2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein and Insulinoma

Previous investigations revealed low activities of lactate dehydrogenase (LDH) and plasma membrane monocarboxylate transporters (MCT) in the pancreatic beta cell. In this study the significance of these characteristics was explored by overexpressing type A LDH (LDH-A) and/or type 1 MCT (MCT-1) in the clonal INS-1 beta cells and isolated rat islets. Inducible overexpression of LDH-A resulted in an 87-fold increase in LDH activity in INS-1 cells. Adenovirus-mediated overexpression of MCT-1 increased lactate transport activity 3.7-fold in INS-1 cells. Although overexpression of LDH-A, and/or MCT-1 did not affect glucose-stimulated insulin secretion, LDH-A overexpression resulted in stimulation of insulin secretion even at a low lactate concentration with a concomitant increase in its oxidation in INS-1 cells regardless of MCT-1 co-overexpression. Adenovirus-mediated overexpression of MCT-1 caused an increase in pyruvate oxidation and conferred pyruvate-stimulated insulin release to isolated rat islets. Although lactate did not stimulate insulin secretion from control or MCT-1-overexpressing islets, co-overexpression of LDH-A and MCT-1 evoked lactate-stimulated insulin secretion with a concomitant increase in lactate oxidation in rat islets. These results suggest that low expression of MCT and LDH is requisite to the specificity of glucose in insulin secretion, protecting the organism from undesired hypoglycemic actions of pyruvate and lactate during exercise and other catabolic states.

Topics: Adenoviridae; Animals; Carrier Proteins; Cell Line; Fluoresceins; Fluorescent Antibody Technique; Gene Expression Regulation; Glucose; Hydrogen-Ion Concentration; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; L-Lactate Dehydrogenase; Lactic Acid; Monocarboxylic Acid Transporters; Pyruvic Acid; Rats; Transfection

L-Lactate, D-lactate, propionate and acetate (all 20 mM) caused a marked intracellular acidification in HIT-T15 cells loaded with 2'7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF), followed by recovery to more alkaline values. The effects of L- and D-lactate, but not propionate or acetate, were inhibited by 5 mM alpha-fluorocinnamate. Both L- and D-lactate caused a marked depolarisation and rise in cytosolic [Ca2+] in HIT cells as assessed by oxonol-V and quin2 fluorescence, respectively. Propionate had similar, though less marked, effects, whereas acetate exerted only a modest influence on membrane potential and cytosolic [Ca2+]. The rate of oxidation of L-lactate by HIT cells greatly exceeded that of D-lactate. alpha-Fluorocinnamate delayed, but did not prevent, the effects of L-lactate on HIT cell membrane potential or cytosolic [Ca2+]. L-lactate diminished the rate of efflux of 86Rb+ from preloaded HIT cells. Inhibition of calcium- and nucleotide-sensitive K+ channels with tetraethylammonium and tolbutamide also reduced the 86Rb+ efflux rate, and prevented any further reduction in response to L-lactate. However, such inhibition of K+ channels did not prevent a further depolarisation and rise in cytosolic [Ca2+] upon the subsequent addition of lactate. It is suggested that the activation of HIT-T15 cells by lactate is not the result of intracellular acidification or increased metabolic flux, and does not require diminished K+ permeability. An alternative mechanism is based upon the possible electrogenic flux of lactate across the plasma membrane.

Topics: Acetates; Aminoquinolines; Animals; Calcium; Cytosol; Fluoresceins; Hydrogen-Ion Concentration; Insulinoma; Islets of Langerhans; Isoxazoles; Lactates; Lactic Acid; Membrane Potentials; Propionates; Tumor Cells, Cultured

The addition of glucose to suspensions of HIT-T15 insulinoma cells caused a small, transient acidification followed by a gradual, progressive alkalinisation, as assessed by the fluorescent pH-sensitive dye 2',7'-biscarboxyethyl-5'-(6')-carboxyfluorescein (BCECF). Treatment of cells with acetate or lactate produced an immediate, marked acidification followed by recovery and a subsequent alkalinisation. In contrast, addition of NH4Cl caused a rapid rise in intracellular pH (pHi) and recovery to resting values. In cells where Na+/H+ exchange was inhibited, either with amiloride or by omission of Na+ from the medium, glucose caused a progressive acidification, whilst recovery from acetate- or lactate-induced acidification was prevented. Under sodium-free conditions, recovery from acidification could be initiated by addition of Na+. Inhibition of HCO3-/Cl- exchange by pretreatment with 4,4'-diisothiocyanatostilbene 2,2'-disulphonic acid (DIDS), or by omission of HCO3- or Cl- from the medium did not affect any of the changes in pHi elicited by the above agents. It is concluded that the principal mechanism responsible for pHi regulation in HIT-T15 cells is the Na+/H+ antiporter and that the HCO3-/Cl- exchange systems make little, if any, contribution.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetates; Adenoma, Islet Cell; Ammonium Chloride; Bicarbonates; Carrier Proteins; Chlorides; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Insulin; Insulin Secretion; Insulinoma; Lactates; Lactic Acid; Pancreatic Neoplasms; Sodium-Hydrogen Exchangers; Tumor Cells, Cultured

Intracellular pH (pHi) was measured in the insulin-secreting HIT-T15 cell line using the pH-sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF). It was observed that the addition of a weak acid (e.g., acetate or propionate) caused a rapid decrease in pHi, followed by a slower recovery to the resting pH value. Conversely the addition of N4Cl caused an increase in pHi followed by recovery. The addition of amiloride caused a fall in pHi; however, in this case no recovery to basal pH levels was observed. Subsequent addition of a weak acid caused a further fall in pHi with no recovery. The addition of glucose caused a transient acidification followed by alkalinization. When glucose was added to cells which had been pretreated with amiloride, the initial acidification was not followed by recovery or alkalinization. Addition of glyceraldehyde, alpha-ketoisocaproate, lactate or pyruvate to HIT cells also resulted in intracellular acidification followed by recovery. Similarly, depolarisation of HIT cells by treatment with high K+ or with Ba2+ was associated with a pronounced fall in pHi, followed by a gradual recovery. Insulin secretion from HIT cells was stimulated by glucose, glyceraldehyde, alpha-ketoisocaproate, lactate, pyruvate and KCl, whilst amiloride and weak acids exerted only modest effects in the absence of glucose, but amiloride in particular markedly potentiated glucose-induced insulin release. Thus, HIT cells appear to have an amiloride-sensitive mechanism for the extrusion of protons, probably Na+-H+ exchange. Whilst intracellular acidification appears to potentiate secretory responses to nutrient stimuli, it seems unlikely that the activation of HIT cells by these nutrients occurs as a result of intracellular acidification. The mechanisms by which various nutrient and non-nutrient stimuli might exert distinct effects on pHi are discussed.

Topics: Acid-Base Equilibrium; Adenoma, Islet Cell; Cytosol; Fluoresceins; Fluorescent Dyes; Glucose; Humans; Insulin; Insulin Secretion; Insulinoma; Pancreatic Neoplasms; Radioimmunoassay; Tumor Cells, Cultured