Gilberto E. Bestetti

Continuous Subtherapeutic Insulin Counteracts Hypothalamopituitary-Gonadal Alterations in Diabetic Rats

In experimental animal models, gonadal axis lesions are probably responsible for reproductive disorders associated with diabetes mellitus. The pathogenesis of these disorders is not yet known, but it is assumed that insulin deficiency plays an important role. To check this hypothesis, we have investigated the hypothalamopituitary-gonadal axis of insulin-treated streptozocin-induced diabetic (STZ-D) rats and compared it with that of untreated diabetic and control animals. Insulin was delivered by subcutaneously implanted osmotic minipumps. Furthermore, to determine whether possible beneficial insulin effects are selectively limited to the gonadal axis or act generally, we also studied retinal microangiopathy. The hypothalamopituitary-gonadal axis of insulin-treated diabetic animals was almost unchanged. On the contrary, retinal microangiopathy was only slightly influenced by subtherapeutic insulin doses. In conclusion, continuous administration of insulin at subtherapeutic doses can successfully counteract most of the effects of diabetes on the gonadal axis. Thus, the gonadal-axis impairment in STZ-D animals appears to be related to the fall of plasma insulin below a critical level. Furthermore, the various organ systems may respond to different plasma insulin threshold levels.

Functional and Morphological Changes in Mediobasal Hypothalamus of Streptozocin-induced Diabetic Rats: In Vitro Study of LHRH Release

To investigate the role of the mediobasal hypothalamus (MBH) in diabetic gonadal axis disorders, the MBHs of adult male streptozocin-induced diabetic (STZ-D) rats were examined after incubation in basal conditions or in K+-enriched medium and compared with those of controls. Diabetes lasted 1 mo. Both luteinizing-hormone-releasing hormone (LHRH) release and MBH morphology were studied. After incubation in basal conditions, the LHRH release was unchanged. By light microscopy, the dilated-axon cross sections were more numerous (P < .01) in the basal arcuate nucleus and in the median eminence. By electron microscopy, the ratio of exocytoses to neurosecretory granules observed in the median eminence axon cross sections was smaller (P < .05). The total LHRH immunoreactivity, the number of labeled axons, and the amount of positive material in the axons were reduced (P < .05). After incubation in K+-enriched medium, the LHRH release was markedly reduced (P < .01). The number and area of dilated-axon cross sections, possibly because of the relation between exocytosis and physiological dilation, were less augmented (P < .01). Whereas the number of exocytoses and the ratio of exocytoses to neurosecretory granules were not decreased, the total LHRH immunoreactivity and the number of labeled axons were reduced (P < .05). The releasable LHRH pool therefore seems to be exhausted in control MBH because of long-term stimulation and reduced in the MBH of STZ-D rats because of diabetes. In conclusion, STZ-D causes functional and anatomical MBH lesions that should be pathogenetically relevant for the disorders of the gonadal axis documented in this animal model.

Technical Aspects in the Study of Pathologic Lesions in the Hypothalamus of the Rat

Adequate techniques are of primary importance for successful morphological studies of lesions in the central nervous system of laboratory animals For investigations of the rat hypothalamus in particular the following points deserve special attention: (a) method of fixation, (b) dissection of tissues, and (c) further processing. Over some years we have developed and employed the techniques herewith described (Bestetti and Rossi 1980, and this volume p. 331; Rossi and Bestetti 1981). They allow accurate study of lesions by combining morphological and immunohistochemical methods.

Effect of growth hormone-releasing stimuli in streptozotocin diabetic rats.

The dynamics of growth hormone (GH) secretion in response to different GH secretagogues has been studied in adult freely moving male rats one month after induction of diabetes by single i.v. injection of streptozotocin (60 mg/kg). Baseline plasma GH concentrations and pituitary GH content were not different in streptozotocin-diabetic (St-D) rats and controls. Clonidine (0.15 mg/kg i.v.), an alpha 2-adrenergic agonist, failed to evoke GH release in St-D rats. Substitution therapy with insulin (1 IU/100 g b.wt.daily) delivered through subcutaneously implanted minipumps, allowed re-institution of a normal GH responsiveness to clonidine. At odds with clonidine, FK 33-824 (0.1 mg/kg i.v.), a potent analog of the opioid peptide Met-enkephalin, induced a similar rise in plasma GH levels in control and St-D rats. Finally, administration of a synthetic replicate of a GH-releasing hormone of human pancreatic origin, hpGRF-40 (2.5 micrograms/kg i.v.) elicited a higher GH response in St-D rats than in controls. These data indicate that in St-D rats: (1) an impaired function of noradrenergic pathways controlling GH release is present; (2) contrary to previous beliefs, an alpha 2-adrenergic mechanism is not involved in the GH-releasing effect of opioid peptides; and (3) pituitary GH responsiveness to hpGRF is increased.

Tuberoinfundibular dopaminergic neurons and lactotropes in young and old female rats.

Aging in female rats is accompanied by several endocrine dysfunctions, such as reproductive decline associated with characteristic hyperprolactinemia, lactotrope hyperplasia, and functional impairment of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. The aim of this morphometrical, immunocytochemical, and densitometrical study was to gain a better anatomical knowledge of TIDA neurons and axons as well as of lactotropes in old female rats with (A) or without (NA) pituitary adenomas, compared with young animals. At the hypothalamic level, we found that tyrosine hydroxylase (TH)-labeled neurons in the arcuate nucleus were comparable in young and old NA yet their size and TH-content were increased in A animals. Also the TH-labeled median eminence axons did not differ significantly between young and old NA but were more numerous in the old A rats. Independently from adenomas, both number of prolactin (PRL)-labeled structures and content of immunoreactive PRL were increased in pituitaries of old rats, the plasma PRL levels, however, were high only in A. Our findings support the documented lactotrope hypertrophy and hyperplasia in old female rats and suggest that TIDA-neuron changes only occur in hyperprolactinemic animals carrier of adenomas.

Functional and Morphological Aspects of Impaired TRH Release by Mediobasal Hypothalamus of STZ-Induced Diabetic Rats

Streptozocin-induced diabetes (STZ-D) in rats is associated with marked hypothyroidism characterized by functional impairment and structural lesions of the pituitary-thyroid axis. Degenerative axonal lesions, which can be prevented by insulin administration, have been reported in the mediobasal hypothalamus (MBH) of STZ-D rats. However, direct evidence connecting anatomic MBH lesions with functional impairment is still missing. We therefore performed a combined functional and morphological investigation in 4-mo-old STZ-D male rats (diabetes lasted 1 mo), applying an in vitro model to study in the same isolated MBH 1) the basal and depolarization-induced thyrotropin-releasing hormone (TRH) release during two successive incubations of 20 min each and 2) morphological and morphometric aspects, including distribution and amount (densitometric evaluation) of immunoreactive TRH in the incubated tissue. In basal conditions, TRH release was much lower in diabetic than control MBH during both incubations (P < .01 vs. P < .05). In depolarizing conditions, TRH release was increased during the second incubation in control (P < .05) and during both incubations in diabetic (P < .01) rats, the percentage increase of the TRH release due to ionic stimulation being much higher in diabetic than control animals (P < .01). As determined by light-microscope morphometry, the total area of dilated-axon cross sections was larger in diabetic than control MBH under basal conditions (P < .01), thus confirming degenerative axonopathy in diabetic rats. By densitometry determination, the amount of immunoreactive TRH was higher in stimulated diabetic MBH compared with both stimulated control and basal diabetic MBH (P < .01). The total area of immunoreactive TRH was increased in stimulated diabetic MBH (P < .05). Our findings show impaired in vitro TRH secretion without reduced TRH content in diabetic MBH. This should play an important role in the pathogenesis of the pituitary and thyroid lesions responsible for the hypothyroidism observed in STZ-D rats.

Reverse hemolytic plaque assay study of luteinizing and follicle-stimulating hormone and thyrotropin secretion in diabetic rat pituitary glands.

Numerous studies indicate that an impaired hypothalamopituitary axis plays an important role in reproductive and thyroid disorders in diabetic humans and animal models. Yet, several questions about the pathogenesis of these diabetic complications have not been answered. To evaluate the basal secretion of single gonadotrophs and thyrotrophs in vitro, uncultured pituitary cells from control rats and 1-mo streptozocin-induced diabetic (STZ-D) rats were studied with a reverse hemolytic plaque assay and morphometry. After light-microscopy immunocytochemistry for gonadotropin and thyrotropin (TSH), we recorded the ratio of plaqueforming to non—plaque-forming cells. The area of plaques produced by luteinizing hormone (LH), folliclestimulating hormone (FSH), and TSH cells and the area of plaque-forming and non—plaque-forming cells were clearly smaller in diabetic than control rats. The plaque area, however, was more severely reduced than the cell area. The percentage of LH-, FSH-, and TSHimmunoreactive plaque-forming cells was greatly decreased in diabetic compared with control animals. In conclusion, our findings demonstrate that the LH-, FSH-, and TSH-secreting cells of diabetic rats released less hormone and were less numerous than the corresponding cells of control rats. Thus, several pathogenetic mechanisms might be involved in reduced gonadotropin and TSH release at the cellular level: 1) anatomical lesions of organelles involved in glycoprotein hormone synthesis and secretion, possibly due to insulin deficiency; 2) decreased gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) receptors on pituitary cells; 3) inadequate GnRH and TRH stimulation; 4) high plasma corticosterone levels; or 5) a combination of points 1–4.

A model for combined morphological and functional investigations on the isolated mediobasal rat hypothalamus

We have developed a model for combined morphological and functional in vitro studies of the isolated mediobasal hypothalamus (MBH) by considering two prerequisites: (1) the tissue must be well preserved, free of morphological artefacts and functionally unimpaired until the end of the in vitro incubation, and (2) the tissue must be processed for morphology in optimal conditions. To test our model we have studied some aspects of the luteinizing hormone-releasing hormone (LHRH) system in 4-month-old male Sprague-Dawley rats. After decapitation the MBH was isolated and put in a flask containing 0.5 ml Hepes-buffered Lockes medium gassed by 5 ml/min of O2/CO2 (95%/5%) and shaken in a water bath at 37 degrees C. After a 10-min washing, the medium was changed twice at an interval of 20 min. After the in vitro incubation the tissue was satisfactorily preserved as judged by light- and electron-microscopic analysis. LHRH, somatostatin and thyrotropin-releasing hormone could be demonstrated by alkaline phosphatase or peroxidase-antiperoxidase immunohistochemistry on semithin sections and by immunogold technique on thin sections. The LHRH secretion was close to basal values after 30 min of incubation (22.1 +/- 4.8 pg/MBH) and then remained constant for another period of 20 min (17.6 +/- 2.6 pg/MBH). During the second 20 min of incubation LHRH secretion increased in presence of 61.6 mM K+ (110.7 +/- 8.7 pg/MBH). Thus the isolated hypothalamus was excitable until the end of the in vitro incubation. We conclude that this model can be successfully used for combined morphological and functional studies.

The age at onset of diabetes influences functional and structural changes in the pituitary-thyroid axis of streptozocin-diabetic male rats.

SummarySevere structural changes leading to marked alterations in secretory activity are known to occur in the pituitary-thyroid axis 1 month after induction of postpuberal streptozocin (SZ)-diabetes. However, SZ-diabetic rats of different age groups have not been compared, nor has the maturity of the pituitary and thyroid glands at the onset of diabetes been correlated with the type and evolution of functional and structural changes. We thus induced diabetes in 1-month (prepuberal or 3-month (postpuberal) old male rats and compared diabetic with control groups 4 and 8 months after SZ or saline injection. We determined: 1) pituitary and thyroid weights, 2) the basal plasma TSH, T3, and T4 concentrations, and 3) several morphometrical measurements in the pituitary and thyroid glands. After 4 months, 1) the pituitary and thyroid weights were decreased, 2) plasma TSH and T3 were unchanged, plasma T4 was reduced, and 3) the number of thyrotropes, degenerative changes of follicle cells, and colloid area were increased, the follicle cell height as well as the number of fused cold follicles decreased, and the follicle area was unchanged in diabetic compared with control rats. The lesions were more conspicuous in prethan in postpuberal diabetic animals. After 8 months, plasma TSH, T3, and T4 were decreased in diabetic compared with control rats. Except for the increased colloid area, all other lesions were similar, though more severe in prepuberal diabetic rats after 8 than 4 months. Few changes were found in postpuberal diabetic rats. We concluded that: 1) the effects of diabetes on the mature hypothalamus, pituitary, and thyroid gland seem to be reversed by aging and 2) the diabetic hypothalamic disorder we previously described appears to play a major pathogenetical role in the development of pituitary and thyroidal lesions.

Hypothalamic-Pituitary-Adrenal Axis of Genetically Obese fa/fa Rats

The genetically obese fa/fa rat, first described by Zucker and Zucker in 1961, is a model of the obesity/type 2 diabetes syndrome. The syndrome is due to an autosomal recessive gene. This animal presents a number of behavioral, metabolic, and endocrine disorders such as hyperphagia, hyperinsulinemia, and hyperlipidemia, all of questionable etiology. Most of these alterations are reversed by adrenalectomy and restored by corticosterone treatment of the adrenalectomized animals. In an attempt to clarify the pathogenesis of the syndrome we investigated morphological and functional aspects of the hypothalamicpituitary-adrenal axis of obese (fa/fa) and lean (FA/?) rats (Bestetti et al. 1990; Guillaume-Gentil et al. 1990).