Inge-Bert Täljedal

Evidence for mediated transport of glucose in mammalian pancreatic β-cells

Abstract Uptake of glucose by microdissected pancreatic islets of obese-hyperglycemic mice was studied at 8°. The use of a double-label procedure permitted correction for label in the extracellular space. The following observations and interpretations were made: 1. 1. l -Glucose was restricted to the sucrose space, whereas d -glucose was uniformly equilibrated over the β-cell membrane. 2. 2. l -Glucose (5–40 mM) had no effect on the uptake of d -glucose (1 mM). 3. 3. The uptake of d -glucose was saturable with a v max of about 400 mmoles/h per kg dry islet, and with a K m around 50 mM. 4. 4. At a medium concentration of 5 mM d -glucose, the uptake of this sugar was almost completely blocked by 10 mM phlorizin. Under similar conditions, 20 mM mannoheptulose had no significant effect on d -glucose uptake. The result contradict the previous hypothesis that the β-cell membrane is freely permeable to d -glucose. It is suggested that the uptake of glucose by these cells is mediated by a membrane-located transport molecule with stereospecificity for d -glucose. Renewed attention should therefore be given to the β-cell membrane as a possible locus for the triggering of insulin release by d -glucose.

Effects of glucose on 45Ca2+ uptake by pancreatic islets as studied with the lanthanum method.

1. Fluxes of 45Ca2+ were studied in pancreatic islets from non‐inbred ob/ob‐mice. Because La3+ blocked the transmembrane fluxes of 45Ca2+ in islet cells, incubations aimed at measuring glucose‐induced changes of the intracellular Ca2+ were ended by washing the islets with 2 mM‐La3+ for 60 min. 2. Uptake of 45Ca2+ progressed for 2 hr; the intracellular concentration of exchangable Ca2+ was about 7 m‐mole/kg dry wt., as estimated from the isotope distribution at apparent equilibrium in islets exposed to 3 mM D‐glucose. Raising the D‐glucose concentration to 20 mM enhanced the 45 Ca2+ uptake whether or not the islets had first been equilibrated with the isotope. The stimulatory effect of D‐glucose was observed in Tris buffer containing no anions but Cl‐ as well as in polyanionic bicarbonate buffer. The effect could not be reproduced with equimolar L‐glucose. 3. The rate of 45Ca2+ release was the same whether the islets had been pre‐loaded in the presence of 3 or 20 mM D‐glucose. Thus the 45Ca2+ that had been taken up in response to 20 mM D‐glucose appeared to be released much more slowly than the bulk of intracellular 45Ca2+. The release of 45Ca2+ was not significantly influenced by D‐glucose during the release period. Incubation for 30 min was require for half of the radioactivity to be released. 4. The rates of insulin secretion were about the same in uni‐anionic Tris buffer as in polyanionic bicarbonate buffer. A marked insulin secretory response to 20 mM D‐glucose was observed in either buffer. 5. It is concluded that 20 mM D‐glucose causes a net uptake of Ca2+ from the extracellular fluid into the interior of the beta‐cells. This uptake is probably not regulated at the level of the plasma membrane but more likely reflects an increased affinity of some intracellular phase or compartment for the ion. Because the observed uptake and release of intracellular 45Ca2+ are slow processes in comparison with the rapid effects of extracellular Ca2+ on insulin secretion, insulin secretion may also depend on a more superficial and La3+‐displacable Ca2+ pool.

The pancreatic β-cell recognition of insulin secretagogues: Comparisons of glucose with glyceraldehyde isomers and dihydroxyacetone

d-Glyceraldehyde stimulated the release of insulin from pancreatic islets of Umea-obob-mice whether or not glucose was present in the medium. Like the action of glucose, that of d-glyceraldehyde was biphasic in time, exhibited a sigmoidal dose-response relationship, was potentiated by theophylline, arginine, iodoacetamide, or l-glyceraldehyde, and was inhibited by epinephrine, 2,4-dinitrophenol, or Ca2+ deficiency. Half-maximum and maximum stimulations were produced by about 3 mm and 10 mm d-glyceraldehyde. Positive interactions were observed between 5 mm d-glyceraldehyde and 5 mm glucose and between 10 mm d-glyceraldehyde and 10 mm leucine. Mannoheptulose (10 mm) or glucosamine (10 mm) did not inhibit but potentiated the effect of 10 mm d-glyceraldehyde. Dihydroxyacetone (2.5–20 mm) also initiated insulin release in the absence of glucose. On the other hand, 5–10 mm l-glyceraldehyde did not initiate secretion but potentiated the effects of 5 mm glucose or 5 mm d-glyceraldehyde. d-Glyceraldehyde or dihydroxyacetone reduced the production of 14CO2 from d-[U-14C]glucose; l-glyceraldehyde had a smaller and statistically insignificant effect. The results suggest that by being phosphorylated and entering glycolysis in the β-cells, d-glyceraldehyde and dihydroxyacetone act as functional analogues of glucose as secretory stimulus. Initiation of insulin release by glucose, d-glyceraldehyde, or dihydroxyacetone may thus depend on the production of a metabolic signal at or below the triose phosphate level.

The pancreatic -cell recognition of insulin secretagogues. IV. Islet uptake of sulfonylureas.

SummaryThe study was aimed at testing the hypothesis that sulfonylureas do not readily penetrate the pancreaticβ-cells but more probably stimulate insulin release by a direct action on theβ-cell plasma membrane. Uptake of radioactively labelled tolbutamide and glibenclamide by microdissected pancreatic islets of obesehyperglycemic mice was compared with the uptake of 3-O-methyl-D-glucose, to which theβ-cells are permeable. In contrast to tolbutamide, glibenclamide was taken up in amounts exceeding the 3-O-methyl-D-glucose space of islets incubated in the absence of serum albumin. Uptake of the sulfonylureas was easily reversible. It was depressed by serum albumin, whereas glucose, leucine or diazoxide had no effects. Antimycin A,p-chloromercuriphenylsulfonic acid and chlorpromazine, all of which increase the uptake of extracellular space markers, strongly stimulated the islet uptake of tolbutamide and glibenclamide but had no effect on the uptake of glibenclamide by subcellular particles of homogenized islets. The results suggest that sulfonylureas bind reversibly to islet tissue but are normally restricted to the outside of theβ-cells.

Prevention of Detrimental Effect of Cyclosporin A on Vascular Ingrowth of Transplanted Pancreatic Islets With Verapamil

The revascularization of pancreatic islet clusters transplanted beneath the renal capsule was studied in a syngeneic mouse model. The degree of vascular ingrowth was visualized by in vivo fluorescence microscopy (fluorescein isothiocyanate-dextran) and judged by a semiquantitative method from coded video recordings. The recipients of isografts were divided into four groups, depending on their daily immunosuppressive treatment: 1) none (controls), 2) 15 mg/kg cyclosporin A (CsA), 3) 0.4 mg/kg verapamil + 15 mg/kg CsA, and 4) 20–30 mg/kg methylprednisolone. In control animals, capillary ingrowth was first demonstrated on day 6, followed by progressive vascularization up to day 34. After 6 mo, the vascular architecture was similar to that seen in normal islets in situ. CsA alone significantly decreased vascular ingrowth on day 14 compared with controls (P < .02). Verapamil prevented the detrimental effect of CsA (P < .01), probably by improving renal subcapsular blood flow. Methylprednisolone did not affect revascularization compared with control animals at day 14. We conclude that CsA inhibits vascular ingrowth into transplanted pancreatic islets, which is likely to have clinical implications. The prevention of CsA vascular ingrowth inhibition by a calcium antagonist indicates a possible approach to the correction of this problem, particularly when the renal capsule is used as the recipients transplant site.

The pancreatic β-cell recognition of insulin secretagogues. II. Site of action of tolbutamide

The distribution of 35S-labelled tolbutamide was studied in microdissected pancreatic islets of obese-hyperglycemic mice. These islets contain more than 90 % β-cells. A comparison with the uptake of 3H-labelled sucrose, mannitol, or 3-O-methyl-D-glucose revealed that tolbutamide did not enter the β-cells but was restricted to the extracellular space. It is suggested that the β-cell plasma membrane contains a tolbutamide receptor, which is responsible for the recognition of sulfonylureas as insulin secretagogues.

Effects of various modifiers of insulin release on the lanthanum-nondisplaceable 45Ca2+ uptake by isolated pancreatic islets.

SummaryThe 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 μmol — 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.

Protection against cyclosporine-induced impairment of renal microcirculation by verapamil in mice.

Fluorescence microscopy was used to examine the effect of cyclosporine (CsA) infusion on renal subcapsular (cortical) blood flow in 53 living mice, using FITC-dextran (MW: 156,000) as a fluorescent marker. CsA (8–19 mg/kg body weight) given i.v. for 1 min induced complete inhibition of blood flow. A complete standstill of flow was also obtained during a continuous infusion with a rate of 0.8–2 mg/kg/min. With lower infusion rates (0.15–0.23 mg/kg/min), blood flow was partially impaired. In all experiments, the decrease in flow occurred after a 15–25 min delay, suggesting a CsA metabolite or exhaustion of a protective mechanism as the causative agent. Pretreatment with an α-blocking agent, phentolamine (1.0 mg/kg), did not prevent the CsA-induced inhibition of blood flow. In contrast, pretreatment with a calcium antagonist, verapamil (0.3–0.4 mg/kg), prevented the impairment of blood flow at low (0.15–0.23 mg/kg/min), and partially at higher (0.8–2.4 mg/kg/min) rates of CsA infusion. Clinical studies are warranted to explore the role of calcium antagonists in the prevention of posttransplant acute cyclosporine-induced nephrotoxicity.

EFFECTS OF ACETYLCHOLINE ON ION FLUXES AND CHLOROTETRACYCLINE FLUORESCENCE IN PANCREATIC ISLETS

1. Acetylcholine potentiated glucose‐stimulated insulin release from ob/ob‐mouse islets in salt‐balanced bicarbonate buffer and to a lesser extent in Tris buffer; basal insulin release at 3 m M-D‐glucose was not affected. Potentiation required the presence of Ca2+.

On insulin secretion.

SummaryAspects of insulin secretory mechanisms and models of diabetogenic B cell damage are discussed. Measurements of fluxes of3H-labelled triphenylmethylphosphonium ion,86Rb+,42K+,22Na+, and42Ca2+ in isolated islets indicate that the triggering of insulin release depends on alterations in the interaction of ions with the B cells. One difficulty in the detailed analysis of these alterations are uncertainties which arise when macroscopic concepts for homogenous phases are applied to microscopic and heterogenous compartments, as exemplified by the meaning of pH in insulin secretory granules and of membrane electric potential. Nonetheless, the importance of an apparent decreased K+ permeability in mediating the insulin-releasing action of glucose, and of an apparent increased Na+ permeability in mediating the potentiating action of acetylcholine is emphasized. Fluorescent probing of Ca2+ by chlorotetracycline revealed effects of glucose alone as well as glucose-dependent and atropine-sensitive effects of acetylcholine. Although acetylcholine, sulfonylureas, and certain thiol-blocking agents may stimulate insulin release by direct effects on the B cell plasma membrane, a high capacity for D-glucose transmembrane transport has probably evolved in order that the interior of the B cells can always sense the circulating glucose concentration. A signal to secretion is thought to be transmitted from glucose metabolism to altered ion fluxes by intervention of reduced pyridine nucleotides and a hypothetical redox protein for which thioredoxin may be a model. The insulin secretory defect in hereditary diabetic C57BL/KsJ-db/db-mice is apparently linked to a decreased basal permeability for K+ and a failure of the B cells to decrease further this permeability in response to glucose. Functioning B cells are acutely damaged when exposed to heterologous serum or alloxan in vitro; cytotoxic activation of complement by the alternative pathway could perhaps occur during islet inflammation. Protection experiments with free-radical scavangers in vitro and in vivo support the theory that hydroxyl radicals are instrumental in the production of alloxan diabetes. Rapid reduction of alloxan by thioredoxin in the presence of molecular oxygen and NADPH leads to strong chemiluminescence from luminol indicative of an intense radical production. The sensitivity of B cells to alloxan may be due to physiological specializations of their plasma membranes, involving the highly effective glucose carrier or the hypothetical oxidation/reduction systems or both.