A new orally bioavailable synthetic androstene inhibits collagen-induced arthritis in the mouse: androstene hormones as regulators of regulatory T cells.

Dehydroepiandrosterone (DHEA) has attracted much interest because of its many antiaging, metabolic and immune-modulating effects in rodents. Synthetic derivatives, such as 5-androstene-16alpha-fluoro-17-one (HE2500) and certain natural metabolites also provide benefit in various animal models of autoimmune and metabolic diseases. But, like DHEA, low potency and low oral bioavailability suggested limited usefulness of these compounds in humans. We hypothesized that HE3286, a novel 17-ethynyl derivative would be orally bioavailable, more potent, and chemically more useful in man than its parent compound. We found that on a dose/mass basis, HE3286 demonstrated up to 25% oral bioavailability in mice. In the DBA mouse model of collagen-induced arthritis (CIA), animals receiving oral treatment with HE3286 (50 mg/kg), beginning at onset of disease, significantly decreased CIA peak scores and daily severity of arthritis scores. Benefit was associated with decreases in: (1) production of TNF-alpha, IL-6, and IL-17; and (2) decreases in joint inflammation, erosion, and synovial proliferation as judged by histological analysis. HE3286 was not found to be immune suppressive in any of the classical models tested, including mitogen-induced proliferation, delayed-type hypersensitivity, or mixed lymphocyte reaction. Instead, benefit was associated with increases in numbers and function of CD4+CD25+FOXp3+CD127- regulatory T cells (T reg). To our knowledge, this is probably the first study to report that an orally bioavailable synthetic analogue of DHEA can ameliorate ongoing disease in a CIA mouse model with relevance to rheumatoid arthritis (RA) and to correlate that finding with decreases in proinflammatory cytokines and increases in T reg cells. Hormones targeting T reg cells hold the intriguing potential to treat autoimmune, infectious, and neoplastic diseases.

Effects of somatostatin antiserum on growth hormone levels in rats with periventricular lesions in the anterior hypothalamus.

To determine whether residual inhibitory control of growth hormone (GH) secretion in rats with lesions of the periventricular nucleus (PVN) and depleted median eminence content of somatostatin (SRIF) is due to SRIF, PVN-lesioned rats were treated with SRIF antiserum. Such treatment, in contrast to normal sheep serum, caused increased (P less than 0.0001) plasma GH in lesioned and control groups. These results indicate that biologically important amounts of SRIF are available in the PVN-lesioned rat.

Release of growth hormone, prolactin and somatostatin during perifusion of anterior pituitary and preoptic-medial basal hypothalamus from male and female rats.

These experiments were designed to determine whether it is possible using in vitro perifusion to identify a sex difference in anterior pituitary (AP) release of growth hormone (GH) and, if so, to determine whether this difference is correlated with a sex difference in hypothalamic release or content of somatostatin (SRIF). Age-matched rats of both sexes were decapitated at approximately 09.00 h, and blood was collected for determination of non-stress plasma concentrations of GH. Each pituitary was rapidly removed and prepared for perifusion of the AP, and each preoptic-medial basal hypothalamus (PO-MBH) was removed and placed in a separate perifusion chamber. The effluent fractions from perifused APs were assayed for GH and prolactin (Prl), and those from PO-MBH blocks were assayed for SRIF. Non-stress plasma GH concentrations were similar in males and females. During perifusion, baseline GH release was higher (P less than 0.001) from male than from female APs. Release of GH from the APs of both sexes was similarly inhibited (P less than 0.001) by a 1-h administration of SRIF (10(-7) M), and high K+ (50 mM) caused larger (P less than 0.05) GH responses from male than from female APs. In contrast, baseline Prl release was higher (P less than 0.01) from female than from male glands, and Prl release was not affected by SRIF. Male and female PO-MBH tissues showed similar baseline release of SRIF and similar responses to high K+. After perifusion, GH content and concentration were higher in APs from males than from females, but SRIF content in the perifused male and female PO-MBH tissues was similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of periventricular lesions on the release of somatostatin during perifusion.

Hypothalamic periventricular (PV) nucleus lesions reduce median eminence (ME) SRIF content by approximately equal to 80% without affecting non-stress plasma growth hormone (GH) levels or the GH response to stress. Our aim was to study the effects of PV lesions on SRIF released during perifusion of preoptic-anterior hypothalamic (PO-AH) tissue. Female rats received anterior or posterior PV lesions; sham-lesioned and intact rats served as controls. Non-stress and stress plasma GH levels were similar in all groups at 2, 4 and 16 weeks after surgery. At 18 weeks after surgery, the perifused PO-AHs of the PV- and sham-lesioned rats released similar amounts of SRIF, and these were higher (P less than 0.001) than amounts released from PO-AHs of intact rats. The PO-AHs from all groups showed similar increases in SRIF release after 56 mM K+. Two rats were chosen randomly from each group to assess ME SRIF content; PV lesions caused almost 80% depletion of SRIF, sham lesions did not. These results confirm that most SRIF neurons in the PV nucleus and 80% of ME SRIF content are not essential for the control of GH levels under non-stress conditions or for the GH response to stress and indicate that PV or sham lesions in the rostral forebrain enhance in vitro SRIF release, perhaps from neurons outside the PV nucleus.

Loss of enhanced nocturnal growth hormone secretion in aging rhesus males.

Enhanced nocturnal release of GH is decreased with aging in man, a change that may implicate GH in a general decline in anabolic metabolism associated with aging. The aim of this study was to determine whether nonhuman primates experience an age-related reduction in plasma GH levels by comparing the 24-h patterns of GH secretion in unrestrained young and aged male rhesus monkeys. Six young (8 yr old) and six aged (22+ yr old) intact rhesus males were fitted with indwelling jugular catheters, cranial platforms, and stainless steel cable tethers. Catheters passed from a swivel device at the top of each cage through a wall to an adjoining room. On four occasions, 1.0-ml blood samples were obtained from each male every 20 min for 24 h for plasma GH RIA. Plasma GH data were analyzed by the PULSAR program to detect hormone peaks. Mean 24-h plasma GH was less (P less than 0.0005) in aged males [0.84 +/- 0.04 ng/ml (+/-SEM)] than in young males (1.37 +/- 0.09 ng/ml). Likewise, the amplitudes of GH pulses were less (P less than 0.001) in aged males than in young males. Although no circadian pattern of GH concentrations was apparent in either age group, young males displayed more (P less than 0.05) nocturnal GH pulses (5.73 +/- 0.41 pulses/12 h) than those occurring during lights-on (3.09 +/- 0.32 pulses/12 h). The numbers of GH pulses in aged males (4.00 +/- 0.63 pulses/12 h) were similar to those in young males during lights-on, but aged males showed fewer (P less than 0.05) nocturnal GH pulses (4.27 +/- 0.47 pulses/12 h) than did young males. The duration of GH pulses in aged males (53.6 +/- 5.0 min) was similar to that in young males (50.6 +/- 5.0 min) during lights-off. Young males showed an extended (P less than 0.001) GH pulse duration (88.8 +/- 8.8 min) during lights-on that was not evident in aged males (66.2 +/- 5.4 min). These data demonstrate that unrestrained young rhesus males experience an enhanced nocturnal release of GH in terms of pulse frequency, and as in humans, this enhanced nocturnal release of GH is diminished with age. In addition, rhesus males experience, as do humans, a reduction in circulating GH levels as a function of age.

The androgen status of aging male rhesus macaques.

Whether the rhesus male experiences changes in the dynamics of testosterone (T) and LH secretion with age was investigated in six young (6-8 yr old; 7-12 kg) and six aged (22+ yr old; 9-11 kg) intact rhesus males. Each male was fitted with an indwelling jugular catheter, which was attached to a cranial platform and stainless steel cable tether. Catheters passed from a swivel device at the top of each cage through a wall to an adjoining room. On four occasions, 1.0-ml blood samples were obtained from each male every 20 min for 24 h for plasma T RIA and plasma LH bioassay. Plasma T and LH data were analyzed by the PULSAR program to detect hormone peaks. Mean 24-h plasma T levels were similar in young and aged males as were the pulse amplitudes of T; however, the pulsatile pattern of T concentrations was different between young and aged males. Young males displayed a marked nocturnal increase in both T concentration and number of T pulses. Aged males demonstrated a significant nocturnal elevation of plasma T concentration, but displayed a significant nocturnal diminution in both concentration and numbers of T pulses compared with young males at night. Although LH concentrations and the total number of LH pulses per 24 h were similar in young and aged males, marked age-related alterations were evident in the pulsatile pattern of LH levels. Unlike young males, aged males failed to display a daytime reduction in the number of LH pulses. Our data point to coexistent changes in hypothalamic sensitivity to the negative feedback effects of T and testicular responsiveness to LH as a function of age in the rhesus macaque.

Hypothalamic aromatase activity in young and old male rats.

We studied the capability of hypothalamic tissue from young (3-4 months) and old (22-24 months) male rats to aromatize androgens to estrogens. Aromatase activity was measured in homogenates of brain tissue by using a radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. Despite the 40% drop in circulating testosterone (T) levels associated with aging in males, we found that hypothalamic aromatase activity was unaffected by age. This finding suggested that chronic exposure to low levels of circulating T can maintain brain aromatase activity in aged male rats. In an experiment designed to examine the acute response of hypothalamic aromatase activity to induction by T, we found a significant positive correlation between circulating T levels and hypothalamic aromatase activity in both age groups. These results demonstrate that T remains an effective regulator of hypothalamic aromatase in old male rats.

Effects of hypothalamic periventricular lesions on pulsatile growth hormone secretion.

The purpose of this study was to determine the effects of destroying somatostatin (SRIF) neurons of the periventricular (PV) nucleus of the hypothalamus on the pulsatile pattern of growth hormone (GH) secretion in female rats. At 6-10 days after placement of PV lesions, blood samples collected every 15 min for 3-4 h showed an elevation in baseline GH levels and an increase in the amplitude of GH secretory peaks; the frequency of pulses was not affected. These changes were associated with an increase in mean integrated plasma GH levels. The alterations appeared transient because nonstress plasma GH levels were normal in two blood samples collected between 6 and 17 weeks after lesion placement and at autopsy at 17 weeks. Stress-induced suppression of GH secretion was also unaffected by the PV lesions. These lesions severely compromised the SRIF system that projects to the median eminence because the median eminence content of SRIF was approximately 85% depleted in the lesioned group. These results confirm that the hypothalamic PV nucleus is essential for maintaining most of the SRIF in the median eminence and demonstrate that damage to the PV nucleus causes transient alterations in the pulsatile pattern of GH secretion. However, the PV nucleus does not appear to play a major role in driving pulsatile GH secretion.

Anterior pituitary prolactin release in vitro after interruption of anterolateral neural connections of the mediobasal hypothalamus in male rats.

These studies examined the effects of surgical interruption of anterolateral neural connections of the medial basal hypothalamus (MBH) on the subsequent ability of the perifused anterior pituitary (AP) to release prolactin (Prl). Anterolateral MBH cuts were made in adult male rats; sham-operated rats served as controls. An in vivo experiment performed at 5 weeks after the two types of surgery showed that the MBH cuts did not alter non-stress plasma Prl concentrations, but this surgery reduced (P less than 0.05) the Prl response to stress. At 4 months after surgery, the perifused APs from rats with MBH cuts released less (P less than 0.001) Prl under basal conditions and when perifused in series with blocks of preoptic-hypothalamic tissue than did APs from the sham-operated controls. The APs from the MBH-cut and control groups released similar amounts of Prl in response to an initial administration of 56 mM potassium (high K+). After 6 h of perifusion, the APs of the group with MBH cuts contained less (P less than 0.001) Prl than did those of the control group. These findings suggest that transecting anterolateral connections of the MBH causes a reduction in Prl synthesis and steady-state release without affecting the pool of Prl that is released in response to high K+.

Anterior pituitary luteinizing hormone secretion during continuous perifusion in aging male rats.

The possibility that the gonadotrope population of the anterior pituitary experiences an age-related alteration in function was investigated by in vivo and in vitro methods in young (4- to 6-month) and old (18- to 20-month) male Sprague-Dawley rats. Plasma concentrations of testosterone were 50% lower in the old rats, but resting concentrations of luteinizing hormone (LH) were similar in the two age groups. After leg-restraint and blood-withdrawal stress, plasma LH levels were significantly elevated in both young and old males; however, LH levels achieved by aged males were 39% less than those achieved by young males. During perifusion of anterior pituitary, release of LH (ng/ml per 10 min) was stable for 7 h; old anterior pituitary released only 52% as much LH as young anterior pituitaries. The anterior pituitary LH content after perifusion was not altered with age. Castration 2 weeks prior to perifusion caused elevations in plasma LH and in LH released from anterior pituitary during perifusion that were similar in the two age groups. Implantation of testosterone-filled Silastic capsules 2 weeks prior to perifusion elevated plasma testosterone and reduced plasma LH levels in both age groups. The in vitro release of LH from anterior pituitaries was similarly reduced in both age groups. Administration of varying doses of LH-releasing hormone (LHRH) during perifusion caused similar releases of LH above baseline levels in young and old rats. These in vitro results show that aged male rat anterior pituitaries release less LH than anterior pituitaries from young males. However, the magnitude of the LH response of anterior pituitaries to LHRH is not reduced with aging. These findings suggest that the decline in androgen status in old rats is not attributable to a deficit in pituitary responsiveness to LHRH. The effects of manipulating testosterone levels failed to implicate a change in anterior pituitary sensitivity to feedback as a cause for the hormonal status of aged male rats.

The steroidogenic capacity of the aging rat testis.

Factors bearing on the steroidogenic capacity of the aging testis have been examined in Sprague-Dawley rats between 3 and 24 months of age. Neither the number nor the binding affinity of testicular receptors for 125I-hCG changed with age. The ability of the aging testis to synthesize cholesterol from octanoate was similarly undiminished. Testicular conversion of cholesterol to pregnenolone, the gonadotropin-responsive, rate-limiting portion of the androgenic pathway, remained near young adult levels with advancing age. The rate at which the testis converted pregnenolone to testosterone actually increased by almost 25% in the oldest rats. Hence, the steroidogenic capacity of the Sprague-Dawley rat testis does not decline with advancing age.

The androgen status of aging male rats.

In male Sprague-Dawley rats between 3-24 months of age, plasma concentrations of testosterone declined by more than 50% while concentrations of LH in plasma remained relatively constant. During the same interval, body weight rose almost 50%, suggesting that total circulating amounts of testosterone, assuming a proportional expansion of plasma volume, remained relatively constant with increasing age and that total LH in the circulation actually increased in older rats. This assumption was justified by demonstrating that blood plasma volume increased in proportion to body weight over the range of ages and weights represented by rats in this study. Plasma testosterone levels achieved after the injection of gonadotropin were significantly lower in the oldest rats, but when adjusted for increased plasma volume, total testosterone added to the circulation in response to injected gonadotropin did not diminish with age. Age-related change was not detected in testosterone secretion by decapsulated rat testes, either under control conditions or after the addition of gonadotropin to the incubation medium. The average volume of individual Leydig cells remained near young adult values with advancing age, while the total number of Leydig cells per testis rose slightly in the oldest rats. Hence, diminished androgen status in old rats could not be attributed to functional or numerical deficits in the Leydig cell population. Instead, low plasma testosterone levels may have resulted from two interrelated extratesticular phenomena, dilution of secreted hormone with the expanded volume of plasma in a significantly larger body mass, and failure of LH levels to rise sufficiently to stimulate additional testosterone secretion. (Endocrinology 108: 712, 1981)

Attrition of the human Leydig cell population with advancing age.

Existing evidence suggests that the aging human male experiences a gradual decline in testosterone production, a phenomenon that should be reflected in the Leydig cell population of the testis. It has been proposed that Leydig cells diminish in number with increasing age, but conflicting claims characterize reports of this topic. We have reinvestigated this possibility by histometric analysis of perfused testes from 25 men ranging from 18 to 87 years of age. Average single Leydig cell volume (2,943 +/- 623 micrometer 3, X +/- S.D.) did not change significantly with increasing age (r = 0.24, P greater than 0.2), suggesting that surviving cells remain active. Total testis weight (43.5 +/- 13.9 g) also did not change with age (r = 0.04, P greater than 0.5). However, both total Leydig cell volume and the absolute number of Leydig cells per individual decreased significantly as functions of age (r = -0.71, P less than 0.002, and r = -0.61, P less than 0.005, respectively). Analysis of relationship between these two parameters indicates that the total volume of Leydig cell cytoplasm contained within the human testis is determined by the number of cells present. Our results show that a pair of young adult testes endowed with more than 700 million Leydig cells at 20 years of age may be expected to undergo an attrition rate of approximately 80 million cells per subsequent decade of life. Thus, Leydig cell attrition is an important correlate of declining androgen status in aging men.

Kidney in the aging cat: hexokinase, aldolase, L-alpha-glycerophosphate dehydrogenase, and lactate dehydrogenase histochemistry.

Hexokinase, aldolase, L-alpha-glycerophosphate dehydrogenase, and lactate dehydrogenase activities were determined in the kidney of the aging cat. Kidneys from 24 domestic cats 2 months to 7.5 years old in 6 age groups were examined by light microscopic and histochemical methods. Enzyme activities in anatomic components of the kidney were assessed on a quantitative basis for evaluation of mean activity between the age groups. In the cats with advancing age, renal components generally had stable activity. A significant (P less than 0.05) increase in hexokinase activity occurred with advancing age in the ascending part of the renal loop (Henle's loop) and in the distal convoluted tubule. Significant (P less than 0.05) increases in aldolase activity with aging were in cortical connective tissue, internal part of the glomerular capsule (podocytes), distal convoluted tubule, and convoluted segment (Pi) of the proximal portion of the nephron tubule. L-alpha-glycerophosphate dehydrogenase and lactate dehydrogenase activity increased significantly with aging in the convoluted (Pi) segment of the proximal portion of the nephron tubule.

Kidney in the aging cat: neutral lipid and adenyl cyclase histochemistry.

Neutral lipid and adenyl cyclase were evaluated to determine their concentrations in the kidney of the aging cat. Kidneys from 24 domestic cats 2 months to 7.5 years old in 6 age groups were examined by light microscopic and histochemical methods. Activity in anatomic components of the kidney was assessed on a quantitative basis for evaluation of mean activity between the age groups. Renal components generally were stable in activity in the cats with advancing age. A significant increase in lipid concentration occurred in the convoluted segments of the proximal tubules with advancing age. In contrast, the straight segments of the proximal tubules displayed a significant decrease in lipid concentration with aging. Significant increases in adenyl cyclase activity were evident with aging in the ascending thick limb of Henle's loop and distal tubules.