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The cellular sequence of intraislet vascular perfusion has been shown to be important in the regulation of islet hormone secretion in the rat and dog islet. In order to test whether a B to A to D sequence of islet cellular perfusion is also present in a nonhuman primate, pancreata from the rhesus monkey, Macaca mulatta, were isolated and perfused in vitro in the presence and absence of anti-insulin gamma globulin. In the presence of the insulin antibody, efflux concentration of insulin decreased rapidly (-95 +/- 1.8%), whereas glucagon and somatostatin concentrations increased (111 +/- 28% and 239 +/- 38%, respectively). These results suggest the presence of a B-A-D cellular sequence of vascular perfusion within the monkey islet. The present results also strongly support the hypothesis that a B-A-D sequence of islet perfusion is important in the regulation of islet hormone secretion and further emphasize the central role of the B-cell in intraislet cellular interactions. The results also suggest that, despite differences in islet anatomy, a B-A-D order of islet cellular perfusion may be the preferred functional sequence among mammalian species.

Hepatic glucose production is stimulated in vitro twice as effectively by pulsatile as by continuous glucagon, given equivalent time-averaged doses. Efficacy studies of pulsatile insulin have yielded conflicting results. In the rat hepatoma cell line H-4-II-E-C3, insulin rapidly (t1/2 15 min) inhibits transcription of the gene and lowers mRNA levels for the gluconeogenic enzyme. PEPCK via a receptor-mediated process. We attached H-4-II-E-C3 cells to Cytodex-3 microcarriers and used a perifusion column system to test whether pulsatile insulin is more or less effective than equivalent time-averaged doses of continuous insulin. PEPCK transcription was induced by inclusion of cAMP analogue 8-(4-chlorophenyl-thio)-cAMP (0.1 mM) and dexamethasone (0.5 microM) in the perifusion medium. Three columns were exposed either to continuous, pulsatile, or no insulin. After 3 h, total nucleic acid was extracted, and mRNA(PEPCK) was measured with a sensitive-solution hybridization assay. Continuous insulin inhibited PEPCK expression in a dose-dependent fashion with EC50 1 x 10(-11) M. Equivalent time-averaged amounts of insulin delivered as pulses achieved significant inhibition but less effectively than continuous insulin. The apparent EC50 for pulsatile insulin increased from 2 x 10(-11) M to 5 x 10(-11) M as the oscillatory period was raised from 5 to 20 min, respectively. These observations suggest that insulin-mediated inhibition of PEPCK gene transcription is diminished by a pulsatile mode of administration in marked contrast to the pulse enhancement demonstrated for glucagon-mediated hepatic glucose production.

Spontaneous in vivo cyclic secretion of insulin and glucagon displays a pulse interval of 10 +/- 0.3 (SE) min and a constant phase relationship in fasting rhesus monkeys. When pancreata from six normal rhesus monkeys were perfused in vitro, the insulin pulse interval averaged 6.3 +/- 0.23 (SE) min. Insulin, glucagon, and somatostatin displayed high-amplitude secretory pulses, and the average pulse interval did not differ among the three islet hormones. The islet pulses are less regular in vitro than in vivo, and the phase relationship among the three hormones is lost. The relative amplitude averaged 142 +/- 10, 110 +/- 18, and 81 +/- 11% of the mean hormone concentrations for insulin, somatostatin, and glucagon, respectively. Similar differences in secretory pattern were observed during perfusion of three baboon pancreata compared with the in vivo pattern in this second primate species. The data suggest that the frequency and phase relationship of the islet pulsatile secretory system is modulated by factors extrinsic to the pancreas in the intact nonhuman primate. The nature of these modulating factors remains to be established. The apparent phase independence of the three islet hormones suggests that each of the major endocrine cell types of the islet possess independent episodic secretory mechanisms.

In 1980 we described an in vivo method for estimating the rate of glucose uptake among selected tissues during an acute insulin response. The method was based upon the same principles as Sokoloff's 2-deoxyglucose (2DG) method. We now report further examination of the basic assumptions of the model and validation of its general applicability by comparing the response of brain and other tissues to prolonged insulin infusion (while glucose is held constant) with their response to a single injection of insulin. The method provides a reproducible estimate of relative insulin response in any tissue that can be anatomically separated at death. Tissues that are minimally sensitive to insulin such as spleen, lung, skin, and gut do not display increments in the calculated value for net rate of tissue uptake of 2DG. Insulin-sensitive tissues display increased rates of uptake that are characteristic for each specific tissue, ranging in magnitude from 1.7- to 17.9-fold over basal among an array of insulin-sensitive tissues. The duration of a unit response to a sub-maximal dose of insulin also varied among the tissues, persisting for 20-30 min after plasma insulin had returned to basal in heart and for 10-20 min in the other insulin-sensitive tissues. The method provides a reproducible measure of glucose metabolism in vivo and has been validated as a means of quantifying relative insulin sensitivity among the peripheral tissues. During steady-state conditions with plasma glucose held constant, brain glucose metabolism was unaffected by a 60-min infusion of insulin.

The dose-response characteristics of three skeletal muscles, three adipose tissue beds, and heart muscle to single i.v. injection of insulin were compared in vivo. Comparisons were made at 8 dose levels spanning the entire range for response by all tissues and for the integrated whole body response as reflected in the rate of disappearance of 3H-2-deoxyglucose from plasma. The insulin-sensitive tissues varied widely with respect to the magnitude of the maximal response and the sensitivity to insulin as judged by the effective dose 50% (ED 50). Among the muscles, a slow-twitch oxidative muscle, soleus, was more sensitive than the fast-twitch glycolytic muscle, extensor digitorum longus (EDL), while a mixed muscle, quadriceps femoris, displayed even lower sensitivity. Heart muscle sensitivity was comparable to EDL. Among the adipose sites, the rank order of sensitivity was subcutaneous greater than epididymal much greater than omental. The threshold for a detectable response to insulin was 0.013 U/kg rat.

The effect of beta-cell deficiency on the spontaneous pulsatile secretory pattern of the islets of Langerhans was studied in the baboon. Measures of beta-cell function were correlated with the secretory pattern before and at intervals after streptozocin administration. The degree of insulin deficiency was variable and ranged from mild to moderate. Highly regular pulses were less prevalent in baboons compared with rhesus monkeys and humans, but the mean frequency was similar and was not affected by treatment. The principal effect of beta-cell destruction was to proportionately reduce the pulse amplitude of insulin (-39%, P less than .003) without detectable change in pulse frequency, interhormonal phase relationship, or the regularity of pulses. Glucagon-pulse amplitude also fell (-19%, P less than .09), but not significantly. However, glucagon-pulse amplitude was strongly correlated with insulin-pulse amplitude (r = -.59, P less than .002), whereas mean fasting plasma concentrations of insulin and glucagon were not significantly changed after treatment. Because streptozocin affects only the beta-cell, the data indicate a major influence of the insulin pulse on the alpha-cell secretory pulse. The data do not support the presence of a separate pacemaker for the alpha-cell but do not eliminate this possibility. The strong correlation of reduction in insulin-pulse amplitude with increasing fasting glucose and decreasing glucose disappearance lends support to growing evidence that the pattern of insulin secretion is an important determinant of normal glucose homeostasis.

Rapid sustained oscillations in basal plasma levels of insulin, glucose, and glucagon (9-12 min/cycle) have been identified in rhesus monkeys and in humans. To assess the possible regulatory role of nutritional state in the control of these plasma fluctuations, 12 chronically cannulated conscious rhesus monkeys were studied at varying intervals following ingestion of a mixed meal. Blood samples were withdrawn at 2-min intervals for periods of 10-30 min. In the postprandial period both absolute and relative amplitudes (half amplitude percent) of oscillations increased up to 16 h postfeeding to maximal amplitudes of +/- 32% for insulin, +/- 4% for glucose, and +/- 22% for glucagon. Periods were consistently in the 9- to 12-min range for insulin and glucose. Progressive deprivation to 88 h produced decreases in amplitudes and more irregularity in fluctuations but did not affect the underlying periodicity of these oscillations in plasma levels of insulin, glucose, and glucagon. Results are consistent with the existence of one or more inherent oscillators in the pancreas that act to synchronize the release of hormones from particular types of islet cells. The amplitude of these oscillations but not the frequencies is associated with changes in liver metabolism and nutritional state.

The effects of cholinergic and adrenergic blocking agents on the period and amplitude of sustained oscillations in plasma levels of insulin and glucose were studied in 16 overnight-fasted conscious rhesus monkeys. Blood samples were withdrawn at 2-min intervals before and during or following administration of drugs that affect neurotransmission. Cholinergic blockade with atropine had no effect on the oscillations. alpha-Adrenergic blockade with phentolamine caused a rise in plasma insulin that lasted less than 10 min and was followed by a slight, although not consistent, decrease in mean insulin and a consistent small decrease in glucose. There was a sustained increase in heart rate, but no effect on the oscillations of insulin or glucose. beta-Adrenergic blockade induced by propranolol led to persistent decreases in mean plasma insulin 60% below the base line, small but variable decreases in glucose, and a sustained decrease in heart rate, but no change in period or relative amplitude of the oscillations in plasma levels of either insulin or glucose. General anesthesia with pentobarbital did not eliminate these oscillations. Our observations on the failure of the blockade of certain putative efferent pathways or depression of higher cortical centers to alter the period of the oscillations of insulin reduce the likelihood that the oscillations are transmitted from a pacemaker in the central nervous system.

Oscillating plasma insulin levels, with periods averaging 9 min, in fasting rhesus monkeys have been previously reported by us. To test whether an oscillator in the central nervous system might be driving these oscillations, we subjected five male rhesus monkeys to morphine, reserpine, and halothane, agents known to affect the central nervous system, in an attempt to either disrupt or change the frequency of the oscillations. We could demonstrate no significant effect of any of the three drugs on the oscillations. We conclude, therefore, that the oscillations in plasma insulin are not driven by an oscillator in the central nervous system. Coupled with the results of others, these data suggest that these oscillations are probably due to an intrinsic pancreatic pacemaker.

Hepatic receptors are normally exposed to discrete pulses of insulin and glucagon at intervals of 8 to 16 min. Using a multicolumn system for perifusing hepatocytes, we investigated the effect of this pattern on the normal processing of the insulin receptor. Surface-receptor binding was measured in acid-washed cells harvested from individual columns. The number of high-affinity surface receptors fell to a nadir 1 min after the end of a 3-min square-wave pulse of insulin. The maximum reduction reached 45% of baseline at amplitudes of 1000 microU/ml or above. The number of surface binding sites returned to baseline 15 min after the end of the pulse, but the affinity constant of the high-affinity receptor was unchanged. The reduction of surface binding was dose dependent, with an ED50 of 251 +/- 34 microU/ml. Prolonging the pulse to 60 min did not affect the nadir or the rate of restoration of the surface-receptor population. The change in surface binding was reduced at 15 degrees C and abolished at 4 degrees C. After a pulse, the pattern of change was a period of rapid decline to a nadir (t1/2 less than or equal to 1 min) that persisted for 3-5 min, followed by restoration of surface binding that reached baseline in 10-15 min. This same pattern was present after six ED95 pulses delivered at intervals of 15 min. These data indicate that the internalization of hepatocyte surface receptors and their recycling and reinsertion into the plasma membrane can be entrained to pulses at the physiologic pulse frequency.

Oscillations in the concentration of plasma glucose were found to reflect large fluctuations in hepatic glucose production. The fluctuations in glucose production were synchronous with fluctuations in the concentration of plasma insulin and glucagon. This synchrony suggests that hepatic pathways are entrained to the islet cycle with a minimal time delay.

In order to separate direct effects of epinephrine on fuel metabolism from those mediated by glucagon, epinephrine (0.1 microng/kg-min) was infused for 120 min in 18- and 65-h fasted, nonanesthetized baboons with and without a concomitant somatostatin infusion. At both stages of fasting, epinephrine stimulated glucagon, secretion, and this was blocked by somatostatin. At 18 h, with epinephrine alone, glucose rose early and remained elevated throughout the infusion. In the glycogen-depleted 65-h fasted animals, there was attenuation of the early glucose rise, with glucose reaching a maximum level at 100-120 min. With somatostatin blockade of glucagon release in the 18-h fasted animals, a pattern of attenuated early glucose rise similar to that of the 65-h fasted animals occurred. Somatostatin also inhibited this early glycogenolytic response when the epinephrine dose was increased fivefold. The behavior of FFA, glycerol, and beta-hydroxybutyrate was unchanged by the addition of somatostatin to epinephrine at either stage of fasting. Thus, glucagon mediates the early glycogenolytic response to epinephrine, but not the delayed hyperglycemia and probably not the lipolysis.

The hypothesis that insulin might promote increased glucose metabolism in putative glucoreceptor areas of the brain was investigated in the rat. Using tritiated 2-deoxyglucose (2-DG), unanesthetized fasted rats were injected with 0.1 U insulin and studied 30 min later. The local uptake of 2-DG into discrete brain areas was examined in serial frozen 400-micrometer sections. Areas 1.1 mm in diameter were punched from the region of the ventral medial, ventral lateral, and dorsal hypothalamus and from a control area from the cerebral cortex. The punched tissue segments were analyzed for total radioactivity and protein content. The results showed that insulin failed to influence the pattern of 2-DG uptake into these discrete brain regions. When the data were analyzed with a simple kinetic model to determine the net fractional rate of uptake of 2-DG by the tissues, brain tissues displayed a 75% rate increase compared to saline-treated controls. Heart muscle collected from the same rats showed a 700% increase after insulin, while lung, an insulin insensitive tissue, displayed a 30% increase. Because the nonsteady state conditions of the model dictate a number of assumptions, the modest increase in the calculated rate of uptake in brain tissue must be verified by a steady state model before it can be accepted as representing a real effect of insulin upon the overall metabolism of glucose in the brain. Regardless of these reservations, it may be concluded from the pattern of response, that insulin does not selectively increase glucose uptake or metabolism in the putative glucoreceptor areas of the hypothalamus under the conditions of these experiments.

Somatostatin, a hypothalamic peptide that inhibits the secretion of pituitary growth hormone, inhibits basal insulin secretion in fasted cats and rats. In fasted baboons both basal and arginine-stimulated secretion of insulin and glucagon are inhibited. Somatostatin appears to act directly on the endocrine pancreas. The action is dose-related, rapid in onset, and readily reversed.

Perfusion of growth hormone inhibitory factor (somatostatin) into rat pancreas inhibited secretion of glucagon and insulin into medium containing 5.5 mM glucose. A 15-min infusion of arginine (20 mM) greatly increased glucagon and insulin secretion. When perfused simultaneously with arginine, somatostatin (55 nM) abolished the increase in glucagon secretion. The acute phase of insulin secretion in response to arginine was attenuated by somatostatin, and subsequent secretion was decreased to control levels. Pretreatment for 5 min with somatostatin blocked even acute-phase insulin secretion in response to arginine. Somatostatin did not affect basal or glucose-stimulated secretion of insulin from rat pancreatic islets isolated by the collagenase technique. Arginine-stimulated secretion of insulin was enhanced by somatostatin in isolated islets. These results demonstrate a direct effect of somatostatin on the pancreas to inhibit secretion of glucagon and insulin. The failure of somatostatin to inhibit insulin secretion in pancreatic islets may be due to alterations in the beta cells produced by the isolation procedure. It is also possible that the effect of somatostatin on insulin secretion may be mediated indirectly.

The previously reported synchronous oscillations in plasma glucose and insulin levels have been further studied to determine whether the phenomenon can be attributed to cyclic secretion or degradation of the hormone. Plasma C-peptide concentrations were measured and found to cycle with the same period as plasma insulin suggesting that the oscillations of insulin arise from changing secretion rather than degradation. The amplitude of C-peptide oscillation was 50 percent that of insulin. Assuming first order kinetics and the same relative rates of disappearance as in humans, this difference in amplitudes in consistent with equal secretion rates for the two peptides.

In overnight fasted rhesus monkeys, synchronous, regular oscillations occurred in the plasma concentrations of glucose, insulin, and glucagon. The oscillations displayed a period averaging 9 minutes. The amplitudes for insulin and glucagon were ten and five times greater than for glucose. Insulin cycled in and glucagon out of phase with glucose. In baboons, oscillations of glucose and insulin were smaller than in rhesus monkeys, while in man, regular oscillations were not observed.

The intravenous infusion of 600 milligrams of glucose over 30 minutes caused a 17 percent fall in the concentration of free fatty acids in arterial blood of subjects who had fasted overnight. This response to glucose was abolished in subjects treated previously with ganglionic or adrenergic blocking agents. Small amounts of insulin were secreted in response to these glucose infusions, but in insulin-dependent diabetics incapable of altering plasma insulin to any great extent, the effect of glucose upon free fatty acids could be obtained provided the subjects were primed with long-acting insulin before the infusions were begun. The response of free fatty acids to doses of glucose which elevated the concentration of glucose in arterial blood by only 3 mg percent and the blockade of this response by autonomic and adrenergic blocking agents suggest that centers in the central nervous system exist which are capable of responding to elevations of arterial glucose by inhibiting the sympathetic tone partially responsible for sustaining lipolysis in fasting.

In obese adult diabetics, the concentration of insulin in venous plasma was unrelated to the degree of hyperglycemia after an overnight fast. However, in these subjects, insulin rose and fell in proportion to the magnitude of change in plasma glucose induced by small intravenous infusions of glucose. The minimal dose of glucose to cause a significant rise in insulin above the fasting level was similar in normal subjects, obese nondiabetic subjects, and in obese, hyperglycemic adult diabetics. This dose lay between infusion of 60 and 100 mg of glucose per min for 30 min. These results suggested that the secretion of insulin was under regulation by changes in blood glucose but was not stimulated in proportion to the stable raised blood glucose concentration of the hyperglycemic diabetic. Artificial hyperglycemia was induced in fasting normal subjects by constant intravenous infusion of glucose at rates of 100-250 mg of glucose per min for periods up to 8 hr. Plasma glucose rose during the 1st hr of infusion and then remained constantly elevated for up to 8 hr. The concentration of plasma insulin paralleled that of plasma glucose. During the period of constant hyperglycemia and elevated insulin, superimposition of a brief additional glucose load resulted in a prompt rise in glucose and insulin, both returning to the previous elevated levels. Thus in normals as well as obese diabetics, stable hyperglycemia does not produce a pancreatic response sufficient to return the blood glucose to an arbitrary normal fasting concentration, yet the beta cells remain readily responsive to a change in plasma glucose. These data suggest that the beta cells do not operate as a control system with an absolute reference point when presented with systemic hyperglycemia. The behavior of the beta cells during hyperglycemia in the fasting obese adult diabetic suggests that the regulation of the basal insulin secretion may not be determined by factors directly related to the prevailing concentration of glucose. It is postulated that the beta cells adapt to hyperglycemia perhaps through the operation of controls directed toward a normal delivery of free fatty acids or some other cellular metabolic substrate during fasting.