Evaluation of the control of glucagon secretion by the parasympathetic nervous system in man.

To evaluate the influence of the parasympathetic nervous system on human glucagon secretion, we have measured the plasma immunoreactive glucagon (IRG) levels after the administration of edrophonium, bethanechol chloride, and 2-deoxyglucose, and have compared the IRG responses to hypoglycemia in normal, atropinized, and vagotomized man. Edrophonium administered i.v. and bethanechol chloride administered s.c. did not affect IRG levels. Two-deoxy-glucose resulted in symptomatic neuroglucopenia with resultant vagal discharge, as evidenced by increased gastric acid secretion; but no changes in IRG concentrations were observed. The IRG response to insulin-induced hypoglycemia in normal subjects was not influenced by the administration of atropine. In seven subjects with a truncal vagotomy and no increased gastric acid secretion during insulin-induced hypoglycemia, the IRG increases were indistinguishable from those of control subjects in terms of timing, peak level obtained, or total glucagon response. We conclude that the cholinergic system is unlikely to play an important role in modulating glucagon secretion in man.     

The parasympathetic nervous system and the thermic effect of glucose/insulin infusions in humans

The thermic effect of glucose/insulin infusions was investigated in seven healthy young men before and during either inhibition (atropine sulphate 10 micrograms/kg bolus; 10 micrograms/kg/h) or stimulation (edrophonium chloride, 10 mg bolus; 0.75 mg/min starting rate) of the parasympathetic nervous system. The thermic effects of glucose/insulin were 6.2% +/- 0.4% and 5.6% +/- 0.7% before atropine and edrophonium, respectively, and increased to 7.1% +/- 0.5% (NS) with atropine and 7.5% +/- 1.2% (P less than .05) with edrophonium. In four subjects atropine or edrophonium was infused before the hyperinsulinemic, euglycemic clamp. A significant increase in resting metabolic rate and plasma norepinephrine concentrations was observed with edrophonium alone. When the thermic effects of glucose/insulin were calculated with respect to the metabolic rates observed during the drug infusions alone, they were 5.9% +/- 1.4% and 3.6% +/- 0.6% (NS) for the clamp + atropine and clamp + edrophonium, respectively. These results demonstrate that the increases in the thermic effect of glucose/insulin infusions observed during inhibition or stimulation of the parasympathetic nervous system were due to atropine or edrophonium increasing the resting metabolic rate rather than increasing the thermic response to glucose-insulin infusions. However, because it has been shown that atropine can decrease the thermic effect of an orally administered meal by approximately 60%, it would appear that the parasympathetic nervous system can influence the thermic effect of food by affecting the rate of digestion, absorption and storage of the ingested nutrients.     

Cyclic changes in the vaginal epithelium of normal rhesus macaques

Studies in nonhuman primates indicate that changes in the thickness and integrity of the vaginal epithelium affect the transmission rates of HIV-1, but few studies have examined the normal variations that may occur in the vagina of normal macaques as a result of aging or changes in the menstrual cycle. This study was conducted to determine if differences occur in the thickness of the vaginal mucosa with age or menses. Vaginal mucosal thickness was compared in 46 rhesus macaques grouped as juvenile (1-3 years old), mature cycling (3-21 years old), and geriatric (> 21 years old). Epithelia of mature cycling macaques were also compared at different stages of the menstrual cycle. Older females (> 21 years) had the thinnest and least keratinized epithelium of all groups, followed by the youngest females (< 3 years). The vaginal epithelium was also thinner in cycling macaques during menses compared to the follicular stage. In addition, young, geriatric, or cycling macaques during menses had minimal keratinization. We hypothesize that normal physiologic changes in the vaginal epithelium of women occur with age and menses, which may affect a woman's susceptibility to HIV-1 transmission and other sexually transmitted diseases. Also, age and menstrual cycle should be considered when designing vaginal transmission experiments in rhesus macaques.     

Influence of the gonads on cortisol secretion in female rhesus macaques

Circadian and ultradian patterns of plasma cortisol were assessed in four intact female rhesus macaques during both the late follicular (days 9-10) and midluteal (days 21-22) phases of the menstrual cycle and compared to patterns in four ovariectomized macaques. Blood samples were drawn from a remote site at 15-min intervals for 24 h via an indwelling catheter. Measures of estrogen and progesterone were obtained for each subject. For purposes of data analyses, group cortisol measurements were collapsed across hourly intervals and submitted to analysis of variance. Pulsatile characteristics of cortisol release were determined for each subject using the PULSAR computer program. Circadian cortisol patterns, present in all three groups, were characterized by a progressive rise during early morning hours (0300-0600 h), followed by a decline of short duration. All groups then displayed an unexpected midday peak (0900-1400 h), at which time cortisol levels reached their daily zenith. In each of the three groups, cortisol levels reached a nadir during late afternoon hours shortly after the light phase ended. The amplitude of circadian changes and daily mean levels of cortisol were significantly reduced by ovariectomy, without alterations in pulsatile characteristics of cortisol secretion. Daily mean cortisol levels decreased from approximately 8 micrograms/100 ml in intact subjects to 4.5 micrograms/100 ml after ovariectomy. No significant differences in the circadian/ultradian periodicity of cortisol secretion were detected between the follicular and luteal groups. When data in the intact female groups were combined and compared to those previously obtained from gonadally intact adult male macaques, similar 24-h patterns of cortisol secretion were detected. Surprisingly, amplitude changes in cortisol concentrations after ovariectomy were temporally and quantitatively similar to those in orchidectomized males. In both male and female animals, circadian patterns of cortisol secretion were reduced by gonadectomy. These results are discussed in terms of the activational influence of gonadal steroids on hypothalamic-pituitary-adrenocortical function.     

Central administration of leptin inhibits food intake and activates the sympathetic nervous system in rhesus macaques

The present study was performed to determine the effects of central administration of leptin on food intake and sympathetic nervous system activity in a nonrodent species, the rhesus monkey. Peripheral administration of leptin at doses (1 and 3 microg/kg, s.c.) that produced increments of circulating leptin concentrations within a physiological range did not inhibit food intake over the subsequent 3 days. In contrast, leptin (1 microg/kg, intracerebroventricularly) had no acute effect on food intake, but caused a significant and sustained suppression (40-50%) of food intake during the entire following day (P < 0.01). In addition, circulating norepinephrine levels increased by 55 +/- 16% (P < 0.02) 1 h after intracerebroventricular leptin administration, but did not increase after artificial cerebrospinal fluid administration. These results indicate that leptin can provide a signal to the central nervous system that decreases food intake in primates and in addition acutely activates the sympathetic nervous system. However, the results showing an acute increase in circulating leptin concentrations after peripheral administration of human leptin suggest that in primates, increases in circulating leptin within the physiological range do not acutely regulate food intake. Leptin may be more important in regulating food intake when there are sustained changes in circulating concentrations of leptin (e.g. with obesity, prolonged energy restriction, or diabetes).     

Changes in gastric secretion following administration of preparations affecting the parasympathetic and sympathetic nervous system.

The changes in gastric secretion following administration of different preparations have been studied in a group of 180 patients with duodenal ulcer and chronic gastritis. In patients with hypersecretion the medication with bishpan and inderal as compared with atropine caused considerable shifts in the indices of gastric secretion. Isoproterenol exerted a stimulating influence both on initial hyper- and hyposecretion, while the other preparations studied were shown not to cause any essential rise in gastric secretion in patients with secretory deficiency. Reliable changes of the stimulated gastric secretion were observed only after inderal. The data obtained can be useful when choosing a rational method for the therapy of gastric scretory disorders.     

The involvement of the sympathetic nervous system in meal-induced thermogenesis in mice.

The acute effect of food intake on the activity of the sympathetic nervous system (SNS) in both heart and brown adipose tissue (BAT) was investigated in mice. Upon delivery to the laboratory mice were housed singly and divided into two groups. Half the mice were accustomed to eat their daily food ration in two meals whereas the other half were given continuous access to food. SNS activity in both heart and BAT was estimated by measuring the accumulation of dopamine (DA) after having blocked the transformation of dopamine into noradrenaline (NA) with 1-cyclohexyl-2-mercapto-imidazole (CHMI). CHMI inhibits the enzyme dopamine beta-hydroxylase. On the day SNS activity was assessed, continuously fed (CF) or meal-fed (MF) mice were injected with either saline or CHMI one hour before being killed. In order to assess the anticipatory effects of being fed, a group of mice already accustomed to the meal-feeding schedule were not allowed to eat after the injections. Additional CF and MF mice were killed without being injected in order to determine the basal levels of both DA and NA. The results show that the accumulation of DA in both heart and BAT was higher in MF than CF mice regardless of whether MF mice were or were not fed after the injection of CHMI. It therefore appears that the intake of food may increase SNS activity in various tissues in mice, and that such a response may be largely of cephalic origin.     

Pulsatile secretion of luteinizing hormone during the menstrual cycle of rhesus macaques

The secretion of LH, PRL, and cortisol was investigated in 4 sexually mature female rhesus macaques with cardiac catheters protected by tethers. Based on endocrine parameters, all 4 of the animals ovulated within 2 months from the time they were tethered, and regular menstrual cycles of 24-34 days were observed. The catheters remained patent for 6-12 months without reposition or repair. Plasma levels of 2 stress-labile hormones, PRL and cortisol, showed diurnal fluctuations comparable to those observed in untethered animals. The frequency of LH secretory episodes was determined by measuring bioactive LH in blood samples collected at 10-min intervals in the follicular phase and at 15-min intervals in the luteal phase of the menstrual cycle. In 10 trials during the follicular phase, we estimated that an average of between 14 and 15 LH pulses occurred every 12 h. The interpulse interval ranged between 20-80 min and averaged 50 min. No change in pulse frequency was observed across the follicular phase. The number of LH pulses decreased after ovulation, and by the end of the luteal phase, the interpulse interval was 4-6 h. One example during the preovulatory LH surge revealed the high frequency, high amplitude nature of LH secretion at that time. Our experience indicates that tethered animals with cardiac catheters show no hormonal indications of stress and represent the best available model for studies requiring frequent and prolonged access to the vascular system. Our data suggest that peripheral LH fluctuations in rhesus monkeys, as in other mammals, are pulsatile, and the frequency of these pulsatile episodes changes with different phases of the menstrual cycle, presumedly in response to varying stimuli to the pituitary from the brain.     

Role of parasympathetic nervous system in glucagon response to insulin-induced hypoglycemia in normal and diabetic rats

Effects of cholinergic mechanisms on glucagon and epinephrine responses to insulin-induced hypoglycemia were examined in diabetic and age-matched control male rats. Atropine did not affect plasma glucose levels or plasma glucagon concentrations, in the basal state, in normal or short-term diabetic rats (10 to 15 days following streptozotocin injection). However, atropine blocked the glucagon response to insulin hypoglycemia in both normal and short-term diabetic rats. Subcutaneous injection of carbachol also failed to alter basal plasma glucose, glucagon, or epinephrine values in both normal and diabetic rats. The lack of glucagon and epinephrine responses to insulin hypoglycemia in long-term diabetic rats (80 to 100 days after streptozotocin injection) was reversed with a single dose of carbachol. Carbachol exaggerated the glucagon response to insulin hypoglycemia in normal and short-term diabetic rats. These results demonstrate that the parasympathetic nervus system plays an important role in the glucagon release in response to insulin hypoglycemia in rats. The lack of glucagon response to insulin hypoglycemia observed in long-term diabetic rats could be due to deteriorated parasympathetic nervous system and also could be corrected with carbachol.     

Central nervous system insulin receptors in normal and diabetic rats.

The presence of specific insulin receptors in homogenates of various parts of the rat brain was demonstrated. Hypothalamic preparations exhibited the greatest insulin binding. Scatchard plots of the binding data were typically curvilinear. Insulin binding to the brain homogenates was influenced by pH, incubation time, temperature, and ionic milieu. The specificity of insulin binding to the brain homogenates was demonstrated by partial displacement of labeled insulin by insulin analogs according to their biological activities and by inhibition of insulin binding by serum containing an antibody to insulin receptors. Insulin binding to brain homogenates from streptozotocin-induced diabetic rats was similar to that from control animals and failed to indicate up-regulation of these receptors.     

Third-ventricular infusion of neuropeptide Y suppresses luteinizing hormone secretion in ovariectomized rhesus macaques

Neuropeptide Y (NPY) has been shown to modulate gonadotropin secretion in an estrogen-dependent manner in the rat and rabbit, and to act centrally in these species to alter GnRH release. The present study examined the ability of centrally administered NPY to affect LH secretion in the primate. Human NPY (hNPY) was administered into the third cerebroventricle of unanesthetized, freely moving, ovariectomized (OVX), or estradiol (E2)-treated OVX rhesus monkeys. LH was measured in blood samples collected remotely at 10-min intervals throughout the experiments. An extensive range of NPY doses was tested in a preliminary study in which OVX monkeys received a 3-h control infusion of Krebs Ringer phosphate buffer (KRP) followed immediately by a 3-h infusion of hNPY (0.1-50 micrograms/h). Only doses greater than or equal to 5 micrograms/h produced a consistent and marked suppression of LH (5 micrograms/h; 35.1 +/- 7.2% reduction, P less than 0.05, n = 4). A longer duration study was performed to better characterize changes in LH pulse frequency and amplitude produced by hNPY treatment. We administered 5 micrograms/h and 15 micrograms/h to OVX (n = 5) and E2-treated OVX (n = 4) monkeys according to the following protocol: 8 h control KRP/8-h hNPY/6-h recovery KRP. In OVX monkeys, LH was suppressed after 2 to 3 h of peptide infusion (P less than 0.01); LH secretion returned to normal after treatment. Both doses of hNPY suppressed mean LH by approximately 55% (P less than 0.05) and LH pulse frequency by approximately 69% (P less than 0.025). LH pulse amplitude was unaffected. In E2-treated OVX monkeys, neither dose of hNPY affected mean LH or LH pulse amplitude. LH pulse frequency was suppressed by approximately 65% (P less than 0.05) during 15-micrograms/h treatment. Because centrally administered hNPY reduced LH pulse frequency and thereby mean LH levels, our results support a central, neural action of NPY to affect the GnRH/LH secretory system. The ability of estrogen feedback to alter the response to NPY treatment supports a physiological role for NPY in controlling reproduction in the primate.     

Effect of the parasympathetic system on secretion of parathyroid hormone

This study evaluated the effect of parasympathetic agonists and antagonists on immunoreactive (i) PTH secretion in vitro and on serum iPTH in vivo in rats. In in vitro studies pilocarpine or bethanechol significantly inhibited PTH secretion. This inhibition was blocked by the simultaneous addition of atropine to the incubation medium. In in vivo studies, the cholinergic agonists pilocarpine and bethanechol and the cholinergic antagonist atropine were administered to rats by IV infusion. Blood was obtained before and again after two hours of infusion for analysis of iPTH. Pilocarpine or bethanechol significantly decreased serum iPTH. This inhibition by either agent was blocked by the simultaneous administration of atropine. Administration of atropine alone significantly increased serum iPTH above baseline. This stimulation of basal serum iPTH by parasympathetic blockade suggests that even basal PTH secretion may be influenced by endogenous parasympathetic tone. Therefore, the following conclusions were reached: (1) parasympathetic influences inhibit PTH secretion, and (2) endogenous parasympathetic tone may be an inhibitory modulator of basal secretion of PTH.     

Islet-cell-to-cell communication as basis for normal insulin secretion

The emergence of pancreatic islets has necessitated the development of a signalling system for the intra- and inter-islet coordination of β cells. With evolution, this system has evolved into a complex regulatory network of partially cross-talking pathways, whereby individual cells sense the state of activity of their neighbours and, accordingly, regulate their own level of functioning. A consistent feature of this network in vertebrates is the expression of connexin (Cx)-36-made cell-to-cell channels, which cluster at gap junction domains of the cell membrane, and which adjacent β cells use to share cytoplasmic ions and small metabolites within individual islets. This chapter reviews what is known about Cx36, and the mechanism whereby this β-cell connexin significantly regulates insulin secretion. It further outlines other less established functions of the protein and evaluates its potential relevance for the development of novel therapeutic approaches to diabetes.     

Nicotinamide and insulin secretion in normal subjects

Summary Nicotinamide has been given both before and after clinical onset of Type 1 (insulin-dependent) diabetes mellitus in an attempt to prolong beta-cell survival. Nicotinic acid, structurally similar to nicotinamide, induces insulin resistance and increases insulin secretion in healthy individuals. It is not known if nicotinamide has similar effects. Since insulin secretion, as measured by the acute insulin response to intravenous glucose, is used to predict diabetes and to monitor therapy, the effects of nicotinamide must be established before trials in individuals at high risk of progression to Type 1 diabetes can be interpreted. Intravenous tolerance tests were performed according to the ICARUS standard protocol in 10 healthy, adult subjects (age 32±5.7 years) before and after 14 days of treatment with nicotinamide 25 mg · kg^–1 · day^–1. The acute insulin response after nicotinamide did not differ from the control study, whether measured as the incremental 0–10 min insulin area (278±142 vs 298±130mU · l^–1 · 10 min^–1) or as the 1±3 min insulin level (78±39 vs 81±44 mU/l). The late insulin response was equally unaffected, as were basal insulin (5.2±1.6 vs 5.6±2.1 mU/l) and glucose (5.0±0.4 vs 4.9±0.2 mmol/l) levels and glucose disposal rates (1.98±0.88 vs 2.04±0.68%/min). Nicotinamide does not affect insulin secretion and glucose kinetics in normal subjects, confirming its suitability for trials designed to delay or prevent the onset of Type 1 diabetes.     

Circadian periodicity in circulating cortisol is absent after orchidectomy in rhesus macaques

Circadian/ultradian patterns of plasma cortisol were assessed in four intact and four gonadectomized male rhesus macaques. Concomitant measures of testosterone (T) were analyzed in the intact animals. Blood samples were drawn every 15 min for 28 h via an indwelling catheter. Plasma concentrations of cortisol and T were determined by RIA. Inverse diurnal patterns of cortisol and T secretion were documented in the control group. The circadian pattern of plasma cortisol was characterized by a progressive rise during the early morning hours (0200-0800 h), followed by a gradual decline until lowest levels were reached at 1600-1900 h. T levels were lowest from 0800-1200 h (2-4 ng/ml) and reached a zenith from 2200-0200 h (8-10 ng/ml). Surprisingly, circadian fluctuations in cortisol were absent after orchidectomy. Cortisol levels in castrates were less (P less than 0.05) than those during peak levels of cortisol secretion in the intact animals (6.25 vs. 10.1 micrograms/100 ml) and elevated (P less than 0.01) compared to concentrations in the intact animals at their nadir (6.4 vs. 2.5 micrograms/100 ml). The loss of circadian fluctuation was not associated with a significant change in frequency of pulsatile cortisol release or a change in the daily mean level. These results are the first in nonhuman primates to demonstrate that the testes modulate circadian activity in the hypothalamic-pituitary-adrenocortical axis. The data differ from previous findings in rodents suggesting that castration increases the synthesis and release of ACTH/corticosterone.