Diurnal variations in testosterone in plasma were studied in two inbred strains of mice, BALB/cBy and C57BL/6By. Blood was taken every 4 h over 24 h from male mice at 70 days of age using a lighting regimen of 12 h light to 12 h darkness (lights on 07.00–19.00 h). Values of testosterone in plasma were transformed to log(testosterone in ng/ml) to reduce inequality of variance between groups. In both strains, the distribution of pooled values over all times of day was bimodal, and bimodality was present at most times of day. Circadian variation was evaluated by dividing the transformed values into high and low modes at each time of day and testing for significant variation in the number of animals in each mode over time using the chi-squared test. Significant circadian variation was found in the BALB/cBy strain of mice but not in the C57BL/6By strain. The highest number of high mode cases for BALB/cBy mice was at 22.00 h and the lowest number of high mode cases was at 10.00 h. The log transformation and bimodality of these values are presented as biological expressions of blood levels of testosterone and of tissue responses to these levels in the male mouse. The strain difference in circadian variation may be related to reported circadian changes in behaviour and to possible genetic effects on sensitivity to environmental change or capacity to express circadian rhythms.

Effects of age, sex and mental condition on the circadian and circannual rhythmicity of plasma prolactin in human subjects were investigated. Circannual changes were recorded on a circadian basis in January, March, June and October in four groups of subjects: seven young men, six elderly men, six elderly women and six senile demented patients (two men and four women). Blood samples were drawn every 4 h over a 24-h period at the four sampling sessions. Circadian rhythms of the hormone were validated in all groups and at all sampling sessions except twice in elderly demented subjects. The 24-h mean levels of prolactin in plasma were approximately the same in young and elderly subjects. The circadian acrophases were most often located in the vicinity of 02.00–04.00 h. The circannual rhythmicity of the hormone showed a sex difference; the rhythm was not validated in either young or elderly men but was detected in the groups of elderly women and elderly demented patients (mainly women). The acrophases were located in May. This paper strongly suggests a sex difference in the circannual rhythmicity of plasma prolactin levels in elderly subjects.

Insulin signaling cascade in peripheral insulin-sensitive tissues regulates whole-body glucose metabolism. Any deregulation in this pathway leads to insulin resistance, ultimately leading to metabolic diseases like type 1 diabetes, type 2 diabetes, and obesity. Insulin signaling in the brain has also been studied for many decades and associated with many primary functions like maintenance of synaptic plasticity, regulation of cognition, and circadian rhythm. Importantly, neuronal insulin signaling has also been associated with the regulation of neuronal glucose uptake. Any impairment in neuronal insulin signaling affecting neuronal glucose uptake has been associated with neurodegenerative disorders like Alzheimer’s disease, the process now being termed as type 3 diabetes. Since the criticality lies in proper signaling cascade, determining important points of deregulation is important. In this review, we have discussed some critical points of such deregulation, dividing them into two classes of enzymes: kinases and phosphatases. We have highlighted their individual roles in neuronal insulin signaling, along with their possible implications in neuronal insulin resistance. Future strategies targeting these nodes in neuronal insulin signaling might be helpful in exploring potential therapeutic opportunities to overcome neuronal insulin resistance and related neurodegenerative diseases.

ABSTRACT

Male Wistar-derived rats (200–250 g) were treated for 14 days with prednisolone 21-sodium succinate at a concentration of 1035 μmol/l in their drinking water. The drug was then replaced with normal tap water and groups of animals were killed at various times during recovery, trunk blood being collected after decapitation. At the same time, hypothalamic slices, anterior pituitary gland fragments and adrenals were removed and their responsiveness assessed by exposure to appropriate stimuli in vitro. Tissues were also extracted to measure changes in content of hormones during recovery. Treatment with prednisolone produced marked reductions in body weight gain, adrenal weight and pituitary ACTH content, but no significant change in hypothalamic corticotrophin-releasing factor (CRF) bio- or immunoreactivity. The ACTH content was restored by 5 days after withdrawal but adrenal weight remained significantly reduced after 9 days of recovery. The responsiveness of the hypothalamus to acetylcholine in vitro was markedly inhibited and was still significantly reduced 7 days after withdrawal. The responsiveness of the anterior pituitary gland to synthetic CRF or arginine vasopressin and that of the adrenal gland to ACTH added in vitro were restored simultaneously after 7 days of withdrawal. In vivo, recovery was assessed by measurement of the response to laparotomy stress. Treatment with prednisolone prevented the increase in the plasma concentrations of ACTH and corticosterone produced by stress, and these responses recovered by 5 days (corticosterone) and 7 days (ACTH) after withdrawal. The abolition of the circadian rhythms of ACTH and corticosterone by treatment was also reversed by 5 days after withdrawal. This pattern of recovery is different from that which we observed after long-term treatment with dexamethasone, where the responsiveness of the hypothalamus and adrenal gland in vitro recovered before that of the anterior pituitary gland.

J. Endocr. (1987) 113, 239–247

ABSTRACT

Young, growing rats which had been chronically (2 weeks) adrenalectomized or parathyroidectomized were used to define the roles of the adrenal and parathyroid glands on the maintenance of normal circadian rhythms of DNA, collagen and non-collagen protein synthesis in the skeleton. The animals were conditioned to food being available ad libitum and to 12 h light: 12 h darkness (lights on from 08.00 to 20.00 h). The pace of DNA, collagen and non-collagen protein synthesis in different regions of the tibia (tibial growth cartilage, metaphysial bone and diaphysial bone) was measured by the in-vivo incorporation of tritiated thymidine (1 h) and radioactive proline (48 h). In intact rats there were no regional differences in the phasing of the circadian profiles; peak DNA and non-collagen protein synthesis occurred at the onset of the dark period while peak collagen synthesis occurred during the middle of the period of light. Adrenalectomy selectively abolished the regional DNA synthesis rhythms without altering the phases of the serum Ca and phosphorus (P) rhythms, which peak at mid-day and at the onset of darkness respectively. Parathyroidectomy abolished the regional rhythms for collagen and non-collagen protein synthesis and serum Ca rhythms, without altering the phase of the serum P and corticosterone rhythms. Dietary Ca-lactate supplements, which raised serum Ca levels towards normal in parathyroidectomized rats, were able to correct serum corticosterone values but did not normalize bone collagen and non-collagen protein synthesis values. These data indicate that the adrenal rhythm governs the proliferative activities of bone and cartilage cells, and that parathyroid hormone is essential to maintain normal collagen and non-collagen protein synthesis rhythms.

J. Endocr. (1984) 103, 49–57

SUMMARY

Six rams of an ancient breed of domesticated sheep (Soay) were subjected to an artificial light régime of alternating periods of long days (16 h light:8 h darkness) and short days (8 h light: 16 h darkness) which induced seasonal development and regression of the testes during a period of 36 weeks. Over 2000 blood samples were taken, and the changes in plasma levels of FSH, LH and testosterone were related to the cycle of testicular activity. During long days plasma levels of gonadotrophins became very low and the testes regressed to about 20% of their maximum size; there was a corresponding reduction in plasma testosterone levels. When the rams were returned to short days reproductive development was again stimulated after 2–3 weeks with a progressive increase in plasma FSH and LH levels and consequent hypertrophy of the testes. It took about 16 weeks of short days for testicular activity to become maximal.

Blood samples collected at hourly intervals for 24 h on ten occasions during the study revealed transitory peaks in plasma FSH and LH levels indicative of episodic release. Changes in gonadotrophin secretion were modulated primarily by alterations in the frequency of episodic release; < 1 spike per 24 h during long days increased to a maximum of 10 spikes/24 h under short daylengths. The peaks of FSH release were of smaller amplitude than those of LH, although during periods of frequent episodic release basal levels of FSH were increased to a greater extent than those of LH.

A circadian rhythm was observed in the plasma levels of FSH, LH and testosterone, which was related to increased gonadotrophin release during the dark phase of the 24 h cycle; changes in blood haematocrit were also observed. The circadian changes appeared to be correlated with the activity cycle of the animals which in turn was dictated by daylight. A possible interrelationship between the circadian cycle and the seasonal cycle is discussed.

In order to determine the temporal relationships between variations in 5-hydroxy-tryptamine (5-HT, serotonin) metabolism in the suprachiasmatic nucleus (SCN) and the cyclic LH surge, and also to check whether implantation of oestradiol capsules might modulate 5-HT metabolism in the SCN, we carried out a parallel study of 5-HT content in the SCN and median eminence, and 5-HT metabolism in the SCN and supraoptic region in vitro. These experiments were performed on intact male rats, ovariectomized females and ovariectomized females implanted with oestradiol.

It was only in ovariectomized rats implanted with oestradiol, in which we have described the existence of a clear-cut circadian rhythm of LH secretion, that we found fluctuations in the content, synthesis and utilization of 5-HT. The content and synthesis were characterized by a peak between 12.00 and 15.00 h, whereas utilization was 50% higher at 09.00 and 19.00 h than at 15.00 h. These fluctuations in 5-HT content and metabolism were specific to the SCN; the median eminence and the supraoptic region did not show such variations. They were also specific to ovariectomized rats implanted with oestradiol, since the patterns of 5-HT content and metabolism in the SCN were the same in males and ovariectomized females and did not differ from those in the median eminence, the supraoptic region or the whole hypothalamus.

These results suggest that 5-HT terminals in the SCN play an important role in the control of cyclic LH secretion at a critical period. Moreover, oestradiol seems to be partly responsible for the fluctuations of 5-HT metabolism in the SCN of ovariectomized rats implanted with oestradiol.

SUMMARY

The concentration of prolactin in serum after oestrogen and progesterone injection into spayed rats was measured by radioimmunoassay.

After a single injection of 5 μg oestradiol benzoate (OB) into long-term ovariectomized rats, serum prolactin concentrations showed a circadian rhythm with high levels in the afternoon and almost no changes in the morning. Peaks of prolactin occurred 2, 3 and 4 days after the injection. Below a dose of 1 μg OB, the response was dose-dependent, but the response was then maximal.

In spayed rats primed with 5 μg OB, the injection of 2 mg progesterone 2, 3 or 4 days later resulted in a significant increase in serum prolactin. This response, in contrast to that of oestrogen, occurred in the morning and in the evening and was found to be dose-dependent. The rise in serum prolactin after injection of 1 mg progesterone also showed a close relationship to the priming dose of OB. Progesterone had no effect in spayed, untreated animals. Maximal levels of prolactin were attained 3–4 h after the s.c. injection of progesterone. The release of prolactin which can be induced either by OB or by progesterone was blocked by the administration of progesterone injected 1 day before the expected release would occur. These results indicate that progesterone exerts both facilitatory and inhibitory effects on prolactin secretion. Male rats were found to be less sensitive to the ovarian steroid treatment.

It is suggested that oestrogen could be responsible for the rise in prolactin observed at pro-oestrus and progesterone for the increase in prolactin in pseudopregnancy and pregnancy.

SUMMARY

The relationship of plasma prolactin concentration and renal electrolyte excretion has been investigated in six normal male volunteers. In two studies, 80 mg frusemide were administered at 18.00 h on Day 1 and followed by dietary sodium restriction. In study A, after 38 h of sodium depletion, a second dose of frusemide was administered at 08.00 h on Day 3. In study B, after 14 h of sodium depletion, the effect of administration of 100 mg spironolactone or 45 mg prorenoate potassium (another aldosterone antagonist) at 08.00 h on Day 2 was compared with that of a placebo.

After the first dose of frusemide in study A, the mean plasma prolactin concentration correlated negatively with the urinary Na and K excretion over 5 h. After 38 h sodium depletion, the plasma prolactin concentration correlated positively with urinary Na excretion following the second dose of frusemide. In study B, after Na depletion for 14 h the plasma prolactin concentration at 08.00 h on Day 2 had a positive correlation with the 24 h urinary log₁₀ Na:K ratio following placebo administration and had negative correlations with the true urinary log₁₀ Na:K ratio following spironolactone and prorenoate potassium administration.

Neither acute Na deprivation nor the administration of single doses of frusemide, spironolactone or prorenoate potassium appeared to affect the normal circadian rhythm of plasma prolactin concentrations which remained constant for each subject throughout the 3 months covered by the investigation.

The correlations of plasma prolactin concentration to renal excretion of electrolytes, with no evidence for a negative feedback control mechanism, suggest an indirect relationship between prolactin and renal function.

Thyroid-stimulating hormone (TSH)-producing cells of the pars tuberalis (PT) display distinct characteristics that differ from those of the pars distalis (PD). The mRNA expression of TSHβ and α GSU in PT has a circadian rhythm and is inhibited by melatonin via melatonin receptor type 1; however, the detailed regulatory mechanism for TSH β expression in the PT remains unclear. To identify the factors that affect PT, a microarray analysis was performed on laser-captured PT tissue to screen for genes coding for receptors that are abundantly expressed in the PT. In the PT, we found high expression of the KA2, which is an ionotropic glutamic acid receptor (iGluR). In addition, the amino acid transporter A2 (ATA2), also known as the glutamine transporter, and glutaminase (GLS), as well as GLS2, were highly expressed in the PT compared to the PD. We examined the effects of glutamine and glutamic acid on TSH β expression and α GSU expression in PT slice cultures. l-Glutamine and l-glutamic acid significantly stimulated TSH β expression in PT slices after 2- and 4-h treatments, and the effect of l-glutamic acid was stronger than that of l-glutamine. In contrast, treatment with glutamine and glutamic acid did not affect α GSU expression in the PT or the expression of TSH β or α GSU in the PD. These results strongly suggest that glutamine is taken up by PT cells through ATA2 and that glutamic acid locally converted from glutamine by Gls induces TSH β expression via the KA2 in an autocrine and/or paracrine manner in the PT.