Uncoupling of proopiomelanocortin (POMC) fragments is related to self-injury

Proopiomelanocortin (POMC) contains several interesting, behaviorally active peptides. Release patterns of these fragments have been related to bizarre episodes of self-injurious behavior (SIB) among autistic individuals. Moreover, elevation in beta-endorphin (betaE) but not ACTH levels was associated with a positive response to an acutely administered, centrally acting opioid blocker among autistic individuals exhibiting SIB. In the present study, POMC fragments were measured in 12 self-injurious patients before and after long term (3 month) treatment with an opiate blocker naltrexone (NTX). POMC fragments were sampled from blood collected at the beginning of the baseline and placebo-controlled treatment phases of the study. Results indicated that the co-release (coupling) of POMC fragments were stable over time and the profile of POMC fragments in plasma predicted the effectiveness of a CNS acting drug in autistic subjects who self-injure.     

Beta-endorphin levels in longtailed and pigtailed macaques vary by abnormal behavior rating and sex

Frequent or severe abnormal behavior may be associated with the release of endorphins that positively reinforce the behavior with an opiate euphoria or analgesia. One line of research exploring this association involves the superhormone, proopiomelanocortin (POMC). The products of POMC appear to be dysregulated in some human subjects who exhibit self-injurious behavior (SIB). Macaque monkeys have POMC very similar to humans, and some laboratory macaques display SIB or frequent stereotypies. We investigated associations between plasma levels of three immunoreactive POMC fragments with possible opioid action and abnormal behavior ratings in macaques. In 58 adult male and female macaques (24 Macaca fascicularis and 34 Macaca nemestrina), plasma levels of intact beta-endorphin (betaE) and the N-terminal fragment (BEN) were significantly higher in animals with higher levels of abnormal behavior. The C-terminal fragment (BEC) was significantly higher in males but unrelated to ratings of abnormal behavior. Levels of ACTH, cortisol, and (betaE-ACTH)/betaE dysregulation index were unrelated to abnormal behavior. None of the POMC products differed significantly by subjects' species, age, or weight. The finding that intact beta-endorphin is positively related to abnormal behavior in two species of macaque is consistent with some previous research on human subjects and nonprimates. The positive relation of the N-terminal fragment of betaE to abnormal behavior is a new finding.     

Diurnal pattern of proopiomelanocortin gene expression in the arcuate nucleus of proestrous, ovariectomized, and steroid-treated rats: a possible role in cyclic luteinizing hormone secretion

Opiate peptides are thought to modulate the pattern of LH release in female rats. We tested the hypothesis that changes in proopiomelanocortin (POMC) gene expression occur in proestrous (PRO) and ovariectomized (OVX) steroid-treated rats which may explain their unique patterns of LH secretion. Using in situ hybridization, we examined whether diurnal changes in POMC gene expression occur in the arcuate nucleus. Four groups of rats were used in this study. 1) PRO rats were used after exhibiting at least two consecutive 4-day estrous cycles; 2) OVX rats were killed 9 days after ovariectomy; 3) estradiol (E2)-treated rats were OVX for 7 days and then treated for 2 days; and 4) E2-progesterone (P4)-treated rats were treated with E2 as described above, and on day 9 at 1030 h, P4 was administered. Rats were killed at 2300, 0300, 1000, 1300, 1500, 1800, or 2300 h, beginning on the evening of diestrous day 2 or day 8 after ovariectomy. POMC gene expression exhibited a diurnal rhythm on PRO. Levels of mRNA rose during the morning, peaked between 0300-1000 h, and decreased by 2300 h. In E2-treated rats, which exhibited a LH surge similar in timing to the PRO surge, POMC mRNA levels exhibited a diurnal rhythm strikingly similar to that observed in PRO animals. OVX abolished the rhythm; however, average POMC mRNA levels across the 24-h period were not significantly different from those in PRO or E2-treated rats. P4 treatment increased POMC mRNA levels by 2300 h compared to those in all other experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Naloxone decreases diurnal variation in pain sensitivity and somatosensory evoked potentials

A group of 35 normal subjects had somatosensory evoked potentials (EPs) recorded and were tested on a psychophysical pain judgment task in morning and afternoon sessions. Subjects tested in the morning were found to be significantly less pain sensitive than those tested in the afternoon on psychophysical judgments. EP measures were consistent with decreased sensitivity in the morning. In a second experiment, a group of 12 normal subjects also received naloxone (2 mg, iv) in a double blind trial, 6 in the morning and 6 in the afternoon. Naloxone hyperalgesia effects on the EP were present in the morning and absent in the afternoon. It has been recently demonstrated that beta-lipotropin, a possible precursor of the endogenous opiate beta-endorphin shares with ACTH a diurnal rhythm. Our results are consistent with the hypothesis that endorphins at least partially mediate diurnal variations in pain appreciation.     

Diurnal Variation in Oral Glucose Tolerance: Blood Sugar and Plasma Insulin Levels Morning,Afternoon, and Evening

Twenty-four subjects received three oral glucose tolerance tests, in the morning, afternoon, and evening of separate days. The mean blood sugar levels in the afternoon and evening tests were similar, and they were both significantly higher than those in the morning test. Plasma immunoreactive insulin levels, however, were highest in the morning test. The pattern of insulin levels during the afternoon and evening tests resembled that described as typical of maturity-onset diabetes.     

The effect of estradiol on output of adrenocorticotropin and prolactin by fetal sheep anterior pituitary cells

We examined the hypothesis that estradiol (E2) would affect fetal anterior pituitary corticotroph and lactotroph function in vitro, and that any effects would be influenced by gestational age. Anterior pituitary cells from fetal sheep at day 129 (n = 4) and at day 139 (n = 5) of gestation were cultured. After 96 h in culture, cells were treated for 18 h with E2 concentrations ranging from 0 to 1000 nM, in the presence or absence of 100 nM of corticotropin-releasing hormone (CRH), cortisol, arginine vasopressin (AVP), or CRH and cortisol, to examine their effects on corticotroph function. Cells were also treated with bromocriptine or increasing concentrations of E2 to study their effects on lactotroph function. Immunoreactive (ir) adrenocorticotropin (ACTH) and prolactin in the culture medium were measured by radioimmunoassay. Levels of cellular pro-opiomelanocortin (POMC) mRNA and prolactin mRNA were determined by in situ hybridization. Immunohistochemistry was used to determine the percentage of cells that were immunopositive for ACTH (corticotrophs) or prolactin (lactotrophs). ACTH output was stimulated by CRH treatment at day 139 but not at day 129 of gestation, and cortisol attenuated this response. ACTH output by cells cultured with 10 nM E2 and 100 nM CRH, at 139 days of gestation, was greater than with CRH alone (p < 0.05). E2 did not affect basal ACTH output or ACTH output with any other treatment or levels of POMC mRNA. Prolactin output was not affected by E2 treatment. Bromocriptine significantly decreased prolactin output but not levels of prolactin mRNA. We conclude that E2 may affect CRH-stimulated fetal sheep pituitary corticotroph function late in gestation, but only within a narrow, physiological range of concentration.     

A comparison of the immediate effects of moderate exercise in the late morning and late afternoon on core temperature and cutaneous thermoregulatory mechanisms

Twelve healthy male subjects each undertook two bouts of moderate exercise (70% VO2max for 30 minutes) in the morning (08:00) and late afternoon (18:00) at least 4 days apart. Measurements were made of heart rate, core (rectal) temperature, sternum skin temperature, and forearm skin blood flow during baseline conditions, during the bout of exercise, and throughout a 30-minute recovery period. Comparisons were made of the changes of heart rate, temperature, and skin blood flow produced by the exercise at the two times of day. Student t tests indicated that baseline values for core temperature (37.15 degrees C +/- 0.06 degrees C vs. 36.77 degrees C +/- 0.06 degrees C) and sternum temperature (33.60 degrees C +/- 0.29 degrees C vs. 32.70 degrees C + 0.38 degrees C) were significantly (p < .05) higher in the late afternoon than the early morning. Two-way analysis of variance (ANOVA) indicated that the increases in core and sternum temperatures during exercise were significantly less (p = .0039 and .0421, respectively) during the afternoon bout of exercise compared with the morning, even though the work loads, as determined by changes in heart rate, were not significantly different (p = .798) at the two times of testing. There were also tendencies for resting forearm skin blood flow to be higher in the afternoon than in the morning and for exercise to produce a more rapid rise in this variable in the afternoon. The possible mechanisms producing these responses to exercise are discussed in terms of those that are responsible for the normal circadian rhythm of core temperature. It is concluded that the body's ability to remove a heat load is less in the early morning, when the circadian system is in a "heat gain" mode, than in the late afternoon, when heat gain and "heat loss" modes are balanced more evenly.     

Ontogenesis of cells producing polypeptide hormones (ACTH,MSH, LPH,GH, Prolactin) in the fetal hypophysis of the rat: Influence of the hypothalamus

Summary The ontogenesis of cells containing polypeptide hormones (ACTH, MSH, LPH, GH and Prolactin) was investigated in the fetal rat hypophysis by immunohistochemistry using the peroxidase-antiperoxidase complex.Corticotrophs, melanotrophs and lipotropic cells were revealed earlier in the pars distalis than in the pars intermedia. In the pars distalis, cells producing LPH were found in the morning of day 15 of gestation using anti-- or anti--LPH sera, and in the afternoon using anti-- or -endorphin sera. Cells containing -MSH were observed from the afternoon of day 15. The cells stainable with the anti--MSH, anti--(17-39)ACTH and anti--(l-24)ACTH sera appeared on day 16. In the pars intermedia, the cells producing -MSH, MSH, - and -endorphin, and -LPH were observed in the morning of day 17, while cells containing ACTH were only revealed in the afternoon of the same day of gestation. Based on the treatment of serial paraffin sections with various antisera, it was clearly shown that MSH, ACTH, and LPH occur in the same cells located in the pars distalis as in the pars intermedia.The development of the corticotrophs, melanotrophs and lipotropic cells does not require the presence of the fetal hypothalamus or other central nervous structures. The pituitary glands of 21 day-old fetuses encephalectomized on day 16 showed as many reactive cells as those of the littermate controls.The somatotrophs were first revealed in the pars distalis in the afternoon of day 19. The cells producing prolactin were not observed before day 21 of gestation. On some cases GH and prolactin were found together in one cell. The cytodifferentiation of GH and prolactin cells is apparently not under hypothalamic control.     

Collegiate rowing crew performance varies by morningness-eveningness

During adolescence and early adulthood, most humans are predisposed developmentally, both biologically and socially, toward evening/night activity. The morningness-eveningness (M-E) tendency to be an evening-preferring (E-type) rather than a morning-preferring (M-type) or intermediate/neither (N-type) "chronotype" may affect athletic performance at various times of day. This study evaluated M-E effects on rowing performance of an intact, experienced, university club crew with near-daily early morning (0500-0700 hours) and late afternoon (1630-1800 hours) training schedules. The hypothesis tested was that chronotype would modify circadian effects during morning and afternoon performances. Eight men and eight women (mean age 19.6 +/- 1.5 years) were tested in a randomized, counterbalanced design. A standard qualifying 2000-m ergometer rowing sprint and a nonroutine standing broad jump task were measured during early morning and late afternoon, separated by 3 days of rest. Each subject's chronotype was determined using two standard self-rating M-E scales, resulting in eight E-type (three women/five men), four M-type (two women/two men), and four N-type (three women/one man) subjects. The rowing results show that E-type and N-type subjects did not differ between morning and afternoon rowing performances, whereas M-type subjects rowed significantly faster in the morning. In contrast, the standing broad jump showed no consistent time-of-day or chronotype effect. These findings suggest that basic performance timing in young athletes is determined to some extent by naturally occurring M-E predispositions. Further, modification of time-of-day influences may be possible by routine practice at the same time each day, as was suggested here by the absence of evening superiority in performances. Understanding their personal M-E tendencies could allow young athletes to arrange training schedules at specific times of day to help counteract any natural circadian influences that might work against their performance.     

Phase-shifted diurnal rhythm of cortisol secretion in a patient with Cushing's syndrome due to adrenocortical adenoma

A 21-year-old woman with Cushing's syndrome was found to have a marked diurnal variation in cortisol secretion. Serum cortisol concentrations and urinary excretion of 17-hydroxycorticosteroids were normal in the morning but clearly increased in the afternoon. The patient was cured by resection of an adrenocortical adenoma. ACTH and prostaglandin E1 stimulated cortisol release from incubated adenoma tissues in vitro. The cause of the abnormal diurnal rhythm of cortisol secretion is unknown.     

One of the two trout proopiomelanocortin messenger RNAs potentially encodes new peptides.

POMC is the precursor for a number of biologically active peptides such as ACTH, alpha-MSH, beta-MSH, and beta-endorphin. It is well known that some of these peptides, especially beta-endorphin, are involved in the regulation of reproductive functions in mammals. In order to investigate the possible role of POMC-derived peptides in the control of fish reproduction, we have cloned and sequenced two different trout POMC cDNAs called POMC A and POMC B. These cDNAs exhibited limited sequence homology (44%). The deduced amino acid sequences also showed weak similarity (43%), despite the high conservation of some peptide sequences (alpha-MSH, beta-MSH, and beta-endorphin). The POMC A coding sequence exhibited an unusual length, generating the longest endorphin ever sequenced. The long carboxy-terminal part of the beta-endorphin A contained three potential dibasic cleavage sites, allowing the occurrence of three new peptides: EQWGREEGEE, ALGE, and YHFQG. Using in situ hybridization, we found that the two POMC genes were expressed in the same pituitary cells. POMC A mRNA was the only one detectable in the hypothalamus of sexually inactive fish, whereas the two POMC genes were expressed in the hypothalamus of sexually active fish. These results indicate that two functional POMC genes are present in the rainbow trout. In POMC neurons, the expression of the POMC B gene is likely to be under the control of sexual steroids.     

Pro-opiomelanocortin synthesized by corticotrophs bears asparagine-linked oligosaccharides terminating with SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha.

We have determined that greater than or equal to 80% of the Asn-linked oligosaccharides on the glycosylated form of mouse adrenocorticotropin (15-kDa adrenocorticotropin (ACTH)) bear one or more branches terminating with the sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha (S4GGnM). Proopiomelanocortin (POMC), the precursor of ACTH, is the first example of a glycoprotein that is not a member of the glycoprotein hormone family to bear such sulfated structures. Like lutropin and thyrotropin, 15-kDa ACTH bears dibranched oligosaccharides terminating with SO4-4-GalNAc; however, at least half of the oligosaccharides on 15-kDa ACTH terminating with SO4-4-GalNAc consist of more highly branched structures that have not previously been described. Both the GalNAc beta 1,4GlcNAc beta 1,2Man-4-sulfotransferase and the glycoprotein hormone-specific GalNAc-transferase are expressed in the corticotroph-derived AtT-20 cell line. A tripeptide recognition sequence, Pro-Val-Lys, similar to the Pro-Leu-Arg sequence required for recognition of glycoprotein hormone alpha- and beta-subunits by the glycoprotein hormone-specific GalNAc-transferase, is present 8 residues amino-terminal to the glycosylated Asn of 15-kDa ACTH. Thus, POMC has the features expected for specific addition of the S4GGnM sequence to its oligosaccharides. The recent discovery of a receptor in hepatic endothelial cells that recognizes oligosaccharides terminating with S4GGnM suggests these sulfated oligosaccharides will regulate the circulatory half-life of glycosylated POMC cleavage products.     

Pro-opiomelanocortin peptides in the human fetal pituitary

Levels of immunoreactive pro-opiomelanocortin (POMC) peptides (N- and C-terminal ACTH, N- and C-terminal LPH and α-MSH) have been measured in pituitary extracts from human fetuses of 12–22 weeks gestation. The levels of ACTH were 30–200 times higher than α-MSH in all fetuses studied. Sephadex G-75 and G-25 chromatography of 8 extracts showed peaks of 34 kilodaltons (K) POMC, 22K ACTH, β-LPH, γ-LPH, β-endorphin, approximately 8K ACTH, 1–39 ACTH, α-MSH and CLIP. The 8K and 22K forms of ACTH are both partly glycosylated.In vitro culture of pituitaries from 2 fetuses (22 and 26 weeks gestation) gave a detectable basal output of ACTH but not of α-MSH. Stimulation of these pituitary cells with human fetal and rat hypothalamic extracts and with synthetic ovine CRF-41 produced a significant increase in ACTH release, and either small or undetectable amounts of α-MSH.These results demonstrate the presence of POMC-related peptides in early gestation human fetal pituitaries and suggest that ACTH, and not α-MSH, is the major corticotrophic hormone at this stage of gestation.     

Dynamics of messenger RNAs encoding inhibin/activin subunits and follistatin in the ovary during the rat estrous cycle

Quantitative changes in ovarian inhibin/activin subunit and follistatin mRNAs during the rat estrous cycle were examined by ribonuclease protection assay using digoxygenin-labeled RNA probes. Levels of ovarian inhibin alpha subunit mRNA remained low throughout estrus, metestrus, and diestrus; abruptly increased on the morning of proestrus; then rapidly decreased when the primary gonadotropin surge occurred. A similar changing pattern was observed in inhibin/activin beta(A) subunit mRNA. On the other hand, inhibin/activin beta(B) subunit mRNA showed a different changing pattern. Levels of beta(B) subunit mRNA remained constant during metestrus and diestrus, abruptly decreased on the afternoon of proestrus, then quickly recovered from the nadir by 1100 h on estrus. Throughout the rat estrous cycle, especially during the periovulatory period, alpha subunit mRNA levels were considerably higher than beta(A) and beta(B) subunit mRNA levels. In addition, changes in plasma concentrations of inhibin A and inhibin B were very similar to that in ovarian beta(A) and beta(B) subunit mRNA levels, respectively, with several-hour delays. These results suggest that levels of beta subunit mRNAs restrict secretion of dimeric inhibins. Levels of follistatin mRNA remained low from the midnight of metestrus to the midnight of diestrus, then increased until initiation of the primary gonadotropin surge. Thereafter, follistatin mRNA decreased, reached the nadir at 0200 h on estrus, then increased abruptly at 1100 h on estrus. Afterward, follistatin mRNA levels remained high until the morning of metestrus. The changing pattern of ovarian follistatin mRNA was similar to, and preceded, the changes in plasma concentrations of progesterone, suggesting that ovarian follistatin may modulate progesterone secretion during the rat estrous cycle.     

Diurnal variation and reliability of the urine lactate concentration after maximal exercise

The postexercise urine lactate concentration is a novel valid exercise biomarker, which has exhibited satisfactory reliability in the morning hours under controlled water intake. The aim of the present study was to investigate the diurnal variation of the postexercise urine lactate concentration and its reliability in the afternoon hours. Thirty-two healthy children (11 boys and 21 girls) and 23 adults (13 men and 10 women) participated in the study. All participants performed two identical sessions of eight 25 m bouts of maximal freestyle swimming executed every 2 min with passive recovery in between. These sessions were performed in the morning and afternoon and were separated by 3–4 days. Adults performed an additional afternoon session that was also separated by 3–4 days. All swimmers drank 500 mL of water before and another 500 mL after each test. Capillary blood and urine samples were collected before and after each test for lactate determination. Urine creatinine, urine density and body water content were also measured. The intraclass correlation coefficient was used as a reliability index between the morning and afternoon tests, as well as between the afternoon test and retest. Swimming performance and body water content exhibited excellent reliability in both children and adults. The postexercise blood lactate concentration did not show diurnal variation, showing a good reliability between the morning and afternoon tests, as well as high reliability between the afternoon test and retest. The postexercise urine density and lactate concentration were affected by time of day. However, when lactate was normalized to creatinine, it exhibited excellent reliability in children and good-to-high reliability in adults. The postexercise urine lactate concentration showed high reliability between the afternoon test and retest, independent of creatinine normalization. The postexercise blood and urine lactate concentrations were significantly correlated in all cases, attesting to the validity of urine lactate as an index of anaerobic metabolism. We conclude that urine lactate, after normalization to creatinine, could be used in training practice either in the morning or in the afternoon. Further research is needed to assess the applicability of this novel exercise biomarker.     

Intragranular processing of pro-opiomelanocortin in the intermediate cells of the rat pituitary glands

Summary Quantitative immunocytochemical studies were done by using the immunogold technique on sections of the intermediate lobe of rat pituitary. Antibodies raised (in rabbits) against the precursor proteins pro-opiomelanocortin (POMC) and ACTH were used. The results clearly indicate that the immature granules are the major site of POMC, as their antigenic density (gold beads/m²) was almost 3 times as high as that of ACTH. In the mature igranules, the antigenic density of ACTH was increased by 2.7-fold compared with the immature granules. Using a computer-assisted method, it was possible to categorize the granules antigenic density according to their size. Using this approach it was found that the antigenic density of POMC remained constant in all mature granules of varied sizes, whereas the antigenic density of ACTH decreased with increasing granule size. The relationship between granule size, degree of maturation, and antigenic density is discussed.     

Diurnal variations of serum beta 2-microglobulin in multiple myeloma

The chronobiological circadian behaviour in serum levels of beta 2-microglobulin has been investigated in three groups of subjects: (A) 6 healthy controls; (B) 6 patients with untreated multiple myeloma; (C) 6 patients with multiple myeloma in complete remission after polychemotherapy. From all subjects, under the same standard life conditions, venous blood samples were drawn at 4-hour intervals starting from midnight during the span of a whole day. Circulating serum beta 2-microglobulin levels were determined by RIA method. The time-related data were analyzed by chronograms and the "mean-group cosinor" method. A significant circadian rhythm for serum beta 2-microglobulin was detected in the control group, with a peak in the morning hours, and in untreated patients, with a peak in the afternoon hours. No significant rhythm was found in treated patients with multiple myeloma. A significant mesor reduction was noted in patients with complete remission, correlated with the absence of circadian rhythm, in respect to untreated patients. These data suggest that serum levels of beta 2-microglobulin could be related to the neoplastic plasma cell proliferation and to the effect of therapy, and that the circadian evaluation could be used as a guide in monitoring myeloma patients.     

Diurnal rhythms of cortisol,ACTH, and beta-endorphin levels in neonates and adults.

To determine whether a diurnal rhythm exists in neonates admitted to neonatal intensive care units where there is continuous artificial lighting and periodic nursing and medical care, plasma cortisol, adrenocorticotropin (ACTH), and beta-endorphin concentrations were measured in two groups of infants and in adult human volunteers. As expected, a diurnal rhythm was seen in adults. A diurnal rhythm was also found for cortisol and endorphin levels in neonates (3 to 4 days postnatally) with minimal stress and in infants who were clinically severely stressed. There was not a significant difference between the morning and afternoon concentrations of ACTH in these infants, but the afternoon concentrations were lower than the morning''s, as would be expected. We found that a diurnal rhythm does exist in neonates within the first few days of postnatal life and that the continuous lighting and medical and nursing interventions do not interfere with this rhythm.     

Diurnal Variation in Response to Intravenous Glucose

Intravenous glucose tolerance tests (25 g) were performed in the morning and afternoon on 13 apparently normal persons. The individual K values (rate of decline of blood sugar) were all higher in the morning tests, and the mean values were significantly higher in the morning. Fasting blood sugar levels were slightly lower in the afternoon. There was no difference between the fasting morning and afternoon plasma insulin levels, but the levels after glucose were lower in the afternoon. Growth hormone levels were low at all times in non-apprehensive subjects and unaffected by glucose. The results suggest that the impaired afternoon intravenous glucose tolerance, like oral glucose tolerance, is associated with impaired insulin release and insulin resistance.