Fasting for 24 hours inhibits glucose-induced insulin secretion from isolated rat pancreatic islets. At glucose 15 raM the inhibition is only present during the initial 30-45 minutes of stimulation. It decreases at higher glucose concentrations and disappears at glucose 37.5 mM. In islets of fed rats, 15 minutes of stimulation with glucose 15 mM induced an almost threefold rise of islet cyclic adenosine 3',5'-monophosphate (cAMP). Both this effect and the secretory response were inhibited by about 65 per cent after 24 hours of fasting and almost completely by 72 hours of food deprivation. Fasting for 24 hours also caused a 56 per cent inhibition of the rise of islet cAMP and insulin secretion induced by D-glyceraldehyde 10 mM, but the inhibition was not further enhanced by 72 hours of fasting. Fasting for 24 and 72 hours did not significantly inhibit the islet cAMP response to 15 minutes of stimulation with glucose 37.5 mM. Fasting for 24 hours impaired neither islet glucose oxidation (14CO2 production) nor over-all glucose utilization (production of 3H-water from [5-3H] glucose) during the initial 30 minutes of stimulation with glucose. After 72 hours of fasting, both over-all glucose utilization and glucose oxidation were reduced by 25 per cent. These findings suggest that inhibition of the islet cAMP response is a primary factor in the early (24 hours) fasting-induced impairment of the insulin secretory response to glucose stimulation at 5-15 mM. Inhibition of islet glucose metabolism seems to be an independent additional phenomenon that becomes manifest after longer periods of food deprivation.

Fasting for 24 or 72 h causes a strong decrease of pancreatic islet calcium content as detected by glyoxal-bis-(2-hydroxyanil), (GBHA). There is strong evidence that GBHA only detects ionized calcium and not total calcium (Wolters et al., 1979). Fasting does not influence the zinc content as detected by dithizone (DZN), and aldehyde-fuchsin (AF) staining intensity is only slightly decreased. After degranulation of islets by tolbutamide (which reduced the insulin content of the pancreas to 10% of the control value) the staining intensities of GBHA, DZN and AF were strongly depressed. Calcium (as well as other elements) were also measured by electron-probe micro-analysis (EPMA). It appeared that 24 or 72 h of fasting did neither affect the total content of Ca nor of Na, P, S, and K of the islets significantly. In exocrine tissue the Ca content increased gradually as a result of fasting. Thus, after 72 h of fasting the Ca content was 25% higher than in fed controls. On the other hand after 72 h of fasting the K content appeared to be decreased. EPMA revealed that after degranulation of islets the Ca content decreased markedly (35%). S appeared to be decreased by only 14%, whereas the content of the other elements was not changed. The results show that GBHA-detectable Ca is only a part of EPMA-detectable Ca. The GBHA-Ca "pool" which contains ionized Ca, is subjected to changes when the animals are fasted, the total Ca content as measured by EPMA does not change. Thus, at least two distinguishable pools of Ca exist within the islets (GBHA-detectable and not-GBHA-detectable). It is suggested that as a result of fasting Ca passes from one pool to another.

Summary. Glucose-stimulated 45calcium uptake and total calcium content of rat pancreatic islets has been studied, using a new fluorometric micro-method to estimate total calcium. Extracellular calcium was separated from incubated tissue by a rapid micro-filtration procedure. Islets incubated up to 60 min with calcium chloride 2.5mmol/1 and glucose 2.5mmol/1 maintained the same calcium content (670 + 7.5 pmol/gg DNA). When the glucose concentration was raised to 15mmol/1 no change in the total calcium content could be detected. On incubation with glucose 2.5 mmol/1 in the absence of calcium, the calcium content decreased to 488 + 27 pmol/gg DNA. On incubation with 4Scalcium chloride 2.5 mmol/1 for 5 or 30min at 2.5 mmol/1 glucose, islets exchanged 21 + 2 and 28 _ 1% of their total calcium content and, at 15 mmol/1 glucose, 30 + 3 and 45 ___ 2%, respectively. Thus, islet calcium has a high turn-over rate. Glucose stimulation results in an increase of the calcium uptake without enhancing the total calcium content and hence must increase the calcium-exchangeable pool.

The nature of tissue calcium, detectable with glyoxal-bis-(2-hydroxyanil), (GBHA), was investigated using gelatin films as model. The results indicate that in the films the procedure detects only the calcium fraction which was ionized in the original gelatin solution. The GBHA staining intensity (absorbance) appeared to be linear with the amount of ionized calcium in the range from 0 to 2 micrograms/cm2. The method allows detection of amounts of ionized calcium as low as 0.15 micrograms/cm2 or 0.0015 pg/mu2. For the measurement of calcium in pancreatic tissue of fed rats, the tissue was subjected to freeze-substitution at -80 degree C in acetone containing 1% oxalic acid. Adjacent sections were stained with either GBHA or aldehyde-fuchsin (AF). Exocrine tissue hardly stained with GBHA whereas islet tissue stained intensely. For GBHA as well as for AF a variation in staining intensity (visual evaluation) between islets was observed. Islet GBHA- and AF-staining intensities did not correlate. The AF-staining intensity but not the GBHA-staining intensity decreased with increasing islet diameter. Also in pancreatic islet tissue the GBHA method appears to be very sensitive and reproducible and small differences in islet GBHA-staining intensity can be detected. The results indicate that between islets differences in ionized calcium content exist. These differences do not correlate with the degree of B-cell granulation.

Pancreatic B-cell calcium as histochemically detectable with glyoxal bis (2-hydroxyanil) = GBHA was studied in isolated islets of fed rats. GBHA has previously been shown by us to detect an ionized or readily ionizable Ca-fraction (GBHA-Ca). In the presence of Ca++ (2.5 mM), high glucose (15 mM) induced a rapid decrease (30%) of islet GBHA-Ca followed by a rise between 30 and 60 min to levels above the initial value. At low glucose (0 or 2.5 mM) GBHA-Ca showed a slight and gradual decline under these conditions. Omission of Ca++ at low glucose rapidly decreased (30%) islet GBHA-Ca. This decrease was markedly inhibited by high glucose, although glucose did not induce insulin secretion under these conditions. Preincubation in the absence of Ca++ (15 min) depleted islet GBHA-Ca, but partial restoration occurred during subsequent incubation with Ca++ at low glucose. By contrast, high glucose completely restored GBHA-Ca within 5 min, followed by a decline and a subsequent rise. Reintroduction of Ca++ also rapidly restored the glucose-induced insulin secretion. These results indicate that islet GBHA-Ca represents a mobile Ca-fraction which is dependent on extracellular Ca++ and which responds very rapidly to glucose stimulation. It is suggested that changes of GBHA-Ca in the B-cells may reflect changes in the Ca pool involved in the insulin secretory mechanism.