Vasopressin stimulation of adrenocorticotropin hormone (ACTH) in humans. In vivo bioassay of corticotropin-releasing factor (CRF) which provides evidence for CRF mediation of the diurnal rhythm of ACTH.

The diurnal response of ACTH release to intravenously administered arginine vasopressin was tested in normal volunteers given consecutively moderate doses of vasopressin every 15 min (0.1, 0.3, 1.0, and 3.0 IU) at 2200 h and again at 0700 h (PM/AM). This protocol was repeated 4 wk later with the times reversed (AM/PM). A dose-related increase in ACTH secretion was observed in all subjects. When the AM response of the AM/PM protocol was compared with the PM response of the PM/AM protocol, the release of ACTH was greater in the morning (P less than 0.05) as evaluated by the following criteria: peak value of ACTH (129.9 +/- 30.4 pg/ml in the AM vs. 57.1 +/- 20.2 in the PM); area under the curve (689 in the AM vs. 259 in the PM); and, sensitivity of the ACTH dose-response curve (first significant increase in ACTH with 1 IU of vasopressin in the AM but not significant even after 3 IU in the PM). In addition, when the AM vasopressin testing followed a previous evening stimulation (PM/AM protocol), there was a blunted ACTH response compared with the AM/PM protocol. Corticotropin-releasing factor (CRF) is probably the major ACTH secretagogue, but since vasopressin acts synergistically with CRF to produce an augmented release of ACTH, we suggest that the ACTH response to administered vasopressin depends upon the ambient endogenous level of CRF. We interpret our data and published data that CRF produces a lesser release of ACTH in the AM as follows: in the morning endogenous CRF is high and administered CRF produces little further release of ACTH, but administered vasopressin acting synergistically with high endogenous CRF causes a greater release of ACTH; conversely, in the evening endogenous CRF is low and administered CRF causes a greater release of ACTH, but vasopressin (a weak secretagogue by itself) gives a low ACTH response. We conclude that vasopressin stimulation of ACTH secretion can be used as an in vivo bioassay of endogenous CRF, and that there is a diurnal rhythm of CRF in hypophyseal portal blood.

Autoantibodies to the insulin receptor. Effect on the insulin-receptor interaction in IM-9 lymphocytes.

The serum of some patients with insulin-resistant "diabetes" contains antibodies that bind to and block the cell membrane receptors for insulin. In this report, we have characterized the effects of the antireceptor antibodies on the interaction of (125)I-insulin with its receptor on the human lymphoblastoid cell line IM-9. Up to 95% of specific insulin binding can be inhibited by pretreatment of the cells with these immunoglobulins. The onset of the inhibitory effect is time- and temperature-dependent, and the effect is reversed extremely slowly if the cells are suspended in a large excess of antibody-free buffer. These features of antibody binding can be easily distinguished from those for insulin binding to its receptor. The inhibitory effect of the antibodies can be reversed by exposure of the cells to conditions known to elute surface immunoglobulins. The three antireceptor sera studied appear to alter the insulin-receptor interaction in different ways. Two antisera markedly reduce receptor affinity through combined effects on the insulin association and dissociation rates, and, additionally, have smaller effects on available receptor number. A third antiserum primarily affects available receptor number and has little effect on receptor affinity. All three antisera inhibit the capacity of insulin to promote negatively cooperative site-site interactions among insulin receptors. The data suggest that these autoantibodies to the insulin receptor bind to different determinants on the receptor and may therefore be useful as unique probes of insulin receptor structure and function.

Effects of autoantibodies to the insulin receptor on isolated adipocytes. Studies of insulin binding and insulin action.

Autoantibodies to the insulin receptor have been detected in the sera of several patients with the Type B syndrome of insulin resistance and acanthosis nigricans. In this study we have used three of these sera (B-1, B-2, and B-3) as probes of the insulin receptor in isolated rat adipocytes. Preincubation of adipocytes with each of the three sera resulted in an inhibition of subsequent [(125)I]insulin binding. 50% inhibition of binding occurred with serum dilutions of 1:5 to 1:7,500. As in our previous studies with other tissues, Scatchard analysis of the insulin-binding data was curvilinear consistent with negative cooperativity. Computer analysis suggested that in each case the inhibition of binding was due to a decrease in receptor affinity rather than a change in available receptor number. In addition to the effects on insulin binding, adipocytes pretreated with antireceptor sera also showed alterations in biological responses. All three sera produced some stimulation of basal glucose oxidation. With serum B-3, maximal stimulation of glucose oxidation occurred at a serum concentration that inhibited binding by only 10-15%, whereas with serum B-2 the dilution curves for inhibition of binding and stimulation of glucose oxidation were superimposable. Serum B-1 behaved as a partial agonist; that is, it inhibited binding more effectively than it stimulated glucose oxidation. Cells pretreated with this serum in a concentration which inhibited binding by 80% also showed a five-fold shift to the right in the dose response of insulin-stimulated glucose oxidation, whereas spermine-stimulated glucose oxidation was unaffected. Serum B-2, which contained the highest titer of antireceptor antibodies, also stimulated 2-deoxy-glucose transport, as well as glucose incorporation into lipid and glycogen. Both the ability of the serum to inhibit binding and stimulate glucose utilization were enriched in purified immunoglobulin fractions and retained in the F(ab')(2) fragment of the IgG. In addition, the bioactivity was blocked by antihuman IgG but not by anti-insulin antibodies. Enzymatic digestion of adipocytes with trypsin resulted in a complete loss of insulin-stimulated bioactivity of serum B-3, but had only minor effects on the glucose oxidation produced by serum B-1 or B-2.These data suggest that the antibodies present in these three sera bind to different determinants on the insulin receptor. Thus, these antibodies may be useful probes of receptor structure and function.

Characterization of antibodies to the insulin receptor: a cause of insulin-resistant diabetes in man.

We have characterized the circulating inhibitor of insulin receptor binding found in several patients with a new syndrome of extreme insulin resistance. The inhibitor is an immunoglobulin by multiple criteria, including precipitation by 33% ammonium sulfate, migration on G-200 Sephadex gel filtration and DEAE chromatography, and immuno-precipitation with specific anti-human immuno-globulins. Although predominantly IgG, some activity is found in the IgM fraction of the immunoglobulins in one patient. The inhibitory immunoglobulins reacted with antisera to both kappa and lambda light chain determinants and are therefore polyclonal. In addition, activity is retained in the F(ab')2 fraction of pepsin-digested IgG. Evidence suggests that these antibodies are directed at determinants on or near the insulin receptor, and that they are responsible for the observed clinical insulin resistance.

Reduced cortisol latency in depressive illness.

Depressed patients commonly have disturbances in their sleep and cortisol secretory patterns. When the sleep-related changes in plasma cortisol concentration were measured in 14 patients with a primary major depressive illness, they differed significantly from the changes measured in 14 age- and sex-matched healthy control subjects. The nadir of the nocturnal plasma cortisol concentration was significantly greater in the group of depressed patients, and the nocturnal increase in the plasma cortisol concentration occurred significantly closer to sleep onset in these patients. The circadian activity within the hypothalamic-pituitary-adrenal axis of these depressed patients showed a subtle but significantly disturbed temporal relationship to sleep onset. This reduced time between sleep onset and the nocturnal increase in cortisol secretion suggests a possible biologic correlate of a depressive illness that might be useful as an illness marker in depressed patients.

Recurrent depression is associated with a persistent reduction in sleep-related growth hormone secretion.

Sleep onset is a powerful physiologic stimulus for growth hormone secretion. Difficulty falling asleep and poor sleep maintenance are prominent symptoms in patients with a major depressive disorder. Much of the disturbance in the sleep electroencephalograms of depressed patients occurs within the first half of the night, the time when growth hormone is usually secreted. Growth hormone secretion was measured during electroencephalographically monitored sleep in 38 patients with a recurrent major depressive disorder and 35 healthy control subjects. Before treatment, depressed patients had a statistically significant reduction in growth hormone secretion during sleep. This reduction, which persisted through treatment and recovery into the drug-free remitted state, may be a trait marker in patients with a recurrent depressive disorder.

Electroencephalographic sleep of younger depressives. Comparison with normals.

The electroencephalographic sleep of younger depressives (aged 20 to 44 years) was compared with that of an age-matched group of normals. The patients demonstrated many of the typical sleep changes reported for older depressed populations: shortened rapid-eye-movement (REM) latency; REM sleep activity alterations, with a shift to the early portion of the night (first REM period); reduced delta sleep; and sleep efficiency reductions marked by sleep-onset difficulties. The traditional scoring procedures were supplemented by automated REM and delta-sleep analyses that provided more precise delineation of these differences between patients and normals, particularly the distributions of REM activity and delta-wave patterning.

Three-year outcomes for maintenance therapies in recurrent depression.

We conducted a randomized 3-year maintenance trial in 128 patients with recurrent depression who had responded to combined short-term and continuation treatment with imipramine hydrochloride and interpersonal psychotherapy. A five-cell design was used to determine whether a maintenance form of interpersonal psychotherapy alone or in combination with medication could play a significant role in the prevention of recurrence. A second question was whether maintaining antidepressant medication at the dosage used to treat the acute episode rather than decreasing to a "maintenance" dosage would provide prophylaxis superior to that observed in earlier trials in which a maintenance dosage strategy was employed. Survival analysis demonstrated a highly significant prophylactic effect for active imipramine hydrochloride maintained at an average dose of 200 mg and a modest prophylactic effect for monthly interpersonal psychotherapy. We conclude that active imipramine hydrochloride maintained at an average dose of 200 mg is an effective means of preventing recurrence and that monthly interpersonal psychotherapy serves to lengthen the time between episodes in patients not receiving active medication.

Corticosteroids in brain tissue.

Total corticosteroid concentrations were determined in the plasma and brains of five species: mouse, rat, cat, monkey and man. Corticosteroid concentrations were measured also in the livers of mice, rats and monkeys. Competitive protein binding techniques were validated and used for the tissue corticosteroid assays. In each species the brain corticosteroid value was less than the total plasma corticosteroid level but greater than the presumed unbound fraction of plasma corticosteroids. A clear circadian variation of brain corticosteroid values was found in mice, together with a rapid elevation of the tissue levels in response to stress. Reduction of both plasma and tissue corticosteroid concentrations was observed after adrenalectomy and in response to dexamethasone treatment of mice. Between 24 and 48 h post morten, mouse brain corticosteroid values decreased greatly. In cat, monkey and human brains all regions examined contained approximately equal amounts of corticosteroids. In particular, brain areas which are not involved in pituitary-adrenocortical regulation contained large amounts of corticosteroids.

Identification of a corticotropin-releasing factor-binding protein in the plasma membrane of AtT-20 mouse pituitary tumor cells and its regulation by dexamethasone.

CRF stimulates the synthesis and secretion of proopiomelanocortin-derived peptides from AtT-20 mouse pituitary tumor cells. This study has shown that there is a specific binding site for CRF located on the plasma membrane of these cells. Both [125I]iodo-Tyr0CRF and noniodinated CRF (10(-11)-10(-7) M) stimulated, in a dose-dependent manner, the secretion of equimolar amounts of beta-endorphin-like immunoactivity from AtT-20 cells. Disuccinimidyl suberate, a cross-linking agent, was used to demonstrate specific binding of [125I]iodo-Tyr0CRF to plasma membranes from these cells. After cross-linking [125I] iodo-Tyr0CRF, the membrane proteins were solubilized with sodium dodecyl sulfate and electrophoresed on a 10% polyacrylamide gel. A single radioactively labeled band, corresponding to a mol wt of 66,000, was identified by autoradiography. [125I]Iodo-Tyr0CRF binding to these membranes was inhibited by 10(-7) M unlabeled CRF or an equimolar concentration of the CRF analog sauvagine. Similar concentrations (10(-7) M) of TRH, GnRH, insulin, [Arg8]vasopressin, somatostatin, and ACTH did not inhibit [125I]iodo-Tyr0CRF binding to the plasma membranes. Incubation of AtT-20 cells for 24 h in the presence of 10 nM dexamethasone reduced [125I]iodo-Tyr0CRF binding by 80% compared to that in untreated cells. Dexamethasone also inhibited the CRF-stimulated beta-endorphin-like immunoactivity secretory response. These data indicate that binding of CRF to a specific membrane protein is an integral component in the stimulation of AtT-20 cells by CRF.

Relationship among selected neuroendocrine and sleep measures in patients with recurrent depression.

Relationships among cortisol, TSH, and EEG sleep variables were examined in a group of 23 outpatients with recurrent unipolar depression at initial assessment prior to treatment for an acute major depressive episode. Blunted TSH responses were found in 39% of the sample (criteria of less than or equal to 7 microU/ml); cortisol nadir of greater than 2.5 micrograms% was rated in 41% of the group; and a reduced REM latency (less than or equal to 60 min) was measured in 57% of the group. Although almost all patients demonstrated abnormalities in at least one major neuroendocrine or sleep category, this preliminary analysis failed to find significant associations among these multiple biologic variables. Further studies on this so-called lack of association in a larger group of recurrent depressives is indicated. At this point caution is urged in assuming that biologic measures will identify a homogeneous group of severely depressed patients.

Acute effect of intravenous clomipramine upon sleep-related hormone secretion in depressed outpatients and healthy control subjects.

Tricyclic antidepressants have been used frequently as pharmacological probes in neuroendocrine studies even though they appear to lack neurochemical specificity. Despite this, the hormonal responses to these drugs have been used to provide evidence that depressed patients have alterations in both noradrenergic and serotonergic tone within the central nervous system. Most studies have been conducted in the morning, which is not a time of high physiological neuroendocrine secretory activity. The present study has used the relatively specific serotonergic probe intravenous clomipramine given to depressed patients and healthy subjects immediately before sleep onset, which is a time of increased neuroendocrine activity. Under these conditions, 12.5 mg clomipramine stimulates the secretion of both cortisol and prolactin, but unlike studies conducted in the morning, clomipramine suppresses the secretion of growth hormone in both groups. These data suggest that serotonergic mechanisms are involved in the regulation of the secretion of these three hormones at the time of sleep onset.

Acute changes in sleep-related hormone secretion of depressed patients following oral imipramine.

Tricyclic antidepressants have acute effects on hormone secretion when given either orally or parenterally in the morning. These drugs also have acute effects on the sleep electroencephalogram (EEG) when given immediately before sleep onset. In particular, imipramine significantly delays the REM-nREM cycle and increases the amount of delta wave activity. This study shows that an oral dose of 50 mg imipramine given at bedtime to depressed patients has little effect on the secretion of prolactin and melatonin, but acutely advances the secretion of growth hormone and cortisol. This suggests that sleep and hormone secretion may only be temporally related, as they can be dissociated pharmacologically.

REM latency in depression: is there one best definition?

The concurrent validity of different definitions of REM latency has been tested by comparing the ability of each definition to discriminate between primary depressives (outpatients and inpatients) and normal controls. In outpatients the percentage of cases correctly identified ranged from 62.5% to 70.8%; in inpatients, from 64.6% to 70.8%. REM latency definitions with the least stringent sleep-onset criteria yielded the lowest specificity. In contrast, the range of sensitivities yielded by different definitions was narrower and not clearly affected by sleep-onset criterion or exclusion/inclusion of wakefulness between sleep onset and first REM period. Furthermore, different definitions of REM latency correlated equally well (p less than 0.01) with Hamilton depression ratings. The shorter REM latencies in both outpatients and inpatients were associated with a later time of NREM sleep onset than in controls, rather than with an earlier REM sleep-onset time.

Effect of nocturnal intravenous cannulation upon sleep-EEG measures.

The effect of an indwelling intravenous cannula on sleep-EEG measures has not been extensively documented despite its relatively frequent use in a variety of clinical studies. When the sleep EEGs from 30 healthy subjects were critically examined, no changes in sleep architecture were found. However, there were significant differences in the distribution of sleep latency, maintenance, and efficiency, with many subjects showing a reduction in the latter two items on the night of cannulation. A decrease in sleep maintenance of greater than 7.5 on the night of cannulation has been used to operationally define an unsatisfactory cannulation study which accounted for 43% of our subjects. It was striking that this group could also be identified by having higher REM densities in each REM period on the previous two nights in the sleep laboratory. This was especially true for the second REM period on the second night when only three of those subjects with subsequent unsatisfactory studies had a REM density less than 0.9. When this criterion was applied to a new group of 19 subjects, 13 were correctly classified with respect to subsequent changes in sleep maintenance before the cannulation study was conducted. It therefore appears possible that REM-sleep measures can be used to predict which subjects will have disrupted sleep on a subsequent cannulation night. Since sleep disruption can significantly mask patterns of hormone secretion, it is essential to monitor the sleep-EEG during such studies.

A reexamination of the relationship between growth hormone secretion and slow wave sleep using delta wave analysis.

Sleep onset growth hormone secretion is a reliable and reproducible finding in young adults and children. Secretion typically occurs during the first non-REM period of sleep and, despite some evidence to the contrary, growth hormone secretion has frequently been associated with the first period of slow wave sleep. By measuring delta wave activity (0.5-2 Hz) instead of slow wave sleep and, accounting for the within subject variability, it has not been possible to demonstrate a consistent or statistically significant linear relationship between delta wave activity and sleep-related growth hormone secretion. This suggests the presence of more complex mediating factors and the possibility that sleep onset and growth hormone secretion are two separate processes which are independently stimulated by events associated with sleep onset.

Growth hormone secretion timing in depression: clinical outcome comparisons.

Growth hormone (GH) secretion in the 100 minutes preceding sleep onset (preSO), as well as in the first half of the night (1st HN), was examined for a group of 13 healthy women and 43 women with recurrent depression who participated in a 3-year maintenance therapy study. GH studies were obtained at several points during treatment and every 3 months during maintenance, during which patients were randomly assigned to active drug or drug-free maintenance treatment cells for 3 years, or until recurrence of depression. Depressed patients were divided into subgroups according to their maintenance treatment assignment (active drug or drug free) and treatment outcome (completing in remission or having a recurrence). Imipramine caused an increase in the GH ratio in all subgroups. Protocol completers had a significantly larger imipramine-induced increase in the GH ratio than did recurrers. The difference in time of GH secretion relative to sleep onset was found to correlate with treatment outcome and was independent of medication status during maintenance.

Minute-by-minute analysis of REM sleep timing in major depression.

Sleep changes described in depressed patients may represent alterations in the timing of rapid-eye-movement (REM) sleep or sleep onset. We examined these variables in groups of healthy control subjects (n = 47), depressed outpatients (n = 98), and depressed inpatients (n = 41). Outpatient depressives had greater severity of clinical symptoms than inpatients using the Hamilton Rating Scale for Depression. The depressed inpatient group had a later mean sleep onset time than the other groups, and the depressed outpatient group had a wider range of good night times than control subjects. REM timing in each group was examined as a relative frequency distribution of REM sleep (FDRS) for each minute across the night. The FDRSs for the three groups were statistically compared using the parameters from a two-component model, which includes a deterministic sinusoidal function and a time series process for errors. The slope of the linear trend in the FDRS rhythm was smaller (less positive) for both depressed groups than for controls. The ultradian FDRS rhythm occurred at an earlier phase, relative to sleep onset, in the inpatient depressed group compared to the control group. The ultradian FDRS rhythm had a longer period in the outpatient group compared to the control and inpatient groups. When referenced to 24-hr clock time in an exploratory analysis, the depressed groups appeared to have less robust FDRS ultradian rhythms than controls, but they did not appear to have a systematic phase alteration compared to controls. Abnormalities of REM sleep timing in groups of depressed patients may reflect a disturbance of sleep initiation and generation, or difficulty in entrainment of REM, rather than a systematic phase alteration in REM sleep propensity.

Prolactin secretion during sleep: a comparison between depressed patients and healthy control subjects.

Although several neuroendocrine abnormalities have been described in depressed patients, relatively little attention has been paid to the pattern of prolactin secretion during sleep. Sleep disturbances are frequently found in depressed patients, and the sleep electroencephalogram (EEG) typically shows significant changes in the first and last 100 min, when prolactin secretion frequently occurs. In this study, carefully defined inclusion criteria were used to ensure comparability in the quality of the sleep maintenance, so that the pattern of sleep-related prolactin secretion in a group of 26 depressed inpatients could be compared to that in a group of 20 healthy control subjects. Starting from sleep onset, the patients did not show any statistically significant difference in either the serum prolactin concentration or the pattern of integrated prolactin secretion relative to the control subjects. A statistically significant relationship between prolactin secretion and the REM-non-REM sleep cycle could not be demonstrated in these subjects.

Sleep, gender, and depression: an analysis of gender effects on the electroencephalographic sleep of 302 depressed outpatients.

Gender-related differences in electroencephalographic (EEG) sleep were examined in 151 pairs of men and women with major depression, all outpatients, matched for age and severity of depression. Across five decades (age 21-69), depressed men had less slow-wave sleep than did depressed women. Gender differences were small with respect to visually scored measures of slow-wave sleep time and percent, but moderate for gender differences in automated measures of slow-wave density. The time constant of the polygraph preamplifier significantly affected both visually scored and automatically scored slow-wave sleep. Other measures such as REM sleep latency, first REM period duration, sleep efficiency, and early morning awakening, showed robust age effects, but no main effects for gender or gender-by-age interactions. Gender effects on slow-wave sleep and delta-wave counts in depression parallel gender effects seen in healthy aging. The possibility of occult alcohol use by depressed male outpatients cannot be definitely excluded as a partial explanation of the current findings. However, covarying for past alcohol abuse did not negate the statistical significance of the observed gender effects on slow-wave sleep and delta-wave density. The possibility of gender differences in slow-wave regulatory mechanisms is suggested, but similarity in temporal distribution of delta-wave density between the first and second non-rapid-eye-movement (NREM) periods does not support gender differences in slow-wave sleep regulation.