The relationship between insulin release and cytoplasmic Ca 2 ϩ concentration ([Ca 2 ϩ ] i ) was studied in isolated pancreatic islets from ob/ob mice. Although [Ca 2 ϩ ] i was low and stable in the presence of 3 mM glucose, basal insulin release exhibited low amplitude pulsatility, with a frequency of 0.32 Ϯ 0.04 min Ϫ 1 . Depolarization by raising K ϩ from 5.9 to 30.9 mM or by the addition of 1 mM tolbutamide caused a pronounced initial insulin pulse followed by declining pulses, but there was no change in frequency. This decline in amplitude of the insulin pulses was prevented in similar experiments performed in the presence of 11 mM glucose.

The role of metabolism in the generation of plasma insulin oscillations was investigated by simultaneous in vivo recordings of oxygen tension (pO 2 ) in the endocrine and exocrine pancreas and portal blood insulin concentrations in the anesthetized rat. At the start of the experiment, the blood glucose concentration of seven rats was 6.2 ؎ 0.1 mmol/l and the arterial blood pressure was 116 ؎ 5 mmHg. These values did not differ from those obtained at the end of the experiment. Islet pO 2 was measured by impaling superficially located islets with a miniaturized Clark electrode. The pO 2 measurements revealed slow (0.21 ؎ 0.03 min ؊1 ) with superimposed rapid (3.1 ؎ 0.3 min ؊1 ) oscillations. The average pO 2 was 39 ؎ 5 mmHg. Simultaneous recordings of pO 2 in the exocrine pancreas were significantly lower (16 ؎ 6 mmHg), but showed a slow and a rapid oscillatory activity with similar frequencies as seen in the endocrine pancreas. Corresponding measurements of portal insulin concentrations revealed insulin oscillations at a frequency of 0.22 ؎ 0.02 min ؊1 . The results are the first in vivo recordings of an oscillatory islet parameter with a frequency corresponding to that of plasma insulin oscillations; they support a primary role of metabolic oscillations in the induction of plasma insulin oscillations. Diabetes 51:699 -703, 2002 T he regular variations in blood concentrations of insulin, with a typical duration of 5-10 min (1-4), are critical to the hormone's effect in lowering blood glucose (5-10). Despite this importance of insulin oscillations, their generation is still unclear. Oscillations of similar duration have been detected in the secretion of insulin from single isolated islets (11,12). Regular plasma insulin variations are therefore believed to be the result of coordinated secretory activities of the islets of Langerhans in the pancreas (10,13-16). In addition, oscillations in the cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] i ) and oxygen tension (pO 2 ) have been shown to be correlated with pulsatile insulin release from isolated islets (12,(17)(18)(19)(20)(21). Therefore, rhythmic changes in ionic fluxes and metabolism are considered to be important for the regulation of plasma insulin oscillations. However, so far these slow in vitro oscillations of [Ca 2ϩ ] i and pO 2 have not been demonstrated in vivo. In the present study, we performed in vivo measurements of pO 2 in rat pancreatic islets and in adjacent exocrine tissue and simultaneously determined portal vein insulin concentrations. RESEARCH DESIGN AND METHODS Materials.Reagents of analytical grade and water purified by a Milli-Q filter (Millipore, Bedford, MA) were used. Tetramethylbenzidine and insulin peroxidase were purchased from Sigma (St. Louis, MO); aprotinin (Trasylol), from Bayer (Leverkusen, Germany); and heparin, from Leo/Løvens (Ballerup, Denmark). The rat insulin standard was obtained from Novo Nordisk (Bagsvaerd, Denmark). IgG-certified microtiter plates were purchased from Nunc (Roskilde, Denmark). The antibodies to insulin...

Pancreatic islets are known to respond to a raise of the glucose concentration with Ca 2 ϩ -induced 2-3-min pulses of insulin release. The reports of cyclic variations of circulating insulin in the fasting state made it important to explore whether insulin release is also pulsatile in the absence of stimulated entry of Ca 2 ϩ . Individual pancreatic islets were isolated from a local colony of ob/ob mice and perifused under conditions allowing dual wavelength recordings of the cytoplasmic Ca 2 ϩ concentration ([Ca 2 ϩ ] i ) with fura-2 and measurements of insulin with ELISA technique. At 3 mM of glucose, [Ca 2 ϩ ] i remained at a stable low level, but insulin was released in pulses with a frequency of 0.41 Ϯ 0.02 min Ϫ 1 , determined by Fourier transformation of original and autocorrelated data. Pulses of basal insulin release were also seen when glucose was omitted and 1 M clonidine or 400 M diazoxide was added to a glucose-free medium. The results indicate that pulsatile insulin release can be generated in the absence of stimulated entry of Ca 2 ϩ . A tentative explanation for this phenomenon is inherent fluctuations in

Neuron activity and insulin release were measured simultaneously from 33 preparations of intrapancreatic canine ganglia and pancreatic parenchyma adjacent to the ganglia. The electrical activity of single neurons of the ganglia was recorded with intracellular microelectrodes, and insulin release from the attached islets was determined with an enzyme-linked immunosorbent assay. Insulin release was 62 ± 18 fmol preparation/min in the presence of 10 mmol/l glucose and pulsatile (3.7 ± 0.4 min/pulse). Corresponding measurements of neuronal electrical activity showed a stable membrane potential of -53.5 ± 0.6 mV. Short, high-frequency (20 Hz) preganglionic nerve stimulation evoked action potentials and, in 46% of the preparations, a threefold rise in the insulin secretory rate associated with increased amplitude of the insulin pulses. The effects were blocked by 10 µmol/l tetrodotoxin (TTX). In other preparations, continuous low-frequency (0.05-0.5 Hz) preganglionic nerve stimulation evoked action potentials and, in 50% of the preparations, a gradual increase of insulin release associated with augmentation of insulin pulse amplitude without alteration of the duration. The effects were blocked by 50 µmol/l hexamethonium (HEX). In the remaining preparations, no change in insulin release was observed during nerve stimulation. In the absence of stimulation, neither TTX nor HEX affected the membrane potential or insulin secretion. These first simultaneous measurements of intrapancreatic ganglion activity and insulin secretion are consistent with amplitude modulation of pulsatile insulin secretion induced by changes in electrical activity in a population of intrapancreatic ganglion neurons. Diabetes 50:51-55, 2001