Effects of dexamethasone on insulin receptor in aging

The aim of this study was to examine the effects of dexamethasone (Dex) on functional properties of the rat insulin receptor (IR). Male Mill Hill hooded rats, 3, 6, 12, 18 and 21 months old, were injected with Dex (4 mg/kg) and rat liver and erythrocytes were used for experiments 18 h after Dex administration. Treatment with Dex lowered the specific binding (SB) of insulin (INS) in the liver of 3- and 18-month-old rats and concentration of INS binding sites (N1, N2) and the dissociation constant of low-affinity binding sites (Kd2) in the liver of 6- and 18-month-old rats. In addition, Dex treatment lowered the liver IR protein level in all analyzed groups, except 21-month-old rats where it remained unchanged, but raised the IR mRNA level in 18-month-old rats. In erythrocytes, treatment with Dex decreased SB and Kd2 (in animals 3 and 6 months old) and N1 (in ones 3 and 18 months old). Following Dex treatment, the INS plasma level increased (in rats 3, 18 and 21 months old), while glucose (Glu) concentration increased in 3 and 12 months old, but decreased in 6- and 21-month-old rats. In summary, Dex exerts the strongest effect on the erythrocyte IR of 3- and 6-month-old rats and the hepatic IR of 18-month-old rats. IR in both tissues is almost insensitive to Dex in 12- and 21-month-old rats. The pattern of age-related changes of IR induced by Dex does not correlate with changes of plasma Glu and INS.     

Increases in the basal secretion rate of follicle-stimulating hormone (FSH) accompany age-associated changes in serum FSH levels on estrus

This laboratory has recently reported that by 5-6 months of age, alterations in the secretion and production of follicle-stimulating hormone (FSH) occur in virgin female rats which precedes the age-related disruption of estrous cycles and attenuation of preovulatory gonadotropin surges. Specifically, circulating immunoreactive FSH levels are higher on estrus in rats 5 months and older compared to levels measured in 2- to 3-month-old rats. Therefore, the present study was conducted to explore a possible mechanism for this age-related increase in FSH levels. At 1400 hr on proestrus, estrus and diestrus-1, groups (n = 6-12 rats/group) of 3- and 7-month-old, cyclic rats were decapitated, trunk blood was collected, and anterior pituitary glands were bisected and placed in incubation flasks containing 1 ml media (medium 199). Following a 30-min preincubation period, hemipituitary fragments were incubated for an additional 2 hr. Media and serum FSH levels were quantified by RIA. Levels of FSH were twofold higher in the serum of 7-month-old rats than 3-month-old rats on estrus. Similarly, the basal secretion rate (BSR) of FSH (expressed as ng FSH/ml/2 hr) was significantly (P less than 0.05) higher from incubated hemipituitary fragments of 7-month-old estrous rats than from fragments obtained from younger estrous rats (7 month: 1637 ng/ml/2 hr vs 3 months: 1253 ng/ml/2 hr). Neither the serum FSH levels nor the BSR of FSH differed between age groups on proestrus or diestrus-1. These results show that age-associated increases in circulating FSH levels on estrus may be attributed to an enhanced basal secretion of FSH from the pituitary gland.     

Immunocytochemical and immuno-electron-microscopical study of growth hormone cells in male and female rats of various ages

Summary Growth hormone (GH) secretory cells were identified by immunogold cytochemistry, and were classified on the basis of the size of secretory granules. Type I cells contained large secretory granules (250\2-350 nm in diameter). Type II cells contained the large secretory granules and small secretory granules (100\2-150 nm in diameter). Type III cells contained the small secretory granules. The percentages of each GH cell type changed with aging in male and female rats of the Wistar/Tw strain. Type I cells predominated throughout development; the proportion of type I cell was highest at 6 months of age, and decreased thereafter. The proportion of type II and type III cells decreased from 1 month to 6 months of age, but then increased at 12 and 18 months of age. The pituitary content of GH was highest at 6 months of age, and decreased thereafter. Estrogen and androgen, which are known to affect GH secretion, caused changes in the proportion of each GH cell type. The results suggest that when GH secretion is more active the proportion of type I GH cell increased, and when GH secretion is less active the proportion of type II and type III cells increased. The type III GH cell may therefore be an immature type of GH cell, and the type I cell the mature type of GH cell. Type II cells may be intermediate between type I and III cells.     

Endocrinological evaluation of GH deficient patient with acromegaloidism showing excessive growth.

In this report we describe the first case of a girl with acromegaloidism in Japan. She had large and coarse facial features with acral enlargement accompanying height overgrowth; these resemble the manifestations of acromegaly and gigantism due to growth hormone (GH) overproduction. However, pituitary function studies revealed a dysfunction of her GH secretion. Moreover, markedly decreased serum somatomedin C (SM-C) levels also indicated impairment of GH secretion. Therefore, GH and SM-C cannot have been responsible for promoting somatic growth. However, serum alkaline-phosphatase (Al-P) and osteocalcin, were increased, indicating that stimulation of bone metabolism was increased without GH and SM-C effects. The patient is a typical case showing growth without GH, and these data suggest the existence of an unidentified growth promoting factor that is independent of GH and SM-C.     

Estrogen antagonism on T3 and growth hormone control of the liver microsomal low-affinity glucocorticoid binding site (LAGS)

Male rat liver microsomes contain a low-affinity glucocorticoid binding site (LAGS) capable of binding all natural glucocorticoids and progesterone with a K_d from 20 to 100 nM. The LAGS level is under endocrine control by T₃, glucocorticoids and GH. These hormones act synergistically at physiological concentrations to increase the LAGS level. Since female rats show a LAGS level that is much lower than the males (0.15 vs 23 pmol/mg protein, respectively), here we investigated whether estradiol could decrease the LAGS in the male rat. Orchiectomized (OX) male rats showed a higher LAGS level than intact rats. This effect was reversed by implanting a Sylastic capsule containing testosterone. When the OX rats were implanted for 20 days with estrogen capsules that provided an estradiol level in serum of 40 pg/ml, their LAGS level decreased from 23 to 0.2 pmol/mg protein. This effect was not observed in intact male rats and can be partially reversed by testosterone implants into OX rats. Both hypophysectomized male rats and hypothyroid-orchiectomized male rats showed very low levels of LAGS. Administration of physiological doses of GH and/or T₃ to these rats greatly increased their LAGS level (from 0.3 to 15 and 16 pmol/mg protein, respectively). Implantation of estrogen capsules to these rats two weeks prior to starting treatment completely inhibited the increase in the LAGS level in response to T₃, and significantly decreased the response to hGH, and to a combination of hGH and T₃. These results suggest that physiological estradiol levels can antagonize the LAGS induction by T₃ and hGH in the male rat, and could be responsible for the low level of LAGS in the female rat. Moreover, estrogen capsules also inhibited the increase in the body and hepatic weights observed after hGH treatment, which suggests a powerful inhibitory effect of low estradiol levels on the male rat liver functions under regulation by T₃ and/or GH.     

Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

The effect of cyclosporine administration on growth hormone release and serum concentrations of insulin-like growth factor-I in male rats.

The aim of this work was to study the effect of cyclosporine on the somatotropic axis. Accordingly, growth hormone (GH) secretion, circulating insulin-like growth factor I (IGF-I) and IGF binding proteins (IGFBPs) in response to cyclosporin A (CsA) treatment were examined in adult male Wistar rats. Cyclosporine administration (5, 10 or 20 mg/Kg daily) over 8 days did not modify the body weight, but it did decrease serum concentration of corticosterone and increased serum IGF-I and GH levels. Rats treated with 5 and 10 mg/Kg of cyclosporine had similar levels of serum IGFBPs to control rats, but there was an increase in circulating IGFBP-3 and IGFPB-1,2 in the group treated with 20 mg/Kg of CsA. The increase in circulating GH correlates with a decrease in pituitary GH content in CsA treated rats, with no modification in hypothalamic somatostatin content, suggesting an increase in pituitary GH release. In order to test this hypothesis, anterior pituitary cell cultures were exposed to different CsA concentrations during a 4 h incubation period. Cyclosporine increased GH secretion in cultured pituitary cells (p<0.05). These data suggest that cyclosporine increases circulating IGF-I and GH by stimulating pituitary GH release.     

Involvement of Nerve Growth Factor and Its Receptor in the Regulation of the Cholinergic Function in Aged Rats

The role of nerve growth factor (NGF) and its receptor (NGFR) in the regulation of cholinergic activity has been studied during the aging process. NGFRs were quantified in cortical membranes using a radioactive binding assay. NGF levels and choline acetyltransferase (ChAT) activity were determined in cortex, hippocampus, neostriatum, and septum. These assays were performed in both adult (6-month-old) and aged (36-month-old) rats. High- and low-affinity 125I-NGF binding sites were present in cortex of adult and aged rats. Furthermore, we observed a decrease in number and affinity of both NGFRs in aged rats. ChAT activity in these rats was lower (approximately 30%) than in adult rats in all the brain regions examined. NGF levels were not modified in cortex and hippocampus and were decreased in neostriatum (55%) and septum (35%). In conclusion, our results suggest that, during the aging process, the cholinergic impairment is related to a decrease in NGF levels in neostriatum but not in cortex and hippocampus. The reduction in level of NGF protein in septum could be due to a decrease in number of high-affinity 125I-NGF binding sites.     

Effects of long-term treatment with GH in the bone mineral density of adults with hypopituitarism and GH deficiency and after discontinuation of GH replacement.

BACKGROUND: Only few previous studies have assessed the effects of long-term growth hormone (GH) replacement therapy on bone mineral density (BMD) in adult patients with GH deficiency. The aim of this study was to investigate the effects of long-term GH therapy on bone metabolism and BMD. MATERIAL AND METHODS: At the start of the study, 20 adults with GH deficiency were randomized to receive either GH, 0.25 IU x kg per week, or placebo. After 6 months, patients in the placebo group were switched to GH therapy, and they received GH for a further 18 months. Of the 20 patients, 14 were male and 6 female with GH deficiency of adult-onset. The mean age of the patients at the start of the study was 40.3+/-10.9 years and the duration of GH deficiency was 10.6+/-6.4 years. Patients deficient in pituitary hormones other than GH had been receiving stable replacement doses of appropriate hormones for at least 6 months before the start of the study. Rates of bone metabolism were assessed by measuring calcium, phosphate, alkaline phosphatase, calciuria, phosphaturia and osteocalcin. BMD was measured by dual X-ray absorptiometry. Body composition was calculated from measurements of bioelectrical impedance. RESULTS: Before GH treatment, BMD in the femoral neck was lower in patients than in controls. The rate of bone resorption markers increased significantly after 6 months and remained stable during the whole treatment period. BMD significantly increased in L2-L4 after 12 months of treatment with an increase of Z-score. The total BMD increase was 4.5+/-6.5%. BMD in the femoral neck increased after 12 months with an increase of Z-score after 18 months. The total increase was 10.4+/-18%. The total BMD increase was not different among patients with or without basal osteopenia. In both groups BMD in L2-L4 and in the femoral neck remained stable after 12 months without GH treatment. Sex, age, BMI and the time in which GH deficiency started, before or after the end of the peak of BMD, did not correlate with BMD. The BMD values and their response to GH treatment did not correlate with other associated deficiencies, and we did not find differences among BMD increase and GH dose, levels of insulin-growth factor-I, insulin growth factor binding protein-3, and parameters of body composition. CONCLUSIONS: The results of the study support previous ones that BMD is subnormal in adults with GH deficiency; that GH replacement therapy stimulates bone turnover with initial biochemical changes; and that in the long term, this stimulation results in a significant augmentation in BMD that continues to increase after 2 years and remains stable after 12 months of GH withdrawal.     

Robust growth hormone (GH) secretion in aged female rats co-administered GH-releasing hexapeptide (GHRP-6) and GH-releasing hormone (GHRH)

Aging is associated with a blunted growth hormone (GH) secretory response to GH-releasing hormone (GHRH), in vivo. The objective of the present study was to assess the effects of aging on the GH secretory response to GH-releasing hexapeptide (GHRP-6), a synthetic GH secretagogue. GHRP-6 (30 micrograms/kg) was administered alone or in combination with GHRH (2 micrograms/kg) to anesthetized female Fischer 344 rats, 3 or 19 months of age. The peptides were co-administered to determine the effect of aging upon the potentiating effect of GHRP-6 on GHRH activity. The increase in plasma GH as a function of time following administration of GHRP-6 was lower (p less than 0.001) in old rats than in young rats; whereas the increase in plasma GH secretion as a function of time following co-administration of GHRP-6 and GHRH was higher (p less than 0.001) in old rats than in young rats (mean Cmax = 8539 +/- 790.6 micrograms/l vs. 2970 +/- 866 micrograms/l, respectively; p less than 0.01). Since pituitary GH concentrations in old rats were lower than in young rats (257.0 +/- 59.8 micrograms/mg wet wt. vs. 639.7 +/- 149.2 micrograms/mg wet wt., respectively; p less than 0.03), the results suggested that GH functional reserve in old female rats was not linked to pituitary GH concentration. The differential responses of old rats to individually administered and co-administered GHRP-6 are important because they demonstrate that robust and immediate GH secretion can occur in old rats that are appropriately stimulated. The data further suggest that the cellular processes subserving GH secretion are intact in old rats, and that age-related decrements in GH secretion result from inadequate stimulation, rather than to maladaptive changes in the mechanism of GH release.     

Induction of lactogenic receptors. I. In the liver of hypophysectomized female rats.

To identify the hormones which affect lactogenic receptors in the liver of chronically hypophysectomized female rats, hormones were injected s.c. for 7 days. Specific binding (%, SB) of labelled ovine prolactin (PRL) in liver membrane preparations (1000,000 X g pellet) of controls was 1%. Estradiol (E2), cortisone (Con), ACTH or bovine growth hormone (bGH) treatment did not induce hepatic binding sites for PRL. Human GH and a single dose of 2mg PRL (but not lower doses) increased SB of PRL. Treatment with oPRL plus ACTH was less effective than hGH plus ACTH (13 vs 28%); combinations of oPRL plus Con as well as administration of oPRL plus ACTH to hypophysectomized and adrenalectomized female rats did not induce SB for PRL. Therapy with oPRL plus hGH (26%) was more potent than oPRL plus bGH (2%). These studies suggest that PRL, GH, and ACTH induce and in concert with sex steroids, modulate the lactogenic receptors in the female rat liver. The effect of ACTH is not due to increased adrenal corticoid secretion.     

Age-associated changes and sex differences in urinary 6-sulphatoxymelatonin circadian rhythm in the rat.

The circadian rhythm of 6-sulphatoxymelatonin (aMT6s) excretion has been determined in male and female rats at 3 weeks and at 2, 8, 14 and 20 months of age. All animals have a pronounced circadian pattern of aMT6s excretion under a 12 hour dark: 12 hour light cycle. A significant increase in aMT6s excretion is observed from 3 weeks to 14 months followed by a decrease at 20 months. There is a highly significant correlation between aMT6s excretion and body weight (r = 0.73 for female rats and r = 0.74 for male rats; p values are all less than 0.001). Thus, a decrease in aMT6s excretion associated with increasing age occurs when body weight is taken into consideration. aMT6s excretion is higher in males at 3 weeks and at 2 and 8 months of ages. Urinary testosterone in male rats and estradiol in female rats increase from 3 weeks to 8 months and decrease at older ages. These data suggest that increase of body weight from 3 weeks to 14 months is an important factor responsible for the age-related alteration. The sex differences in aMT6s excretion in younger rats may be associated with their sex hormonal milieu.     

Distribution of the kinin-breakdown activity in areas of the rat brain in the dynamics of spontaneous hypertension

Kinin-damaging activity (KDA) has been studied in 8 brain areas of normotensive rats and rats with spontaneous hypertension, aged 3 to 12 months. A significant depression in KDA was revealed in midbrain, striatum, thalamus, and pituitary body of normotensive rats 6 months of age, as compared to 3-month-old animals. A tendency towards KDA increase was noted in the hypothalamus. In 12-month-old normotensive rats KDA level returned to baseline (3 months of age). Comparison of KDA in rats with spontaneous hypertension aged 3 to 12 months has revealed no age-dependent differences and it is, therefore, believed that rats with spontaneous hypertension lack certain mechanisms inducing a considerable decrease of KDA in 6-month-old normotensive rats.     

Mast cell density: a quantitative index of acute liver inflammation

OBJECTIVE: To evaluate mast cell (MC) density, in liver tissues taken from young and aging rats treated with carbon tetrachloride (CCl4) or untreated, as a quantitative marker of acute liver inflammation and to investigate whether the density of MCs varied with the rats' age. STUDY DESIGN: Rats aged 2, 6, 12 and 19 months treated intraperitoneally with CCl4 were killed 2 and 24 hours after intoxication. Hepatocellular damage was established by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity. Four histologic sections of 12 specimens from each age group were stained with toluidine blue to identify the MCs, which were counted using a computer-assisted image analysis system. RESULTS: Histology showed hepatocellular necrosis with inflammatory infiltration both 2 and 24 hours after intoxication. Serum AST levels were high in the 6- and 12-month-old rats, whereas ALT levels were high in the those aged 2 and 19 months. Two and 24 hours after intoxication, MC density increased considerably in young rats but less so in rats aged 19 months. CONCLUSION: MC density can be a useful marker of acute liver inflammation. The greater density in young rats suggests that older rats have a reduced immune response or recruit fewer MCs.     

Age-related decline in chaperone-mediated autophagy

Intracellular protein degradation rates decrease with age in many tissues and organs. In cultured cells, chaperone-mediated autophagy, which is responsible for the selective degradation of cytosolic proteins in lysosomes, decreases with age. In this work we use lysosomes isolated from rat liver to analyze age-related changes in the levels and activities of the main components of chaperone-mediated autophagy. Lysosomes from "old" (22-month-old) rats show lower rates of chaperone-mediated autophagy, and both substrate binding to the lysosomal membrane and transport into lysosomes decline with age. A progressive age-related decrease in the levels of the lysosome-associated membrane protein type 2a that acts as a receptor for chaperone-mediated autophagy was responsible for decreased substrate binding in lysosomes from old rats as well as from late passage human fibroblasts. The cytosolic levels and activity of the 73-kDa heat-shock cognate protein required for substrate targeting to lysosomes were unchanged with age. The levels of lysosome-associated hsc73 were increased only in the oldest rats. This increase may be an attempt to compensate for reduced activity of the pathway with age.     

Alteration of somatostatin but not growth hormone-releasing factor pituitary binding sites in obese Zucker rats.

The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [125I-Tyr10]hGRF(1-44)NH2 as radioligand and [127I-Tyr10]hGRF-(1-44)NH2 as competitor, and in pituitary membrane preparations, using [125I-Tyr0, D-Trp8]SRIF14 as radioligand and [127I-Tyr0, D-Trp8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.     

Impaired glucose priming of insulin secretion from perfused pancreas in aged female rats.

Effects of age and glucose levels on insulin secretion and synthesis were studied in the perfused pancreas of young (2-month-old) and older (10-month-old) female Wistar rats. Insulin secretion induced by 16.7 mM glucose showed a triphasic pattern: an early spike and fall (first phase, 0-6 min), followed by a sustained gradual increase (second phase, 7-120 min) and a gradual decreased release thereafter (third phase, 121-360 min) during the perfusion period of 360 min. First and second phase insulin secretion, but not third phase, were lower in older rats than in young rats. Insulin synthesis in old rat pancreas perfused with 16.7 nM glucose for 360 min was much greater than that of young rats. Second phase insulin secretion was restored to comparable levels by 28 mM glucose in older rats. Repeated pulses of 28 mM glucose potentiated subsequent insulin secretion in young rats, but not in older rats. These findings provide further evidence that sensitivity to glucose in pancreatic B cells is altered by aging.