PreproTRH(158-183) fails to affect pituitary-adrenal response to CRH/vasopressin in man: a pilot study.

Non-glucocorticoid inhibitors of the HPA-system are of utmost interest in the treatment of diseases with impaired regulation of this system, like the metabolic syndrome and depression. In rats, a fragment of the thyreotropin-releasing hormone (TRH) preprohormone, preproTRH((178-199)), has been demonstrated to inhibit basal and stimulated secretion of cortisol. Our pilot study aimed to explore the first time similar effects of the homologue peptide preproTRH((158-183)) in healthy humans. In a double-blind within-subject comparison, eight healthy young men were infused intravenously with placebo and preproTRH((158-183)) at varying doses of 5, 10, 25 and 50 mg/kg of body weight. After 15 min of infusion a corticotropin-releasing hormone (CRH)/vasopressin-test was performed. Plasma concentrations of pituitary hormones and free thyroxine, blood pressure, heart rate and feelings of activation and mood were assessed repeatedly at close intervals. Individual hormone profiles and collapsed data across all doses did not reveal any effects of preproTRH((158-183)) on HPA-activity, although it increased TSH and fT4, stimulated the release of GH and increased systolic blood pressure in the course of the experiment (p<0.05, for all effects). Self-reports indicated enhanced feelings of activation and general well-being following preproTRH (p<0.05). Our data exclude a substantial inhibitory effect of preproTRH((158-183)) on HPA secretory activity and, thus, contrast with findings in rats. In humans, the peptide appears to even exert an albeit weak stimulatory effect on autonomic stress systems as indicated by increased cardiovascular activity in combination with enhanced subjective arousal.

The principle of homeostasis in the hypothalamus-pituitary-adrenal system: new insight from positive feedback.

Feedback control, both negative and positive, is a fundamental feature of biological systems. Some of these systems strive to achieve a state of equilibrium or "homeostasis". The major endocrine systems are regulated by negative feedback, a process believed to maintain hormonal levels within a relatively narrow range. Positive feedback is often thought to have a destabilizing effect. Here, we present a "principle of homeostasis," which makes use of both positive and negative feedback loops. To test the hypothesis that this homeostatic concept is valid for the regulation of cortisol, we assessed experimental data in humans with different conditions (gender, obesity, endocrine disorders, medication) and analyzed these data by a novel computational approach. We showed that all obtained data sets were in agreement with the presented concept of homeostasis in the hypothalamus-pituitary-adrenal axis. According to this concept, a homeostatic system can stabilize itself with the help of a positive feedback loop. The brain mineralocorticoid and glucocorticoid receptors-with their known characteristics-fulfill the key functions in the homeostatic concept: binding cortisol with high and low affinities, acting in opposing manners, and mediating feedback effects on cortisol. This study supports the interaction between positive and negative feedback loops in the hypothalamus-pituitary-adrenal system and in this way sheds new light on the function of dual receptor regulation. Current knowledge suggests that this principle of homeostasis could also apply to other biological systems.

Causes of obesity: looking beyond the hypothalamus.

The brain takes a primary position in the organism. We present the novel view that the brain gives priority to controlling its own adenosine triphosphate (ATP) concentration. It fulfils this tenet by orchestrating metabolism in the organism. The brain activates an energy-on-request system that directly couples cerebral supply with cerebral need. The request system is hierarchically organized among the cerebral hemispheres, the hypothalamus, and peripheral somatomotor, autonomic-visceromotor, and the neuroendocrine-secretomotor neurons. The system initiates allocative behavior (i.e. allocation of energy from body to brain), ingestive behavior (intake of energy from the immediate environment), or exploratory behavior (foraging in the distant environment). Cerebral projections coordinate all three behavioral strategies in such a way that the brain's energy supply is guaranteed continuously. In an ongoing learning process, the brain's request system adapts to various environmental conditions and stressful challenges. Disruption of a cerebral energy-request pathway is critical to the development of obesity: if the brain fails to receive sufficient energy from the peripheral body, it compensates for the undersupply by increasing energy intake from the immediate environment, leaving the body with a surplus. Obesity develops in the long term.

The selfish brain: competition for energy resources.

Although the brain constitutes only 2% of the body mass, its metabolism accounts for 50% of total body glucose utilization. This delicate situation is aggravated by the fact that the brain depends on glucose as energy substrate. Thus, the contour of a major problem becomes evident: how can the brain maintain constant fluxes of large amounts of glucose to itself in the presence of powerful competitors as fat and muscle tissue. Activity of cortical neurons generates an "energy on demand" signal which eventually mediates the uptake of glucose from brain capillaries. Because energy stores in the circulation (equivalent to ca. 5 g glucose) are also limited, a second signal is required termed "energy on request"; this signal is responsible for the activation of allocation processes. The term "allocation" refers to the activation of the "behavior control column" by an input from the hippocampus-amygdala system. As far as eating behavior is concerned the behavior control column consists of the ventral medial hypothalamus (VMH) and periventricular nucleus (PVN). The PVN represents the central nucleus of the brain's stress systems, the hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). Activation of the sympatico-adrenal system inhibits glucose uptake by peripheral tissues by inhibiting insulin release and inducing insulin resistance and increases hepatic glucose production. With an inadequate "energy on request" signal neuroglucopenia would be the consequence. A decrease in brain glucose can activate glucose-sensitive neurons in the lateral hypothalamus (LH) with the release of orexigenic peptides which stimulate food intake. If the energy supply of the brain depends on activation of the LH rather than on increased allocation to the brain, an increase in body weight is evitable. An increase in fat mass will generate feedback signals as leptin and insulin, which activate the arcuate nucleus. Activation of arcuate nucleus in turn will stimulate the activity of the PVN in a way similar to the activation by the hippocampus-amydala system. The activity of PVN is influenced by the hippocampal outflow which in turn is the consequence of a balance of low-affinity and high-affinity glucocorticoid receptors. This set-point can permanently be displaced by extreme stress situations, by starvation, exercise, hormones, drugs or by endocrine-disrupting chemicals. Disorders in the "energy on request" process will influence the allocation of energy and in so doing alter the body mass of the organism. In this "selfish brain theory" the neocortex and the limbic system play a central role in the pathogenesis of diseases, such as anorexia nervosa, obesity and diabetes mellitus type II. From these considerations it appears that the primary disturbance in obesity is a displacement of the hippocampal set-point of the system. The resulting permanent activation of the feedback system must result in a likewise permanent activation of the sympatico-adrenal system, which induces insulin resistance, hypertension and the other components of the metabolic syndrome. Available therapies for treatment of the metabolic syndrome (blockade of alpha- and beta-adrenergic receptors, insulin and insulin secretagogues) interfere with mechanisms, which must be considered compensatory. This explains why these therapies are disappointing in the long run. New therapeutic strategies based on the "selfish brain theory" will be discussed.

High plasma VEGF relates to low carbohydrate intake in patients with type 2 diabetes.

OBJECTIVE: Vascular endothelial growth factor (VEGF) has been suggested to enhance glucose transport across the blood-brain barrier, thereby increasing brain glucose supply. Increased brain glucose concentration is known to suppress food intake and to decrease body mass via action on hypothalamic regulation centers. Based on the crucial role of VEGF on brain glucose supply, we hypothesized that higher VEGF concentrations are associated with lower food intake and body mass in humans. METHODS: Intending to investigate subjects with high variance of blood glucose, we examined patients with type 2 diabetes mellitus. Our hypothesis was tested in a population-based cohort of 190 subjects with type 2 diabetes. Plasma VEGF levels in conjunction with other parameters known to modulate food intake were measured and subsequently correlated with food intake patterns at a breakfast buffet as well as with body mass. RESULTS: We found that subjects with higher concentrations of plasma VEGF had 17% less carbohydrate intake (P=0.003) and 4.8% lower body mass (P=0.017) than those with lower VEGF concentrations. Intake of protein and fat did not correlate with VEGF concentrations. These associations of plasma VEGF were confirmed in multiple linear regression analyses controlling for several parameters interacting with food intake. CONCLUSION: We conclude that high plasma VEGF concentrations are associated with less carbohydrate intake and lower body mass in type 2 diabetes. The role VEGF plays in facilitating glucose access to the brain represents a new aspect of food intake regulation and energy homeostasis, with relevance for diseases with body mass disturbances.

Acute effects of aldosterone on the autonomic nervous system and the baroreflex function in healthy humans.

Aldosterone has been reported to impair the baroreflex response in animal models. The present study aimed to investigate the acute effects of aldosterone on the autonomic nervous system and the baroreflex control of muscle sympathetic nerve activity (MSNA) and heart rate in healthy humans. Nine healthy subjects were examined in a double-blind, placebo-controlled, cross-over study design, receiving either i.v. aldosterone (100 microg) or placebo on the experimental day. Heart rate variability (HRV) was measured at rest, whereas blood pressure, heart rate and MSNA (assessed by microneurography from the peroneal nerve) were monitored both at rest and during baroreflex tests. Baroreceptor stimulation and deactivation was induced by i.v. infusion of incremental doses of phenylephrine and sodium nitroprusside. HRV indices at rest were specifically changed by aldosterone with a significant increase in standard deviation of RR intervals and total power, and a trend towards increased time domain parameters indicating parasympathetic predominance in heart rate regulation. Basal MSNA, blood pressure and heart rate remained unaffected by aldosterone administration. Sodium nitroprusside decreased diastolic blood pressure and increased MSNA as well as heart rate in both the aldosterone and placebo experiments. However, the tachycardic response to arterial baroreceptor deactivation was more pronounced in the aldosterone experiments. By contrast, baroreflex control of MSNA and heart rate during phenylephrine infusion was not affected by aldosterone. Thus, our study demonstrates that, in healthy humans, aldosterone tends to increase cardiac vagal activity and enhances the heart rate response to nitroprusside whereas MSNA remains unaffected.

Sleep loss and the development of diabetes: a review of current evidence.

Emerging evidence suggests that short duration of sleep and sleep disturbances increase the risk of developing diabetes. The mechanism of this presumed adverse influence of sleep loss on glucose metabolism is not well understood yet. However, in diabetes research and diabetes care, the multitude of influences of sleep and sleep loss on glucose regulation has been largely neglected so far. Here, we provide a short overview of the current epidemiological and experimental evidence for a potential contribution of sleep loss to the development of diabetes.

Melatonin does not inhibit hypothalamic-pituitary-adrenal activity in waking young men.

The pineal hormone melatonin is mainly secreted during night-time which, in humans, is the normal time of sleep. It has been proposed that, during this period, melatonin exerts an inhibitory influence on secretory activity of the hypothalamic-pituitary-adrenal (HPA) system, although there is little evidence for this view in humans. In blind humans, a single oral dose of melatonin at bed time suppressed nocturnal cortisol secretion. However, suppression could have been secondary to an improved sleep after melatonin in these experiments. In the present study, we examined whether melatonin exerts a similar inhibitory effect on HPA activity in waking subjects. Fourteen healthy young men were tested at bed time, but kept awake throughout the experimental epoch. Thirty minutes after oral ingestion of 5 mg melatonin, activity of the HPA-system was stimulated through a standard insulin-induced hypoglycaemia. Adrenocorticotrophin hormone and cortisol concentrations under basal conditions before insulin injection, as well as in response to insulin-induced hypoglycaemia, were almost identical for the melatonin and placebo control conditions (P > 0.5). However, melatonin increased plasma prolactin concentrations (P < 0.01) and reduced systolic blood pressure in the time interval following hypoglycaemia (P < 0.05). Based on a review of the literature and our results, we conclude that melatonin per se has no substantially suppressing effect on HPA secretory activity, although such an effect can be gated by sleep-related processes.

Relationship between metabolic parameters, blood pressure, and sympathoendocrine function in healthy young adults with low birth weight.

The association between low birth weight (LBW) and elevated blood pressure has been attributed to disturbances in the endocrine and sympathetic nervous system. The present study focussed on parameters of cardiovascular and sympathetic function and on adrenocortical activity in 24 healthy subjects aged 20 - 30 years with a birth weight of less than 2500 g at term and a control group of 24 subjects with a normal birth weight (NBW; 3200 - 3700 g) who were thoroughly matched for gender, body mass index (BMI), and age. Blood pressure, heart rate, and insulin resistance (calculated according to the homeostasis model assessment) were determined. Additionally, free salivary cortisol was measured at 08 : 00 am and 11 : 00 pm. In 13 subjects of each group, muscle sympathetic nerve activity (MSNA) was measured microneurographically at rest and after baroreflex stimulation by nitroprusside (12 NBW and 9 LBW subjects). Metabolic parameters, blood pressure, and salivary cortisol did not differ between LBW and NBW subjects. MSNA was significantly lower in the LBW group. In both groups insulin resistance correlated positively with BMI and negatively with morning cortisol. In the LBW group, but not the NBW group, systolic and diastolic blood pressure correlated positively with BMI and insulin resistance, and negatively with morning salivary cortisol. A correlation between morning salivary cortisol and the MSNA was only found in NBW subjects. This positive correlation strengthened when MSNA was stimulated by nitroprusside administration. However, in the same maneuvre a negative correlation between morning salivary cortisol and MSNA was observed in the LBW group. The data indicate that insulin resistance depends on the same factors in LBW and NBW subjects. In LBW subjects the interplay between adrenocortical and sympathetic activity is altered. Furthermore, LBW subjects differ from the NBW group in their significant interrelationship between blood pressure and metabolic factors.

Antecedent hypoglycaemia attenuates vascular endothelial growth factor response to subsequent hypoglycaemia in healthy men.

AIM: The plasma concentration of vascular endothelial growth factor (VEGF) has recently been shown to increase sharply in response to hypoglycaemia and, thus, has been proposed as having a role in hypoglycaemia counter-regulation. Many counter-regulatory hormones show a reduced response after antecedent hypoglycaemia. We therefore investigated whether this decrease in responsiveness with repetitive hypoglycaemia also pertains to VEGF. METHODS: Three hypoglycaemic clamp experiments were performed on two consecutive days in 15 healthy men. VEGF response was assessed during the first and last hypoglycaemic period. RESULTS: As expected, plasma VEGF concentrations rose markedly during the clamps (P < 0.001). The increase was distinctly blunted during the third (+13 +/- 8 pg/ml) as compared with the first (+54 +/- 18 pg/ml) hypoglycaemic clamp (P = 0.046). CONCLUSION: This data confirms that circulating VEGF concentrations increase acutely during hypoglycaemia. Like the counter-regulatory hormones, the hypoglycaemia-induced rise in VEGF is attenuated after antecedent hypoglycaemia. The origin of increased systemic VEGF concentration during hypoglycaemia and its physiological role remains to be defined.

Serum adiponectin concentrations during a 72-hour fast in over- and normal-weight humans.

OBJECTIVE: Adiponectin is currently considered an important link between obesity and insulin resistance, since circulating levels of this insulin sensitizing hormone have consistently been found to be reduced in obese subjects. However, until now it is not known how the secretion of adiponectin is regulated in response to acute metabolic changes. Here, we assessed the influence of complete fasting for 72 h on serum adiponectin levels. DESIGN: Between group comparison of repeated measurements. SUBJECTS: In total, 18 normal-weight (mean+/-s.e.m. BMI: 22.2+/-0.4 kg/m(2); age: 39.2+/-4.4 y) and nine over-weight (BMI: 33.2+/-1.8 kg/m(2); age: 36.9+/-4.5 y) subjects. MEASUREMENTS: Serum adiponectin levels were measured every 4 h during a 72-h fasting period. Additionally, concentrations of plasma glucose and serum insulin and leptin were assessed at the beginning and in the end of the fasting experiment. Insulin resistance was estimated using the homeostasis model assessment (HOMA). RESULTS: While concentrations of glucose, insulin, and leptin decreased across the fasting period by 31.0, 33.1 and 60.0%, respectively (all P<0.005), adiponectin levels remained unchanged (P=0.817). Overall, over-weight subjects exhibited slightly lower adiponectin levels than normal-weight subjects (P=0.092), but there was no difference in the time course of adiponectin levels during fasting between these two groups (P=0.970). Although, averaged adiponectin levels before and after fasting did not systematically differ, individual changes in adiponectin levels across fasting displayed a slight but significant inverse correlation with changes in plasma glucose concentration (r=-0.42, P=0.03). DISCUSSION: The data show that serum adiponectin concentrations remain remarkably stable during 72 h of fasting in normal- and over-weight subjects. Thus, adiponectin appears to reflect primarily long-term changes in body weight with little evidence for a dependence on short-term regulatory influences.

Chronic fentanyl application induces adrenocortical insufficiency.

We report a case of a 64-year-old man with secondary adrenocortical insufficiency who has been on a chronic transdermal fentanyl treatment because of sciatic pain syndrome. Shortly before admission to our hospital, the patient had discontinued his hydrocortisone medication. Adrenal crisis was assumed and during therapy with hydrocortisone infusion, the patient recovered. We suspected an opiate-induced suppression of the hypothalamus-pituitary-adrenal (HPA) axis. Therefore, we gradually reduced the opiate dosage. After 1 week, HPA axis function was markedly improved. We conclude that opiate medication may inhibit - in a life-threatening way - the organism's ability to respond to physical, emotional or metabolic stressors.

Differential regulation of human blood glucose level by interleukin-2 and -6.

While the acute phase reaction to infection is associated with hyperglycemia, during progressing infection hypoglycemia can develop. The cytokines regulating the dynamics of host defense may concurrently contribute to blood glucose regulation. To examine this hypothesis, changes in blood glucose concentrations in healthy men were compared following administration of interleukin-2 (IL-2) and IL-6 representing, respectively, major mediators of the adaptive and the early innate immune response to bacterial infection. Doses of 10 000 IU/kg IL-2 and 0.5 microg/kg IL-6 (vs. placebo) were administered subcutaneously in two groups of men (n = 18 and 16) at 1900 h before a period of nocturnal rest allowing an assessment of changes under basal conditions. Serum concentrations of glucose and of various hormones were assessed every 60 min. Despite generally lowered glucose concentration at night, IL-2 induced a transient but distinct decrease in blood glucose concentration most consistent 8 - 9 hours following injection (p < 0.01). The hypoglycemic response to IL-2 was not accompanied by changes in serum insulin, C-peptide or cortisol. In contrast to IL-2, IL-6 led to an increase in cortisol, followed by a pronounced increase in blood glucose again peaking about 8 hours after injection (p < 0.001). Results indicate a differential regulation of blood glucose concentration by cytokines. Contrasting with the hyperglycemic effects of the acute phase regulator IL-6, the T-cell cytokine IL-2 seems to support glucose uptake and utilization by immune cells.

Differential adaptation of neurocognitive brain functions to recurrent hypoglycemia in healthy men.

Antecedent hypoglycemia is known to attenuate hormonal and symptomatic responses to subsequent hypoglycemia. Whether this pertains also to hypoglycemia-induced cognitive dysfunction is controversially discussed. Neurocognitive adaptation might essentially depend on the type of function. Here, we compared the influence of recurrent hypoglycemia in 15 healthy men on counterregulatory hormones, subjective symptoms of hypoglycemia, short-term memory performance (word recall), and performance on an auditory attention task (oddball). The attention task was also used to record event-related brain potential (ERP) indicators of stimulus processing. In each subject, three consecutive hypoglycemic clamps were performed, two on day 1 and the third on day 2. Neurocognitive testing was performed during baseline and at two different hypoglycemic plateaus (2.8 and 2.5 mmol/l) during the first and last clamp. As expected, hormonal responses were significantly reduced to the last as compared to the first hypoglycemia indicating adaptation. Subjective symptoms also decreased in response to recurrent hypoglycemia. Short-term memory performance deteriorated distinctly on the first hypoglycemic clamp, but maintained the normal level on the last clamp (P=0.006). Likewise, the impairment in reaction time (P=0.022) and response accuracy (P=0.005) was distinctly smaller on the last than first hypoglycemia. In parallel, the hypoglycemia-induced decrease in P3 amplitude (P=0.019) and the increase in P3 latency (P=0.049) were diminished with recurrent hypoglycemia, indicating that late stages of controlled stimulus processing likewise adapted. In contrast, the distinct decrease in amplitudes of the N1 and P2 components of the ERP (preceding the P3) was closely comparable in response to the first and last hypoglycemia (P>0.3). Together results indicate an adaptation to recurrent hypoglycemia for signs of controlled stimulus processing presumably involving hippocampo-prefrontocortical circuitry, while earlier automatic stages of processing appear to be spared.

[Circadian rhythms in endocrinology]. / Zirkadiane Rhythmen in der Endokrinologie.

The function and fission properties of every cell in the organism follow a distinct rhythm. The synchronization of all these peripheral rhythms is controlled by a superordinate "master-clock", which is located in the nucleus suprachiasmaticus of the hypothalamus. Endocrine systems, aside from the autonomic nervous system, serve as efferent signals to transmit the rhythm to peripheral organs. The measurement of time-dependent changes in circulating hormone concentrations allows conclusions concerning the functionality of circadian rhythms and therefore represents a central approach in endocrine research. Regarding the interpretation of diurnal changes, one has to keep in mind that apart from the light/dark cycle other factors like the sleep/wake cycle or food intake also clearly influence the secretory activity of many endocrine organs. The dissociation of intrinsic circadian rhythms from other influencing factors requires the performance of costly and well-controlled experimental studies. Moreover, because of the complex interactions between hormones it is necessary to assess diurnal changes in activity of various endocrine systems in parallel. Only such an approach enables to understand the impact of endocrine rhythms on the complexly regulated homeostasis of e.g. glucose metabolism or the immune system.

Glucocorticoids and melanocortins in the regulation of body weight in humans.

The last decade witnessed a dramatic increase in knowledge concerning regulation of body weight and obesity. According to recent concepts constancy of body weight is a side product of regulatory events which ensure constant glucose fluxes to the brain. Within these control systems glucocorticoids and melanocortins play a fundamental role at several sites. The melanocortin neurons in the arcuate nucleus are important mediators of the feedback effects of leptin and insulin. Glucocorticoid and mineralocorticoid receptors in hippocampal neurons are crucial as they define the balance between glucose allocation processes and food intake. Thereby, the hippocampal structures determine the setpoint for bodyweight regulation. Novel approaches to treatment of obesity must aim at manipulating these brain structures.

The selfish brain: competition for energy resources.

The brain occupies a special hierarchical position in the organism. It is separated from the general circulation by the blood-brain barrier, has high energy consumption and a low energy storage capacity, uses only specific substrates, and it can record information from the peripheral organs and control them. Here we present a new paradigm for the regulation of energy supply within the organism. The brain gives priority to regulating its own adenosine triphosphate (ATP) concentration. In that postulate, the peripheral energy supply is only of secondary importance. The brain has two possibilities to ensure its energy supply: allocation or intake of nutrients. The term 'allocation' refers to the allocation of energy resources between the brain and the periphery. Neocortex and the limbic-hypothalamus-pituitary-adrenal (LHPA) system control the allocation and intake. In order to keep the energy concentrations constant, the following mechanisms are available to the brain: (1) high and low-affinity ATP-sensitive potassium channels measure the ATP concentration in neurons of the neocortex and generate a 'glutamate command' signal. This signal affects the brain ATP concentration by locally (via astrocytes) stimulating glucose uptake across the blood-brain barrier and by systemically (via the LHPA system) inhibiting glucose uptake into the muscular and adipose tissue. (2) High-affinity mineralocorticoid and low-affinity glucocorticoid receptors determine the state of balance, i.e. the setpoint, of the LHPA system. This setpoint can permanently and pathologically be displaced by extreme stress situations (chronic metabolic and psychological stress, traumatization, etc.), by starvation, exercise, infectious diseases, hormones, drugs, substances of abuse, or chemicals disrupting the endocrine system. Disorders in the 'energy on demand' process or the LHPA-system can influence the allocation of energy and in so doing alter the body mass of the organism. In summary, the presented model includes a newly discovered 'principle of balance' of how pairs of high and low-affinity receptors can originate setpoints in biological systems. In this 'Selfish Brain Theory', the neocortex and limbic system play a central role in the pathogenesis of diseases such as anorexia nervosa and obesity.

A regulatory role of prolactin, growth hormone, and corticosteroids for human T-cell production of cytokines.

The release of the pituitary hormones, prolactin and growth hormone (GH), and of adrenal corticosteroids is subject to a profound regulation by sleep. In addition these hormones are known to be involved in the regulation of the immune response. Here, we examined their role for in vitro production of T-cell cytokines. Specifically, we hypothesized that increased concentrations of prolactin and GH as well as a decrease in cortisol, i.e., hormonal changes characterizing early nocturnal sleep, could be responsible for a shift towards T helper 1 (Th1) cytokines during this time. Whole blood was sampled from 15 healthy humans in the morning after regular sleep and was activated in vitro with ionomycin and two concentrations of phorbol myrestate acetate (PMA, 8 and 25 ng/ml) in the absence or presence of prolactin, prolactin antibody, GH, glucocorticoid receptor (GR) antagonist RU-486, or mineralocorticoid receptor (MR) antagonist spironolactone. Hormones were examined at physiological concentrations. Production of T-cell derived cytokines was measured at the single cell level using multiparametric flow cytometry. Generally, effects were more pronounced after stimulation with 8 rather than 25 ng/ml PMA. The following changes reached significance (p <.05): prolactin (versus prolactin antibody) increased tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) producing CD4+ and CD8+ cells and interleukin-2 (IL-2) producing CD8+ cells. Compared with control, prolactin antibody decreased, whereas GH increased IFN-gamma+CD4+ cells. RU-486 increased TNF-alpha, IFN-gamma, and IL-2 producing CD4+ and CD8+ cells. Surprisingly strong effects were found after MR blocking with spironolactone which increased TNF-alpha, IFN-gamma, and IL-2 producing CD4+ and CD8+ cells. No effects on IL-4+CD4+ cells were observed, while the IFN-gamma/IL-4 ratio shifted towards Th1 after spironolactone and after RU-486 plus GH. Results suggest that enhanced prolactin and GH concentrations as well as low cortisol levels during early nocturnal sleep synergistically act to enhance Th1 cytokine activity.