liver model has been used to examine effects of vasoactive intestinal peptide (VIP), somatostatin, bombesin, and thyrotropin-releasing hormone (TRH) on bile flow and bile acid transport. No changes were seen following bolus administration of bombesin (3×10^–8–1.5×10^–6 M) or TRH (3×10^–7–3×10^–6 M), while somatostatin (6×10^–6 M) produced a small decrease in bile flow without any change in bile acid output. VIP (3×10^–7 M) caused a highly significant increase in both volume of bile flow (0.85±0.8 to 1.11±0.09 l/min/g liver,P<0.001) and bile acid output (31.6±1.5 to 43.2±1.7 nmol/min/g liver,P<0.001). Elimination of Ca²⁺ from liver perfusate did not prevent VIP-induced increases in bile flow and bile acid output, and no synergistic effect of concomitant theophylline administration was observed. While effects of VIP on bile flow appear to be due to alterations in hepatic transport of bile acids, the exact mechanism(s) producing these changes remains to be elucidated.This work was supported by National Institutes of Health grant AM-32208 and the Research Service of the Veterans Administration.     

Effect of phenformin on hepatic balances of gluconeogenic substrates in man

Summary The effect of a five day pretreatment with phenformin (3 x 50 mg daily) on hepatic metabolism was studied in six healthy volunteers. Arterial and hepatic venous concentrations of substrates and hepatic blood flow were estimated during a basal period and during a low-dose lactate infusion (0,03 mmol · kg^–1· min^–1). The results have been compared with those obtained from untreated normal subjects in a previous study (16). During the baseline period arterial concentration of alanine and the hepatic venous concentration ratios of alanine: pyruvate and-hydroxybutyrate: acetoacetate were significantly increased with phenformin treatment, while the balances of carbon dioxide and glucose and the fractional extraction of alanine were decreased compared to the values obtained in untreated subjects. During lactate infusion mean arterial lactate concentration was significantly increased and hepatic lactate extraction was decreased compared to untreated persons under the same conditions. In the phenformin-treated group lactate infusion resulted in hepatic output of pyruvate and the hepatic glucose balance remained unchanged compared to baseline. Since the rate of hepatic blood flow was not increased during lactate infusion a significantly smaller glucose output and lactate uptake was obtained with phenformin. These findings support the present view that the hypoglycaemic effect of biguanides is due, at least in part, to inhibition of hepatic gluconeogenesis.     

Effect of lactate and H+ on structure and function of rat intestine

Segments of rat ileum and colon were infusedin vivo to test if 0.1 M lactate, 10^–4 M H⁺ or both altered mucosal structure and function. In the first series of experiments, lactate concentration was kept at 0.1 M while H⁺ was varied from 10^–4 to 10^–7 M. Lactate and 10^–4 M H⁺ in the colon, and lactate and 10^–5 M H⁺ in the ileum depressed net water transport and caused sloughing of superficial absorptive cells. In the second series of experiments, H⁺ concentration was kept at 10^–4 by using carboxymethylcellulose, rather than organic acids, to buffer the infusion mixture; the concentration of lactate was varied from 0 to 0.1 M. Mucosa remained normal after infusions of 10^–4 M H⁺ alone. Addition to the 10^–4 M H⁺ infusion of 0.1 M, lactate in the colon or 0.075 M lactate in the ileum caused increased mucosal sloughing. Thus lactate plus H⁺ (or unionized lactic acid) alters colonic and ileal mucosa. Because such high concentrations of lactate and H⁺ are found in subjects malabsorbing carbohydrates, the present experiments support the contention that H⁺ and organic acids are etiological factors in some cases of chronic fermentative diarrhea.This project was funded by Research Grant AM 16059, United States Public Health Service, National Institutes of Health.     

Determination and kinetic analysis of non-insulin mediated glucose uptake in Type 1 (insulin-dependent) diabetes mellitus

Summary In man, total glucose uptake is the sum of insulin mediated glucose uptake and non-insulin mediated glucose uptake. The latter pathway has not been examined in Type 1 (insulin-dependent) diabetes mellitus. In order to assess non-insulin mediated glucose uptake in Type 1 diabetes, we measured steady-state rates of glucose uptake during glucose clamps at 5.27, 9.71 and 12.5 mmol/l using low (0.25 mU· kg^–1·min^–1), intermediate (0.75 mU·kg^–1·min^–1) and high (1.50 mU·kg^–1·min^–1) insulin infusion rates in 10 subjects with Type 1 diabetes. For insulin infusion rates of 0.25, 0.75 and 1.50 mU·kg^–1·min^–1 as plasma glucose rose from 5.27 to 9.71 mmol/l, total glucose uptake increased by 35, 43 and 52 percent respectively (p<0.05 for each insulin infusion rate). For all three insulin infusion rates, there was no significant increase in total glucose uptake as plasma glucose increased from 9.71 to 12.5 mmol/l. At each glycaemic level, glucose uptake correlated significantly with plasma free insulin (r=0.81, p<0.01 at 5.71 mmol/l; r=0.84, p<0.01 at 9.71 mmol/l; r=0.73, p<0.02 at 12.5 mmol/l). Linear regression analysis to a point corresponding to plasma free insulin equalling zero, yielded values for non-insulin mediated glucose uptake (mmol·kg^–1·min^–1) of 0.11,0.14,0.18 at plasma glucose of 5.27, 9.7 and 12.5 mmol/l respectively. Thus, increasing plasma glucose concentrations were associated with increasing rates of non-insulin mediated glucose uptake. For each insulin infusion rate used, the percent of total glucose uptake accounted for by non-insulin mediated glucose uptake remained independent of plasma glucose concentration, but decreased as insulin infusion rate increased. During the insulin infusion at 0.25 mU·kg^–1·min^–1, this percentage ranged from 83.7 to 91.4%. Analysis of glucose uptake data derived for theoretical plasma insulin levels of 0, 40, 80 and 160 U/ml yielded linear Eadie-Hofstee plots (r=– 0.83 to – 0.99), suggesting that insulin increased Vmax but did not alter Km. Hence, in these subjects with Type 1 diabetes, glucose uptake, both insulin mediated and non-insulin mediated can be described by Michaelis-Menten kinetics. Comparison of values obtained for Vmax and Km in the present studies of Type 1 diabetes with those obtained from non-diabetic subjects indicates that non-insulin dependent glucose uptake in Type 1 diabetes is quantitatively similar to that of non-diabetic subjects.     

Effects of various modifiers of insulin release on the lanthanum-nondisplaceable45ca2+ uptake by isolated pancreatic islets

Summary The uptake of⁴⁵Ca²⁺ by a lanthanum-nondisplaceable pool in pancreatic islets was studied. Raising the extracellular D-glucose concentration from 3 to 20 mM stimulated the⁴⁵Ca²⁺ uptake in hand-dissected islets of ob/ob-mice as well as in collagenase-isolated islets of ob/ob or normal mice. The effect was dose-dependent in the range of 0–20 mM D-glucose and was seen throughout a wide range of extracellular calcium concentrations (16 mol — 2.56 mmol of Ca²⁺ added per litre of medium). The⁴⁵Ca²⁺ uptake was also enhanced by other known insulin secretagogues (D-mannose, L-leucine, tolbutamide) and was uninfluenced by compounds lacking insulinreleasing capacity (3-O-methyl-D-glucose, L-glucose, D-galactose, D-leucine). The stimulatory effect of D-glucose was blocked by inhibitors of glucoseinduced insulin release (D-mannoheptulose, diazoxide, L-adrenaline). The results support the view that the lanthanum-nondisplaceable calcium pool is related to the insulin-releasing mechanism, although the exact nature of this relationship is still unclear.On leave from the Institute of Pharmacology and Toxicology, University of Göttingen, D-34 Göttingen, Germany Recipient of a research grant from the Deutsche Forschungsgemeinschaft (Le 348/1)     

Peripheral and hepatic insulin sensitivity in subjects with impaired glucose tolerance

Summary Recent evidence suggests that the post-prandial hyperglycaemia in impaired glucose tolerance is primarily due to impaired suppression of basal hepatic glucose output. This in turn appears to be secondary to decreased first phase insulin secretion, although decreased hepatic insulin sensitivity, which is a feature of non-insulin-dependent diabetes mellitus, might also play a role. Eight mildly overweight subjects with impaired glucose tolerance and eight closely matched control subjects with normal glucose tolerance underwent an intravenous glucose tolerance test to assess first phase insulin secretion. Insulin sensitivity was examined by a 150-min hyperinsulinaemic-euglycaemic clamp. Somatostatin was infused from 150 min to suppress endogenous insulin secretion, and glucagon and insulin were replaced by constant infusion. Glucose with added dideuterated glucose (labelled infusion technique) was infused to maintain euglycaemia. First phase insulin secretion ( 0–10 min insulin area ÷ 0–10 min glucose area) was significantly decreased in the subjects with impaired glucose tolerance (median [range]: 1.2 [0.2–19.4] vs 9.1 [2.6–14.5] mU·mmol^–1; p<0.01). During the clamp, circulating insulin (93±8 [mean±SEM] and 81±10 mU·l^–1) and glucagon (54±4 and 44±6 ng·l^–1) levels were comparable. Total glucose disposal was decreased in subjects with impaired glucose tolerance (2.78±0.27 vs 4.47±0.53 mg·kg^–1·min^–1; p<0.02), and was primarily due to decreased non-oxidative glucose disposal. However, hepatic glucose output rates were comparable during the clamp (0.38±0.10 and 0.30±0.18 mg·kg^–1·min^–1). Therefore, the main defects in subjects with impaired glucose tolerance are decreased first phase insulin secretion and peripheral non-oxidative glucose disposal, but hepatic glucose output shows normal responsiveness to insulin.Abbreviations FPIS First phase insulin secretion - PG plasma glucose - NIDDM non-insulin-dependent diabetes mellitus - IGT impaired glucose tolerance - HGO hepatic glucose output - IVGTT intravenous glucose tolerance test - OGTT oral glucose tolerance test     

Importance of glucagon in the control of futile cycling as studied in alloxan-diabetic dogs

Summary In order to determine the role of glucagon in futile or substrate cycling in diabetes, we measured tracer determined glucose kinetics during a combined infusion of 2-³H-glucose (total glucose production) and 6-³H-glucose (glucose production) in six alloxan-diabetic dogs. The animals received either a 420 min infusion of (1) somatostatin alone (0.3 g·kg^–1· min^–1), (2) somatostatin with insulin replacement (100 U·kg^–1min^–1) or (3) glucagon (6 ng·kg^–1· min^–1) together with somatostatin and transient insulin replacement. When somatostatin was given alone, plasma glucagon (p<0.004) and insulin (p<0.0001) were suppressed. Glucose production and disappearance and plasma glucose concentrations fell (p<0.0001), but the metabolic clearance of glucose did not change significantly. In the basal state, futile cycling comprised 29±4%, 33±4% and 33±3% of total glucose production in the three goups of studies, which is high compared to normal dogs. The absolute rate of futile cycling fell slightly but significantly from 10.0±1.7 to 8.3±1.7 mol·kg·^–1min^–1 (p<0.0008). When insulin replacement was given during somatostatin infusion to correct for the small somatostatin-induced insulin suppression, there were similar changes in plasma glucagon, glucose concentrations and glucose kinetics as seen during the infusion of somatostatin alone. Futile cycling decreased to a slightly greater extent from 12.8±2.8 to 9.5±1.7mol·kg^–1·min.^–1 (p<0.02). When glucagon was infused together with somatostatin and insulin replacement, plasma glucagon (p<0.0002) increased and plasma glucose levels rose (p<0.001) due to a transient increase in glucose production. Metabolic clearance of glucose did not change significantly. There was a marked increase in futile cycling from 12.2±1.7 to 21.7±1.7mol· kg^–1·min^–1 (p<0.0001) in response to exogenous glucagon excess. There was a slight (p<0.01) drop in free fatty acid levels with somatostatin. Free fatty acid levels nearly doubled (p<0.025) with the infusion of glucagon together with somatostatin. In conclusion, (a) futile cycling was increased in alloxan-diabetic dogs; (b) glucagon suppression can suppress futile cycling only if total insulin deficiency is prevented; and (3) hyperglucagonaemia increases futile cycling, and this effect is more pronounced during insulin deficiency.     

Effect of metformin on insulin-stimulated glucose transport in isolated skeletal muscle obtained from patients with NIDDM

Summary Metformin has been demonstrated to lower blood glucose in vivo by a mechanism which increases peripheral glucose uptake. Furthermore, the therapeutic concentration of metformin has been estimated to be in the order of 0.01 mmol/l. We investigated the effect of metformin on insulin-stimulated 3-0-methylglucose transport in isolated skeletal muscle obtained from seven patients with non-insulin-dependent diabetes mellitus (NIDDM) and from eight healthy subjects. Whole body insulin-mediated glucose utilization was decreased by 45% (p<0.05) in the diabetic subjects when studied at 8 mmol/l glucose, compared to the healthy subjects studied at 5 mmol/l glucose. Metformin, at concentrations of 0.1 and 0.01 mmol/l, had no effect on basal or insulin-stimulated (100 U/ml) glucose transport in muscle strips from either of the groups. However, the two control subjects and three patients with NIDDM which displayed a low rate of insulin-mediated glucose utilization (<20 mol·kg^–1·min^–1), as well as in vitro insulin resistance, demonstrated increased insulin-stimulated glucose transport in the presence of metformin at 0.1 mmol/l (p<0.05). In conclusion, the concentration of metformin resulting in a potentiating effect on insulin-stimulated glucose transport in insulin-resistant human skeletal muscle is 10-fold higher than the therapeutic concentrations administered to patients with NIDDM. Thus, it is conceivable that the hypoglycaemic effect of metformin in vivo may be due to an accumulation of the drug in the extracellular space of skeletal muscle, or to an effect of the drug distal to the glucose transport step.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - KHB Krebs-Henseleit's bicarbonate buffer - HEPES N-2, hydroxyethyl-piperazine-N'-2-ethanesulfonic acid - BSA bovine serum albumin - RIA radioimmunoassay - HbA_1c glycated haemoglobin A_1c - BMI body mass index (kg/m²) - GLUT 1 HepG2/erythrocyte - GLUT 4 insulin-regulatable glucose transporter     

Human islet amyloid polypeptide at pharmacological levels inhibits insulin and phorbol ester-stimulated glucose transport in in vitro incubated human muscle strips

Summary Human islet amyloid polypeptide, at concentrations of 1–100 nmol/l, has been demonstrated to inhibit the insulin-stimulated increase in rat muscle glycogen content. However, at physiological concentrations (1–10 pmol/l) of islet amyloid polypeptide, no effects have been reported. We tested the effect of a wide range of concentrations of human islet amyloid polypeptide on insulin- and phorbol ester-stimulated 3-0-methylglucose transport in in vitro incubated human skeletal muscle. Muscle specimens from the quadriceps femoris muscle were obtained from 23 healthy subjects with the use of a newly-developed open muscle biopsy technique. Human islet amyloid polypeptide at a concentration of 100 nmol/l had no effect on basal glucose transport, but inhibited the stimulatory effect of a maximal insulin concentration (1000 U/ml) by 69% (p<0.001). The presence of human islet amyloid polypeptide at 1, 10 and 100 nmol/l decreased the effect of 100 U/ml of insulin on glucose transport by 61% (p<0.05), 78% (p<0.05) and 76% (p<0.05), respectively. Similarly, human islet amyloid polypeptide at a concentration of 10 nmol/l inhibited phorbol ester-stimulated glucose transport by 100% (p<0.05). The inhibitory effects of human islet amyloid polypeptide on glucose transport were present in the muscle strips despite no net changes in glycogen content. Human islet amyloid polypeptide at 10 and 100 pmol/l had no effect on the rate of insulin-stimulated glucose transport. In conclusion, pharmacological concentrations of human islet amyloid polypeptide inhibit insulin as well as phorbol ester-stimulated glucose transport in human skeletal muscle, while physiological concentrations do not exert an inhibitory effect. Furthermore, these results suggest that the inhibitory effect of human islet amyloid polypeptide on glucose transport is located at a point distal to the insulin binding process.     

Multiple defects of both hepatic and peripheral intracellular glucose processing contribute to the hyperglycaemia of NIDDM

Summary Non-insulin-dependent diabetic (NIDDM) patients were studied during a modified euglycaemic state when fasting hyperglycaemia was normalized by a prior (–210 to –150 min) — and later withdrawn (–150–0 min) — intravenous insulin infusion. Glucose metabolism was assessed in NIDDM patients (n=10) and matched control subjects (n=10) using tritiated glucose turnover rates, indirect calorimetry and skeletal muscle glycogen synthase activity determinations. Total and non-oxidative exogenous glycolytic flux rates were measured using appearance rates of tritiated water. A+180 min euglycaemic hyperinsulinaemic (40 mU·m^–2·min^–1) clamp was performed to determine the insulin responsiveness of the various metabolic pathways. Plasma glucose concentration increased spontaneously during baseline measurements in the NIDDM patients (–120 to 0 min: 4.8±0.3 to 7.0±0.3 mmol/l; p<0.01), and was primarily due to an elevated rate of hepatic glucose production (3.16±0.13 vs 2.51±0.16 mg·kg FFM^–1·min^–1; p<0.01). In the NIDDM subjects baseline glucose oxidation was decreased (0.92±0.17 vs 1.33±0.14 mg·kg FFM^–1·min^–1; p<0.01) in the presence of a normal rate of total exogenous glycolytic flux and skeletal muscle glycogen synthase activity. The simultaneous finding of an increased lipid oxidation rate (1.95±0.13 vs 1.61±0.07 mg·kg FFM^–1·min^–1; p=0.05) and increased plasma lactate concentrations (0.86±0.05 vs 0.66±0.03 mmol/l; p=0.01) are consistent with a role for both the glucose-fatty acid cycle and the Cori cycle in the maintenance and development of fasting hyperglycaemia in NIDDM during decompensation. Insulin resistance was demonstrated during the hyperinsulinaemic clamp in the NIDDM patients with a decrease in the major peripheral pathways of intracellular glucose metabolism (oxidation, storage and muscle glycogen synthase activity), but not in the pathway of non-oxidative glycolytic flux which was not completely suppressed during insulin infusion in the NIDDM patients (0.55±0.15 mg·kg FFM^–1·min^–1; p<0.05 vs 0; control subjects: 0.17±0.29; NS vs 0). Thus, these data also indicate that the defect(s) of peripheral (skeletal muscle) glucose processing in NIDDM goes beyond the site of glucose transport across the cell membrane.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - FFM fat free mass - HGP hepatic glucose production - R_d peripheral glucose disposal (uptake) rate - G6P glucose 6-phosphate - UDPG uridine diphosphate glucose - FV fractional velocity