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K Tornheim

Publications and source records attributed to K Tornheim.

At least 37 records · Page 2Linked to original sources
PubMed1996

Temporal sequence of metabolic and ionic events in glucose-stimulated clonal pancreatic beta-cells (HIT).

Stimulation of insulin release by glucose requires increased metabolism of glucose and a rise in cytosolic free Ca2+ in the pancreatic beta-cell. It is accompanied by increases in respiratory rate, pyridine and flavin nucleotide reduction state, intracellular pH and the ATP/ADP ratio. To test alternative proposals of the regulatory relationships among free Ca2+, mitochondrial metabolism and cellular energy state, we determined the temporal sequence of these metabolic and ionic changes following addition of glucose to clonal pancreatic beta-cells (HIT). Combined measurements of the native fluorescence of reduced pyridine nucleotides and oxidized flavin, intracellular pH, and free Ca2+ were performed together with simultaneous measurement of O2 tension or removal of samples for assay of the ATP/ADP ratio. The initial changes were detected in three phases. First, decreases occurred in the ATP/ADP ratio (<3 s) and increases in pyridine (2 +/- 1 s) and flavin (2 +/- 1 s) nucleotide reduction. Next, increases in the O2 consumption rate (20 +/- 5 s), the ATP/ADP ratio (29 +/- 12 s) and internal pH (48 +/- 5 s) were observed. Finally, cytosolic free Ca2+ rose (114 +/- 10 s). Maximal changes in the ATP/ADP ratio, O2 consumption and pyridine and flavin nucleotide fluorescence preceded the beginning of the Ca2+ change. These relationships are consistent with a model in which phosphorylation of glucose is the initial event which generates the signals that lead to an increase in respiration, a rise in the ATP/ADP ratio and finally influx of Ca2+. Our results indicate that Ca2+ does not function as the initiator of increased mitochondrial respiration.

Adenosine Diphosphate↗
PubMed1996

Temporal patterns of changes in ATP/ADP ratio, glucose 6-phosphate and cytoplasmic free Ca2+ in glucose-stimulated pancreatic beta-cells.

Closure of ATP-sensitive K+ (K(ATP)) channels is part of the stimulus-secretion coupling mechanism in the pancreatic beta-cell, leading to membrane depolarization and influx of Ca2+ through voltage-sensitive L-type Ca2+ channels. The elevated ATP/ADP ratio seen in the presence of high levels of glucose has been postulated to mediate the glucose-induced closure of the K(ATP) channels and rise in cytoplasmic free Ca2+ concentration ([Ca2+]i), or alternatively to be a consequence of activation of mitochondrial dehydrogenases by the increase in [Ca2+]i. To distinguish between these two possibilities, the time course of the change in the ATP/ADP ratio was determined in comparison with that of [Ca2+]i. We here show that a severalfold rise in the ATP/ADP ratio occurs rapidly on stimulation of suspensions of mouse pancreatic beta-cells with glucose. The change in the ATP/ADP ratio is an early event that begins within 20-40 s and precedes the rise in [Ca2+]i. The temporal relationship indicates that the adenine nucleotide changes cannot be a consequence of the [Ca2+]i changes and may indeed be the connecting link between glucose metabolism and [Ca2+]i changes. When the cells were sequentially treated with high glucose concentration, clonidine and finally high extracellular Ca2+ concentration to induce synchronized oscillations in [Ca2+]i in the cell suspension, corresponding oscillations in the ATP/ADP ratio were observed. Glucose 6-phosphate levels oscillated out of phase with the ATP/ADP ratio. These results support the hypothesis that the Ca2+ oscillations previously observed in glucose-stimulated single islets or beta-cells may reflect oscillations in the ATP/ADP ratio that accompany oscillatory glycolysis.

Adenosine Diphosphate↗
PubMed1996

Glucose-induced oscillatory insulin secretion in perifused rat pancreatic islets and clonal beta-cells (HIT).

Normal insulin secretion is oscillatory in vivo and from groups of perifused islets. Stimulation of rat islets with different glucose concentrations gave insulin oscillations of similar period (5-8 min) but increasing amplitude. It has been assumed that oscillatory secretion is due to oscillations in intracellular free Ca2+, as seen in single islets and single pancreatic beta-cells. However, when islets were perifused with diazoxide and high KCl to maintain high intracellular free Ca2+, insulin oscillations of similar amplitude and period still occurred on glucose stimulation, although superimposed on elevated basal secretion. Several likely possibilities for a diffusible synchronizing factor were tested, including pyruvate, lactate, ATP, and insulin itself; nevertheless, perifusion with high concentrations of these did not prevent insulin oscillations. Clonal pancreatic beta-cells (HIT) and dissociated islets also exhibited oscillatory insulin secretion, but with the 5- to 8-min period oscillations superimposed on 15- to 20-min period oscillations. These results indicate that the mechanisms for generating and synchronizing insulin oscillations reside in the beta-cell, although the structure of the islet may modulate the oscillation pattern.

Animals↗
PubMed1996

Oxidative stress increases glyceraldehyde-3-phosphate dehydrogenase mRNA levels in isolated rabbit aorta.

We have recently shown that inhibition of endogenous Cu,Zn superoxide dismutase (SOD) by diethyldithiocarbamate (DDC) increased superoxide anion levels in isolated rabbit aortic rings, describing a useful experimental model to examine the effects of oxidative stress on the vessel wall. The present study examined the effects of oxidative stress on the steady-state mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). Aortic rings were incubated in physiological salt solution at 37 degrees C for up to 6 h. DDC (2 mM) decreased total SOD activity to < 5% of control levels and increased superoxide anion level ninefold. Steady-state mRNA levels of GAPDH were increased under comparable conditions. Although decreased biological activity of endothelium-derived nitric oxide was indicated by lower basal guanosine 3',5'-cyclic monophosphate levels in aortic rings treated with DDC compared with those in control rings (1.2 +/- 0.1 vs. 1.9 +/- 0.3 fmol/microgram protein, P < 0.05), neither endothelium denudation nor NG-nitro-L-arginine methyl ester had any effects on the steady-state mRNA levels of GAPDH. The cell.permeable iron chelator 1,10-phenanthroline completely prevented the increases in GAPDH mRNA levels induced by DDC. These results suggest that oxidative stress resulting from inhibition of endogenous Cu,Zn SOD causes induction of GAPDH gene expression and that the hydroxyl radical, produced through the iron-catalyzed Haber-Weiss reaction, is the intracellular reactive oxygen species responsible for the DDC-stimulated increase in GAPDH mRNA.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed1996

Altered contractility of rabbit penile corpus cavernosum smooth muscle by hypoxia.

PURPOSE: To investigate the effects of severe hypoxia on trabecular smooth muscle contractility. MATERIALS AND METHODS: Strips of rabbit corpus cavernosum were mounted in organ chambers to measure isometric tension. In some experiments intracellular free Ca2+ concentration and tension were measured by the intracellular fluorescent dye FURA-2 and isometric tension recording simultaneously. RESULTS: Contractions elicited by norepinephrine, endothelin-1 or potassium were attenuated under hypoxic conditions (pO2 approximately 10 mm. Hg). Strips contracted with 20 mM. K+ under normoxic conditions and then exposed to hypoxia consistently lost the potassium-induced tone. The hypoxia-induced relaxation was not affected by the removal of the endothelium, by treatment with the cyclooxygenase blocker, indomethacin, or with the guanylate cyclase blocker, methylene blue. The potassium channel opener, cromakalim, and adenosine relaxed potassium contracted strips; however, the potassium channel blockers glibenclamide, apamin, barium chloride and charybdotoxin or the adenosine receptor antagonist 8-(p-sulfonyl)theophylline were unable to prevent hypoxia-induced relaxation. Tetraethylammonium, a potassium channel blocker, and the depolarizing agents ouabain and high potassium (80 mM.), partially prevented hypoxia-induced relaxation. The calcium ionophore, ionomycin, had no effect on hypoxia-induced relaxations. Hypoxia, within 2 to 6 minutes, caused a large accumulation in intracellular calcium, concomitant with a loss of trabecular smooth muscle tone. Both these effects could be reproduced by inhibiting oxidative phosphorylation with 2,4,dinitrophenol. Reoxygenation resulted in an immediate recovery of both tone and intracellular calcium levels. Tissues under hypoxic conditions for 30 minutes had a 24% and 67% decrease in the ATP/ADP ratio and in creatine phosphate concentrations, respectively. CONCLUSION: Hypoxia causes a simultaneous increase in intracellular calcium and relaxation which, we propose, is the consequence of inhibition of oxidative phosphorylation with loss of high energy phosphates, necessary for the homeostasis of Ca2+ and the contractile mechanism of the cavernosal smooth muscle.

Adenosine Diphosphate↗
PubMed1995

An NADPH oxidase superoxide-generating system in the rabbit aorta.

Superoxide anion can modulate vascular smooth muscle tone and potentially affect the growth response in vascular disease. The present studies were undertaken to characterize the source of superoxide in rabbit aorta. Rings of aorta (5 mm) were incubated in physiological salt solution (PSS) for 30 min at 37 degrees C in the presence of 10 mM diethyldithiocarbamate (DDC) with or without inhibitors of superoxide-generating systems. Rings were then placed in PSS containing 250 microM lucigenin at 37 degrees C in the presence or absence of inhibitors, and changes in amounts of superoxide were determined by measuring chemiluminescence (units). The inhibitors of xanthine oxidase, oxypurinol (300 microM), and of mitochondrial NADH dehydrogenase, rotenone (50 microM), had no significant effect on superoxide levels. An inhibitor of NADPH oxidase, iodonium thiophen, caused a concentration-dependent inhibition of superoxide anion (12.49 +/- 1.48 vs 5.27 +/- 1.81 and 2.30 +/- 0.36 units, control vs 7 microM and 70 microM iodonium thiopen, respectively). A structurally related iodonium compound, diphenyleneiodonium (20 microM), caused a 78% reduction in basal and DDC-evoked superoxide levels. In the presence or absence of DDC, exogenous administration of NADPH (10 microM-1 mM), but not NADP (1 mM), elicited a concentration-dependent rise in superoxide levels that was inhibited by iodonium thiophen. Particulate fractions of whole aortic tissue exhibited NADPH-dependent superoxide production that was inhibited by 1 microM diphenyleneiodonium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Phosphofructokinase isozymes in pancreatic islets and clonal beta-cells (INS-1).

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal beta-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the beta-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the beta-cell. Western analysis of PFK subunits in isolated rat islets and INS-1 cells showed the presence of M-type, as well as C-type and perhaps lesser amounts of L-type. Kinetic studies of PFK activity in INS-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1995

Changes of corneal redox state in diabetic animal models.

Metabolic and biochemical changes of the corneal epithelium and endothelium were studied in experimental diabetic animal models. Ocular redox fluorometry was used to noninvasively determine tissue reduction-oxidation (redox) changes in organ cultured rabbit corneas incubated in high glucose concentration media, alloxan-induced diabetic rabbits, and nonobese diabetic mice. The ratio of autofluorescence from reduced pyridine nucleotide to oxidized flavoproteins (PN/Fp) was used as the indicator of the redox state. Chemical assays for NADH and NAD+ were performed on in vitro materials. Analysis of corneal endothelial morphology using specular microscopy was performed to study possible correlations with metabolic changes. Both the PN/Fp and NADH/NAD+ ratios increased in the corneal endothelium under all experimental conditions. Changes in redox state were not observed in the corneal epithelium in any of the models. Morphologic analysis of the corneal endothelium revealed no significant changes. These results indicate that redox changes occur in the diabetic corneal endothelium, but not the corneal epithelium. Ocular redox fluorometry is capable of detecting changes in the corneal endothelial redox state noninvasively.

Animals↗
PubMed1994

Changes in internal pH caused by movement of fatty acids into and out of clonal pancreatic beta-cells (HIT).

Cells require a constant influx of free fatty acids for lipid resynthesis and metabolic energy. Fatty acids also act as second messengers and modulate channel activities. In the pancreatic beta-cell, fatty acids have both acute and chronic effects on insulin secretion. We show that the addition of fatty acid to pancreatic beta-cells in vitro produces a persistent decrease in intracellular pH, which begins immediately after the addition of fatty acid and has an exponential time course with t1/2 approximately 60 s. The pH drop can be largely reversed by the addition of albumin. The observed pH effect can be explained by passive diffusion ("flip-flop") of un-ionized fatty acid across the plasma membrane. Acidification by a fatty acid dimer and alkalinization by an alkylamine also favor the flip-flop mechanism of transport rather than a protein-mediated mechanism. Our method provides for the first time a real-time measurement of fatty acid import into cells. The significant pH change may be important in mediating some of the regulatory effects of fatty acid, such as inhibition of glycolysis.

Biological Transport↗
PubMed1994

Kinetic applications using high substrate and competitive inhibitor concentrations to determine Ki or Km.

Conventional procedures for determining Km or Ki values generally employ subsaturating concentrations of substrate and competitive inhibitor; however, this is impractical in some cases if a significant percentage would be enzyme-bound or metabolized. Applications employing high and competing concentrations of substrates and inhibitors are described. Ki or Km values can be calculated from the equations. Ki = [I]/((V0)/V - 1)(1 + [S]/Km)) Km = [S](V0/V - 1)/(1 - V0/V + [I]/Ki), where V0 and V are the rates in the absence and presence of inhibitor. This is illustrated by measurements of inhibition of human urinary kallikrein by synthetic peptides.

Kallikreins↗
PubMed1994

Glucose-stimulated increase in cytoplasmic pH precedes increase in free Ca2+ in pancreatic beta-cells. A possible role for pyruvate.

The temporal relationship of glucose-induced increases in cytoplasmic pH (pHi) and cytoplasmic free Ca2+ was studied in single mouse pancreatic beta-cells and suspensions of clonal beta-cells (HIT). In both preparations of cells the increase in pHi preceded the cytoplasmic free Ca2+ increase. Therefore the alkalinization cannot be a consequence of the Ca2+ influx. A potential metabolic mechanism for the increase in pHi, involving stimulation of pyruvate transport and oxidation, was demonstrated in a model system of liver mitochondria incubated with pyruvate, ATP, and hexokinase to which glucose was then added to initiate ATP use. The involvement of this mechanism in beta-cells is suggested by the observation that the alkalinization was prevented in most cells by incubation with 3-hydroxycyanocinnamate, a mitochondrial pyruvate transport inhibitor. On the other hand, the inhibited cells exhibited normal Ca2+ responses to glucose stimulation. This indicates that neither pyruvate metabolism nor the alkalinization is of critical importance for the Ca2+ signal, though pyruvate oxidation or its metabolites may be important in downstream regulation of secretion.

Animals↗
PubMed1994

Glycolytic pH oscillations in a flow reactor.

A new type of flow reactor (UCSTR) has been developed that uses anisotropic ultrafiltration membranes in a continuous flow stirred tank reactor (CSTR) to facilitate the study of nonlinear enzyme catalyzed reactions. The design allows the study of enzymes with subunit molecular weights > or = 9000 dalton and protein concentrations up to at least 2 mg/ml under flow conditions with a residence time of 3 min or more, in a reactor of volume 1.67 ml. The UCSTR allows continuous potentiometric or spectrophotometric measurement without design change. Calibration of reactor performance was carried out by reproducing pH oscillations in the ferrocyanide-hydrogen peroxide reaction. Experimental verification of oscillatory glycolysis in the UCSTR was carried out with extract of rat skeletal muscle. Input feeds were fructose-6-phosphate and ATP with low concentrations of phosphate as buffer. Oscillations in pH, sustained for over eight hours, were observed. A six-step mechanism, including product activation and substrate inhibition, seven concentration variables, and four enzymes sufficed simulate the pH oscillations observed in the UCSTR.

Animals↗
PubMed1993

Differential effect of metabolic fuels on the energy state and Na(+)-K(+)-ATPase in isolated cerebral microvessels.

Isolated bovine cerebral microvessels (ICMV) were incubated with different metabolic fuels to determine their ability to support microvessel Na(+)-K(+)-ATPase (quantitated as ouabain-sensitive 86Rb+ uptake) and the ATP/ADP ratio. In comparison with ICMV incubated with glucose, Na(+)-K(+)-ATPase activity was reduced by 55% after a 3-h incubation in fuel-free medium and by 30-40% after incubation with beta-hydroxybutyrate, acetoacetate, or glutamate. However, Na(+)-K(+)-ATPase activity was not significantly decreased in ICMV incubated with pyruvate or oleate plus carnitine. In contrast, only glucose was able to maintain the ATP/ADP ratio. To evaluate the effect of endogenous fatty acid metabolism on these parameters, ICMV were incubated with bromostearate, an inhibitor of fatty acid oxidation. Bromostearate decreased both Na(+)-K(+)-ATPase activity and the ATP/ADP ratio, even in the presence of glucose. These results indicate that the varying effects of different fuels on Na(+)-K(+)-ATPase in ICMV cannot be explained solely by their effects on the ATP/ADP ratio or on glycolytic ATP generation. They suggest that other fuel-modulated factors play a key role in regulating this enzyme.

3-Hydroxybutyric Acid↗
PubMed1993

Superoxide anion production by rabbit thoracic aorta: effect of endothelium-derived nitric oxide.

Rabbit thoracic aorta was assessed for the influence of the endothelium and nitric oxide (NO) on superoxide anion (SO) levels in the presence and absence of an inhibitor of superoxide dismutase. Aortic rings (0.5 cm) were incubated for 30 min at 37 degrees C in the presence or absence of diethyldithiocarbamate (DDC, 10 mM), a CuZn superoxide dismutase inhibitor. Rings were then placed in a solution containing lucigenin (250 microM) at 37 degrees C, and changes in amounts of SO over 10 min were determined by measuring chemiluminescence under basal and acetylcholine-stimulated conditions. Treatment with DDC markedly enhanced basal levels of SO, and the DDC-evoked levels were significantly reduced by the SO scavenger, Tiron (10 mM). Addition of acetylcholine (10 microM) to the assay did not significantly affect the levels of SO in either control or DDC-treated rings. Also, mechanical removal of the endothelium or pretreatment of the rings with the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) (300 microM), did not significantly affect the levels of SO in DDC-treated rings. In contrast, exogenous NO at 1 and 10 microM reduced the DDC-evoked SO levels by 54 and 77%, respectively. These data imply that the predominant sources of SO in the rabbit aorta are vascular components other than the endothelium and that endogenous superoxide dismutase modulates the level of SO. Although exogenous NO reduced aortic SO levels, neither basal nor acetylcholine-stimulated production of endogenous NO appears sufficient to reduce SO levels.

Acetylcholine↗
PubMed1992

Acyl-CoA esters modulate intracellular Ca2+ handling by permeabilized clonal pancreatic beta-cells.

Cytosolic free Ca2+ rises in pancreatic beta-cells in response to glucose stimulation and is part of the coupling to insulin secretion. This study evaluates a possible role for cytosolic long chain acyl-CoA esters in modulating Ca2+ handling by clonal beta-cells (HIT). Intact cells incubated with 20 microM free palmitic acid exhibited a 40% decrease in basal cytosolic free Ca2+. In contrast, acyl-CoA esters, up to a chain length of 16, but not the corresponding fatty acids, significantly lowered the Ca2+ set point maintained by cells permeabilized with saponin. The maximum response to the various acyl-CoA esters increased with increasing chain length, with no differences in the half-maximally effective concentration of 0.5 microM. Long chain acyl-CoA esters caused a 40-50% increase in 45Ca2+ influx into a non-mitochondrial pool in the permeabilized HIT cells, consistent with a stimulatory effect on the endoplasmic reticulum Ca(2+)-ATPase activity, but did not affect inositol 1,4,5-trisphosphate-induced Ca(2+)-efflux. Thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase activity, blocked the decrease in the Ca2+ set point caused by acyl-CoA esters. The ability of acyl-CoA esters to lower the Ca2+ set point depended on the ATP/ADP ratio (or free ADP); the Ca2+ set point was lowered by 36 +/- 3.6% at an ATP/ADP ratio of 90 and by 14 +/- 1.9% at an ATP/ADP ratio of 7. Depletion of cellular protein kinase C did not prevent the acyl-CoA-induced lowering of the Ca2+ set point. These findings suggest that the increases in long chain acyl-CoA esters may play a role in restoring cytosolic free Ca2+ through activation of Ca(2+)-ATPases.

Acyl Coenzyme A↗
PubMed1992

Endothelium-dependent inhibition of Na(+)-K+ ATPase activity in rabbit aorta by hyperglycemia. Possible role of endothelium-derived nitric oxide.

Hyperglycemia has been shown to diminish Na(+)-K+ ATPase activity in rabbit aorta. To examine the basis for this effect, aortic rings were incubated for 3 h in Krebs-Henseleit solution containing 5.5 or 44 mM glucose, and Na(+)-K+ ATPase activity was then quantified on the basis of ouabain-sensitive (OS) 86Rb-uptake. Incubation with 44 mM glucose medium caused a 60% decrease in Na(+)-K+ ATPase activity in rings with intact endothelium (from 0.22 +/- 0.01 to 0.091 +/- 0.006 nmol/min per mg dry wt; P less than 0.01). Similar decreases (45%; P less than 0.01) in Na(+)-K+ ATPase activity were seen when rings incubated with 5.5 mM glucose were exposed to NG-monomethyl L-arginine (300 microM), an inhibitor of endothelium-derived nitric oxide (EDNO) synthesis or when the endothelium was removed (43% decrease). The decrease in Na(+)-K+ ATPase activity induced by hyperglycemia was totally reversed upon adding to the medium either L-arginine, a precursor of EDNO biosynthesis or sodium nitroprusside, which bypasses endothelium and directly activates the soluble guanylate cyclase in vascular smooth muscle. A decrease in Na(+)-K+ ATPase activity (42%; P less than 0.05), only seen in the presence of endothelium, was also observed in aortas taken directly from alloxan-induced diabetic rabbits. These studies suggest that the decrease in vascular Na(+)-K+ ATPase activity induced by hyperglycemia is related, at least in part, to a decrease in the basal release of EDNO. They also suggest that alterations in basal EDNO release and possibly Na(+)-K+ ATPase activity contribute to the impairment in vascular relaxation caused by hyperglycemia and diabetes.

Animals↗