Dopamine production in the caudate putamen restores feeding in dopamine-deficient mice.

Dopamine-deficient (DD) mice cannot synthesize dopamine (DA) in dopaminergic neurons due to selective inactivation of the tyrosine hydroxylase gene in those neurons. These mice become hypoactive and hypophagic and die of starvation by 4 weeks of age. We used gene therapy to ascertain where DA replacement in the brain restores feeding and other behaviors in DD mice. Restoration of DA production within the caudate putamen restores feeding on regular chow and nest-building behavior, whereas restoration of DA production in the nucleus accumbens restores exploratory behavior. Replacement of DA to either region restores preference for sucrose or a palatable diet without fully rescuing coordination or initiation of movement. These data suggest that a fundamental difference exists between feeding for sustenance and the ability to prefer rewarding substances.

Cross-sectional association of endogenous steroid hormone, sex hormone-binding globulin, and precursor steroid levels with hemostatic factor levels in postmenopausal women.

Essentials Endogenous hormone levels' influence on hemostatic factor levels is not fully characterized. We tested for associations of endogenous hormone with hemostatic factor levels in postmenopause. Estrone levels were inversely associated with the natural anticoagulant, protein S antigen. Dehydroepiandrosterone sulfate levels were inversely associated with thrombin generation. SUMMARY: Background Oral use of exogenous estrogen/progestin alters hemostatic factor levels. The influence of endogenous hormones on these levels is incompletely characterized. Objectives Our study aimed to test whether, among postmenopausal women, high levels of estradiol (E2), estrone (E1), testosterone (T), dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), and androstenedione, and low levels of sex hormone-binding globulin (SHBG), are positively associated with measures of thrombin generation (TG), a normalized activated protein C sensitivity ratio (nAPCsr), and factor VII activity (FVIIc), and negatively associated with antithrombin activity (ATc) and total protein S antigen (PSAg). Methods This Heart and Vascular Health study cross-sectional analysis included 131 postmenopausal women without a prior venous thrombosis who were not currently using hormone therapy. Adjusted mean differences in TG, nAPCsr, FVIIc, ATc and PSAg levels associated with differences in hormone levels were estimated using multiple linear regression. We measured E2, E1, total T, DHEAS, DHEA and androstenedione levels by mass spectrometry, SHBG levels by immunoassay, and calculated the level of free T. Results One picogram per milliliter higher E1 levels were associated with 0.24% lower PSAg levels (95% Confidence Interval [CI]: -0.35, -0.12) and 1 µg mL-1 higher DHEAS levels were associated with 40.8 nm lower TG peak values (95% CI: -59.5, -22.2) and 140.7 nm×min lower TG endogenous thrombin potential (ETP) (95% CI: -212.1, -69.4). After multiple comparisons correction, there was no evidence for other associations. Conclusions As hypothesized, higher E1 levels were associated with lower levels of the natural anticoagulant PSAg. Contrary to hypotheses, higher DHEAS levels were associated with differences in TG peak and ETP that suggest less generation of thrombin.

Ganglioneuromas and renal anomalies are induced by activated RET(MEN2B) in transgenic mice.

Multiple endocrine neoplasia type 2B (MEN2B) is an autosomal dominant syndrome characterized by the development of medullary thyroid carcinoma, pheochromocytomas, musculoskeletal anomalies and mucosal ganglioneuromas. MEN2B is caused by a specific mutation (Met918-->Thr) in the RET receptor tyrosine kinase. Different mutations of RET lead to other conditions including MEN2A, familial medullary thyroid carcinoma and intestinal aganglionosis (Hirschsprung disease). Transgenic mice were created using the dopamine beta-hydroxylase promoter to direct expression of RET(MEN2B) in the developing sympathetic and enteric nervous systems and the adrenal medulla. DbetaH-RET(MEN2B) transgenic mice developed benign neuroglial tumors, histologically identical to human ganglioneuromas, in their sympathetic nervous systems and adrenal glands. The enteric nervous system was not affected. The neoplasms in DbetaH-RET(MEN2B) mice were similar to benign neuroglial tumors induced in transgenic mice by activated Ras expression under control of the same promoter. Levels of phosphorylated MAP kinase were not increased in the RET(MEN2B)-induced neurolglial proliferations, suggesting that alternative pathways may play a role in the pathogenesis of these lesions. Transgenic mice with the highest levels of DbetaH-RET(MEN2B) expression, unexpectedly developed renal malformations analogous to those reported with loss of function mutations in the Ret gene.

Alteration of neurotransmitter phenotype in noradrenergic neurons of transgenic mice.

The normal complement of neurotransmitters in noradrenergic neurons was altered by expressing the structural gene for the enzyme phenylethanolamine-N-methyltransferase (PNMT) under the control of the dopamine-beta-hydroxylase gene promoter in transgenic mice. This resulted in accumulation of large amounts of epinephrine in neurons of the sympathetic nervous system (SNS) and central nervous system (CNS) but did not reduce norepinephrine levels. Adrenalectomy reduced PNMT levels in the SNS and CNS, suggesting that the transgene is positively regulated by adrenal steroids. Epinephrine levels were unaffected by this treatment in the CNS, suggesting that PNMT is not rate limiting for epinephrine synthesis. However, catecholamines were elevated in a sympathetic ganglion and a target tissue of the SNS, perhaps due to up-regulation of tyrosine hydroxylase in response to adrenalectomy. These transgenic mice also reveal a marked difference in the ability of chromaffin cells and neurons to synthesize epinephrine.

Fasting-induced increases in food intake and neuropeptide Y gene expression are attenuated in aging male brown Norway rats.

Aging in man is associated with alterations in food intake (FI) and body weight (BW). To establish a model of age-related alterations in FI and BW regulation, FI and BW were determined in young (3-month-old), middle-aged (12-month-old), and old (24-month-old) male Brown Norway rats during ad libitum feeding and after 72 h of fasting. FI was reduced with aging both during ad libitum feeding and after fasting. With fasting, young rats lost more BW than older rats, but regained BW more rapidly during refeeding. To determine whether age-related impairments in FI and BW regulation are mediated by neuropeptide Y (NPY), a potent stimulator of FI, we compared arcuate nucleus prepro-NPY (ppNPY) messenger RNA (mRNA) by in situ hybridization in fasted and ad libitum-fed (fed) young, middle-aged, and old rats. ppNPY mRNA declined with aging in both fed and fasted rats. Although ppNPY mRNA increased with fasting in all age groups, this response was attenuated with aging. In conclusion, impaired FI and BW recovery after fasting is associated with reduced NPY responsiveness to fasting in aging rats. Impaired activation of the hypothalamic NPY pathway may, therefore, contribute to age-related alterations in FI and BW regulation.

Age-related decrease in neuropeptide-Y gene expression in the arcuate nucleus of the male rat brain is independent of testicular feedback.

Neuropeptide-Y (NPY) is thought to modulate reproductive function and food intake. NPY neuronal activity is modulated by sex steroids, and NPY secretion declines with aging. We hypothesized that reduced NPY secretion with aging is due to decreased NPY gene expression, and that this decrease is independent of testicular feedback. To test this hypothesis, arcuate nucleus (ARC) prepro-NPY (ppNPY) mRNA levels, determined by in situ hybridization, and serum testosterone levels, determined by RIA, were compared in sham-operated and orchidectomized young (3 months old), middle-aged (13 months old), and old (23 months old) male Brown Norway (BN) rats. Hybridization area and average optical density (OD) were used as indices of ARC ppNPY mRNA content. In sham-operated rats, both ppNPY mRNA hybridization area and OD decreased progressively with aging, whereas serum testosterone levels were decreased only in old, not in middle-aged or young, rats. In orchidectomized rats, ppNPY mRNA hybridization area also decreased significantly with aging, although OD did not change significantly. The ppNPY mRNA hybridization area was lower in orchidectomized than in intact young and middle-aged rats, whereas OD was unchanged by orchidectomy. Furthermore, the effects of aging and orchidectomy on ppNPY mRNA levels were not localized to a specific region of the ARC. We conclude that in the male BN rat, ARC NPY gene expression is decreased with aging independently of the effects of testicular feedback. This reduction in NPY synthetic capacity may contribute to altered reproductive function and food intake with aging.

Aging and the neuroendocrine regulation of reproduction and body weight.

Aging in men is associated with a decline in trophic factors such as testosterone (T), alterations in body composition and impaired energy and body weight regulation. We performed studies to investigate the mechanisms underlying age-related changes in the neuroendocrine control of testis function, body composition, food intake and body weight in the Brown Norway (BN) rat. We found that similar to aging men, male BN rats demonstrate both primary and secondary testicular failure with aging without confounding age-related tumors, hormonal changes and systemic illnesses. With aging, these animals have blunted circadian variations in luteinizing hormone (LH) and T, and decreased hypothalamic gonadotropin-releasing hormone (GnRH) synthetic capacity with preserved pituitary gonadotropin responses to GnRH. We found that aging male BN rats have increased peripheral and visceral adiposity associated with increased insulin and leptin levels, and decreased relative lean body mass and muscle mass. We found that these rats exhibit reduced food intake and body weight gain associated with decreased hypothalamic neuropeptide Y (NPY) gene expression in the arcuate nucleus (ARC), both during ad-libitum feeding and after a 72-h fast. Recently, we found that old male BN rats treated chronically with troglitazone, an insulin sensitizer, lowered high insulin and leptin levels, decreased body fat, and corrected the blunted food intake and body weight gain response to fasting without affecting basal ARC NPY gene expression. These findings suggested that hyperinsulinemia and/or hyperleptinemia associated with aging may contribute to the age-related impairment in energy and weight regulation. Our studies suggest that the aging male BN rat is an excellent model to investigate the mechanisms underlying the age-associated changes in the neuroendocrine control of body composition, energy intake and body weight.

Cross-sectional and longitudinal analysis of age-associated changes in body composition of male Brown Norway rats: association of serum leptin levels with peripheral adiposity.

Aging-associated alterations in body composition are accompanied by changes in the endocrine system. We evaluated, in male Brown Norway rats, the effects of aging on body composition and the association with serum levels of leptin, insulin, and testosterone. Body composition was assessed cross-sectionally in male rats (3, 8, 17, and 29 months) by a combination of dual energy x-ray absorptiometry (DEXA) and dissection of specific muscles and adipose depots. Longitudinal changes in body composition were quantified by DEXA before and after 3 months of ad-libitum feeding. Body weight, lean mass, absolute and percentage fat increased with age, whereas percentage of lean mass decreased. Leptin and insulin levels increased with age in proportion to adiposity; the increase in leptin with age was related to increased total and peripheral, but not visceral, fat. Testosterone decreased with age, and was associated with decreased lean and skeletal muscle mass. These findings suggest that alterations in body composition with age may be due to decreased trophic and increased lipogenic hormones. Relative to other rodent models, Brown Norway rats undergo shifts in body composition and in the hormonal milieu that are consistent with changes seen in aging humans.

Age-related decrease in hypothalamic gonadotropin-releasing hormone (GnRH) gene expression, but not pituitary responsiveness to GnRH, in the male Brown Norway rat.

As is the case in humans, aging male Brown Norway (BN) rats exhibit both primary and secondary (hypothalamic/pituitary) testicular failure. We hypothesized that secondary testicular failure in aging BN rats is due to alterations in both hypothalamic and pituitary function. In order to determine whether gonadotropin-releasing hormone (GnRH) gene expression is altered with aging, we compared hypothalamic preproGnRH (ppGnRH) mRNA by in situ hybridization histochemistry and GnRH peptide content in microdissected brain areas by radioimmunoassay in intact (or sham-operated) young, middle-aged, and old male rats. In addition, we determined hypothalamic-pituitary responsiveness to the removal of testicular feedback by comparing ppGnRH messenger RNA (mRNA) and gonadotropin levels in sham-operated and orchidectomized young, middle-aged, and old rats. In sham-operated rats, both the cellular ppGnRH mRNA content and the number of neurons expressing ppGnRH mRNA were lower in old compared with young and middle-aged rats. In addition, GnRH content decreased with aging in intact rats in 2 of the 3 brain areas examined, and GnRH content tended to decrease with aging in the third region. Morning serum luteinizing hormone (LH) levels were unchanged with aging, whereas follicle-stimulating hormone (FSH) was significantly increased in old compared with younger intact rats. The cellular ppGnRH mRNA content also decreased with aging in orchidectomized rats, although the number of neurons expressing ppGnRH mRNA was unchanged with aging in these rats. Within age groups, the cellular ppGnRH mRNA content was higher in orchidectomized than in sham-operated rats, though there was no effect on the number of neurons expressing GnRH. In a second study, we compared pituitary responsiveness to GnRH by measuring serum LH and FSH levels after GnRH administration in intact BN rats of different ages. The LH response to GnRH was unchanged with aging, whereas the FSH response to GnRH tended to increase with aging. Despite similar LH responses, the testosterone (T) response to GnRH declined progressively with aging. A third study assessed age-related changes in the circadian rhythm of circulating LH, T, and corticosterone (B) levels. LH levels over a 24-hour period decreased with aging and tended to be lower in the morning hours in all age groups, and circadian rhythmicity was blunted in middle-aged and old compared with young rats. T levels over 24 hours declined progressively with aging, and these levels showed a bimodal diurnal variation in young rats, a variation that was not evident in older animals. B levels over a 24-hour period were lower in old than in younger animals, and with aging, there was dampening of the amplitude of the circadian rhythm of B. Taken together, these findings suggest that secondary testicular failure in aging male BN rats is due in part to decreased GnRH gene expression rather than to decreased pituitary responsiveness to GnRH. This reduction in GnRH gene expression with aging is not dependent on testicular feedback factors. Finally, the blunted circadian rhythmicity of LH and T secretion with aging provides further evidence of altered hypothalamic regulation of gonadal hormone secretion in old animals.

Androgen synthesis in the gonadotropin-suppressed human testes can be markedly suppressed by ketoconazole.

CONTEXT: The concentration of intratesticular testosterone (IT-T) required for human spermatogenesis is unknown because spermatogenesis can persist despite the markedly reduced IT-T concentrations observed with LH suppression. Methods to lower IT-T further are needed to determine the relationship between IT-T and spermatogenesis. OBJECTIVE: The objective of the study was to determine the effect of inhibiting the synthesis and metabolism of testosterone (T) on IT-T in gonadotropin-suppressed human testes. DESIGN/SETTING/PATIENTS: Forty normal men participated in a blinded, placebo-controlled, randomized trial at an academic center. INTERVENTION/OUTCOME MEASURES: All men were first administered the GnRH antagonist acyline to suppress LH. Forty-eight hours after acyline administration, subjects were randomly assigned to placebo, ketoconazole (to inhibit T synthesis) at 400 or 800 mg, dutasteride (to inhibit T metabolism) 2.5 mg, or anastrazole (to inhibit T metabolism) 1 mg, daily for 7 days (n = 8/group). Intratesticular steroid concentrations were measured 48 hours after acyline administration alone and again after 7 days of combination treatment. RESULTS: After 7 days of combination treatment, the median IT-T (25th, 75th percentile) in the placebo group was 14 (8.0, 21.2) ng/mL. IT-T was reduced to 3.7 (2.5, 7.1) ng/mL in the ketoconazole 400 mg group and 1.7 (0.8, 4.0) ng/mL in the ketoconazole 800 mg group (P < .001 vs placebo for both comparisons). IT-T concentrations in the dutasteride and anastrazole groups were similar to placebo. CONCLUSION: Combining inhibition of steroidogenesis with gonadotropin suppression lowers IT-T more than gonadotropin suppression alone. This combination might be useful to determine the minimum IT-T concentration necessary for human spermatogenesis, information essential for developing male hormonal contraceptives.

The effect of gonadotropin withdrawal and stimulation with human chorionic gonadotropin on intratesticular androstenedione and DHEA in normal men.

INTRODUCTION: Concentrations of intratesticular (IT) testosterone (T) are known to be 100-200 times those of serum T; however, the IT concentrations of T's precursors, their testicular to serum gradients, gonadotropin dependence, and response to stimulation with human chorionic gonadotropin (hCG) have not been studied in detail. We hypothesized that serum and IT androstenedione (ADD) and IT dehydroepiandrosterone (DHEA) would be significantly suppressed by the administration of a GnRH antagonist and increased when stimulated by hCG, without a similar suppression of serum DHEA. METHODS: We suppressed gonadotropins in 23 normal men with the GnRH antagonist acyline and randomly assigned them to one of four doses of hCG, 0, 15, 60, or 125 IU sc every other day for 10 d. Blood and IT fluid for the measurement of serum and IT hormones were obtained at baseline and after 10 d of treatment. RESULTS: Baseline IT ADD [median (25th, 75th percentile)] was 629 (308, 860) nmol/liter, and IT DHEA was 564 (411, 879) nmol/liter, which were 175 and 27 times higher than their respective serum concentrations. IT ADD and IT DHEA were suppressed by 98 and 82%, respectively, by acyline and significantly increased with hCG administration. Likewise, serum ADD was suppressed by 50%, but serum DHEA was unchanged. DISCUSSION: ADD and DHEA are highly concentrated within the human testes compared with serum. Serum and IT ADD and IT DHEA are markedly suppressed with GnRH administration and stimulated by hCG, but serum DHEA is not, suggesting that most circulating DHEA is not of testicular origin.

Dose-dependent increase in intratesticular testosterone by very low-dose human chorionic gonadotropin in normal men with experimental gonadotropin deficiency.

CONTEXT AND OBJECTIVE: In men with infertility secondary to gonadotropin deficiency, treatment with relatively high dosages of human chorionic gonadotropin (hCG) stimulates intratesticular testosterone (IT-T) biosynthesis and spermatogenesis. Previously we found that lower dosages of hCG stimulated IT-T to normal. However, the minimal dose of hCG needed to stimulate IT-T and the dose-response relationship between very low doses of hCG and IT-T and serum testosterone in normal men is unknown. DESIGN, SETTING, PATIENTS, AND INTERVENTION: We induced experimental gonadotropin deficiency in 37 normal men with the GnRH antagonist acyline and randomized them to receive one of four low doses of hCG: 0, 15, 60, or 125 IU sc every other day or 7.5 g daily testosterone gel for 10 d. Testicular fluid was obtained by percutaneous aspiration for steroid measurements at baseline and after 10 d of treatment and correlated with contemporaneous serum hormone measurements. RESULTS: Median (25th, 75th percentile) baseline IT-T was 2508 nmol/liter (1753, 3502 nmol/liter). IT-T concentrations increased in a dose-dependent manner with very low-dosage hCG administration from 77 nmol/liter (40, 122 nmol/liter) to 923 nmol/liter (894, 1017 nmol/liter) in the 0- and 125-IU groups, respectively (P<0.001). Moreover, serum hCG was significantly correlated with both IT-T and serum testosterone (P<0.01). CONCLUSION: Doses of hCG far lower than those used clinically increase IT-T concentrations in a dose-dependent manner in normal men with experimental gonadotropin deficiency. Assessment of IT-T provides a valuable tool to investigate the hormonal regulation of spermatogenesis in man.

Restoration of norepinephrine and reversal of phenotypes in mice lacking dopamine beta-hydroxylase.

Mice with a targeted disruption of the dopamine beta-hydroxylase (DBH) gene are unable to synthesize norepinephrine (NE) and epinephrine. These mice have elevated levels of dopamine in most tissues, although the levels are only a fraction of those normally found for NE. It is noteworthy that NE can be restored to normal levels in many tissues after a single injection of the synthetic amino acid precursor of NE, L-threo-3,4-dihydroxyphenylserine (DOPS). In other tissues, NE can be restored to normal levels after multiple injections of DOPS, whereas in the midbrain and cerebellum, restoration of NE is limited to 25-30% of normal. NE levels typically peak approximately 5 h after DOPS administration and are undetectable by 48 h. Epinephrine levels are more difficult to restore. The elevated levels of dopamine fall modestly after injection of DOPS. S(-)-Carbidopa, which does not cross the blood-brain barrier, inhibits aromatic L-amino acid decarboxylase and effectively prevents restoration of NE by DOPS in the periphery, while allowing restoration in the CNS. Ptosis and reductions in male fertility, hind-limb extension, postdecapitation convulsions, and uncoupling protein expression in dopamine beta-hydroxylase-deficient mice are all reversed by DOPS injection.

Induction of stereotypy in dopamine-deficient mice requires striatal D1 receptor activation.

Motor stereotypies are abnormally repetitive behaviors that can develop with excessive dopaminergic stimulation and are features of some neurologic disorders. To investigate the mechanisms required for the induction of stereotypy, we examined the responses of dopamine-deficient (DD) mice to increasing doses of the dopamine precursor L-DOPA. DD mice lack the ability to synthesize dopamine (DA) specifically in dopaminergic neurons yet exhibit robust hyperlocomotion relative to wild-type (WT) mice when treated with L-DOPA, which restores striatal DA tissue content to approximately 10% of WT levels. To further elevate brain DA content in DD mice, we administered the peripheral L-amino acid decarboxylase inhibitor carbidopa along with L-DOPA (C/l-DOPA). When striatal DA levels reached >50% of WT levels, a transition from hyperlocomotion to intense, focused stereotypy was observed that was correlated with an induction of c-fos mRNA in the ventrolateral and central striatum as well as the somatosensory cortex. WT mice were unaffected by C/L-DOPA treatments. A D1, but not a D2, receptor antagonist attenuated both the C/L-DOPA-induced stereotypy and the c-fos induction. Consistent with these results, stereotypy could be induced in DD mice by a D1, but not by a D2, receptor agonist, with neither agonist inducing stereotypy in WT mice. Intrastriatal injection of a D1 receptor antagonist ameliorated the stereotypy and c-fos induction by C/L-DOPA. These results indicate that activation of D1 receptors on a specific population of striatal neurons is required for the induction of stereotypy in DD mice.

Feeding behavior in dopamine-deficient mice.

Mice that cannot make dopamine (DA), a condition caused by the selective inactivation of tyrosine hydroxylase in dopaminergic neurons, are born normal but gradually become hypoactive and hypophagic, and die at 3 weeks of age. We characterized the feeding and locomotor responses of these DA-deficient (DA-/-) mice to 3, 4-dihyroxy-L-phenylalanine (L-DOPA) to investigate the relationship between brain DA levels and these complex behaviors. Daily administration of L-DOPA to DA-/- mice stimulated locomotor activity that lasted 6 to 9 hr; during that time the mice consumed most of their daily food and water. The minimal dose of L-DOPA that was sufficient to elicit normal feeding behavior in the DA-/- mice also restored their striatal DA to 9.1% of that in the wild-type (WT) mice at 3 hr; then DA content declined to <1% of WT levels by 24 hr. This dose of L-DOPA induced locomotor activity that exceeded that of treated WT mice by 5- to 7-fold, suggesting that DA-/- mice are supersensitive to DA. Unexpectedly, DA-/- mice manifested a second wave of activity 24 to 48 hr after L-DOPA treatment that was equivalent in magnitude to that of WT mice and independent of DA receptor activation. The DA-/- mice approached, sniffed, and chewed food during this second period of activity, but they ate <10% of that required for sustenance. Therefore, DA-/- mice can execute behaviors necessary to seek and ingest food, but they do not eat enough to survive.