Janove Sehlin

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.

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.

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+.

The susceptibility to nephrotoxicity of streptozotocin-induced diabetic rats subchronically exposed to cadmium chloride in drinking water

Streptozotocin-induced diabetic rats and normal non-diabetic (ND) rats were exposed to cadmium chloride in drinking water in doses of 0, 50 and 100 ppm for 90 days. There was a dose-related increase in urinary protein and enzymes in the diabetic group, but an increase in proteinuria only in the high exposure subgroup of the ND group. It is suggested that diabetic rats induced by streptozotocin are more susceptible to cadmium nephrotoxicity than normal (ND) rats. Metallothionein synthesis in liver was estimated to be similar in both the diabetic and non-diabetic groups after exposure to cadmium. Less excretion of cadmium in urine and greater accumulation of cadmium in kidney were observed in the diabetic group, and this may be one of the mechanisms underlying the susceptibility of diabetic animals to the effects of cadmium. Further biochemical and histological studies are required in order to explain the detailed events involved in inducing such changes in the toxicokinetics of cadmium.

Transport and storage of 5-hydroxytryptamine in pancreatic β-cells

Abstract To elucidate the role of biogenic amines in insulin secretion, pancreatic islets rich in β-cells were microdissected from obese-hyperglycemic mice and were incubated with 14 C-labelled 5-hydroxytryptamine (5-HT). The saturability of uptake and the fact that 5-HT was accumulated to high levels indicated that the β-cells possess a transport system with great capacity for this amine. The initial uptake was not sensitive to glucose or diazoxide. Efflux of radioactivity from islets preloaded with 14 C-labelled 5-HT exhibited complex kinetics suggesting incorporation of the amine into some less mobile compartment of the β-cells. This compartment may be the insulin-containing secretory granules, since homogenization and centrifugation of preloaded islets revealed closely parallel sedimentation profiles for insulin and 14 C. The apparent co-sedimentation of insulin and 5-HT probably reflects the ultrastructural organization of the β-cells, as insignificant radioactivities were recovered in the sediments after adding 14 C-labelled 5-HT to homogenized islets. Furthermore, gel filtration of insulin on 5-HT-equilibrated Sephadex G-50 did not indicate any great affinity of insulin for the amine. Neither glucose nor glibenclamide could be shown to mobilize granule-bound 5-HT from intact β-cells. In these experiments insulin release was slow despite a glucose concentration as high as 20 mM. It seems possible that co-storage of insulin and 5-HT reduces the ability of β-granules to undergo normal emiocytosis.

Measurements of serum glucose using the luciferin/luciferase system and a liquid scintillation spectrometer

A single-step assay for serum glucose measurements is described. The assay is based on the phosphorylation of D-glucose by glucokinase and the measurement of ATP consumption by firefly luciferase. The luminescence is recorded in an ordinary liquid scintillation spectrometer. The use of stable reagents and a stable final signal (light emission) makes it possible to analyze a large number of samples in each assay run. The assay is of particular value when repeated serum glucose determinations are performed on samples from small laboratory animals.