Linda L. Bellush

Protection against diabetes-induced nephropathy in growth hormone receptor/binding protein gene-disrupted mice.

To further investigate the role of GH in diabetic nephropathy, experimental diabetes was induced with streptozotocin (STZ) in mice in which the GH receptor/binding protein gene was disrupted. Body weight, blood glucose, and renal histology and morphometry were studied 10 weeks after diabetes induction in wild-type (+/+) mice and in mice heterozygous (+/−) and homozygous (−/−) for the disruption. Equivalent levels of hyperglycemia developed in all diabetic groups. Normal weight gain was absent in +/+ and +/− diabetic groups, and− /− diabetics lost weight during the study. Diabetic +/+ and +/− groups both showed evidence of glomerulosclerosis, increases in glomerular volume, and increases in the ratio of mesangial area to total glomerular area, whereas diabetic −/− mice showed none of these pathological changes. These results extend our previous findings of protection against diabetes-associated kidney damage in transgenic mice expressing a GH antagonist. Taken together, the results argue for an important r...

Evidence for Growth Hormone (GH) Autoregulation in Pituitary Somatotrophs in GH Antagonist-Transgenic Mice and GH Receptor-Deficient Mice

Growth hormone (GH) modulates the hypothalamic release of somatostatin and GH-releasing hormone; however, there has been no evidence of GH autoregulation on the pituitary somatotroph. To determine the effects of GH on its own regulation, we examined the pituitaries of giant transgenic mice expressing a GH agonist (E117L), dwarf transgenic mice expressing a GH antagonist (G119K), and dwarf mice devoid of the GH receptor/binding protein (GHR/BP). In the E117L transgenic mice, the number and distribution of pituitary GH-immunoreactive cells were unchanged from nontransgenic littermate controls; an ultrastructural examination revealed typical, densely granulated somatotrophs. In contrast, the pituitaries of the G119K mice contained both moderately granulated somatotrophs and a sparsely granulated (SG) population with well-developed synthetic organelles and a distinct juxtanuclear globular GH-staining pattern. GHR/BP-deficient mice exhibited a marked reduction in the intensity of cytoplasmic GH immunoreactivity; however, prominent GH staining in the juxtanuclear Golgi was seen. GH-immunoreactive cells were increased in number, and the reticulin network pattern was distorted; stains for proliferating cell nuclear antigen confirmed mild hyperplasia. Electron microscopy showed that the somatotrophs were hyperactive SG cells with prominent endoplasmic reticulum membranes, large Golgi complexes, and numerous mitochondria. These findings are consistent with synthetic and secretory hyperactivity in pituitary somatotrophs due to the reduced GH feedback regulation. The changes are most striking in animals that are devoid of GHR/BP and less marked in animals expressing a GH antagonist; both models had reduced insulin-like growth factor-I levels, but the more dramatic change in the GHR/BP animals can be explained by abrogated GH signaling. This represents the first evidence of direct GH feedback inhibition on pituitary somatotrophs, which may have implications for the use of GH analogs in different clinical settings.

The functional significance of biochemical alterations in streptozotocin-induced diabetes

These experiments examined the effects of restraint stress on dopamine (DA) and 5-hydroxytryptamine (5-HT) and their principal metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA), respectively, in 4 brain regions, as well as on plasma corticosterone concentration (CORT) and behavior in streptozotocin-induced diabetic rats and nondiabetic controls. Diabetic rats had widespread reductions in DA and 5-HT turnover (DOPAC/DA and 5-HIAA/5-HT ratios). Restraint led to equivalent increases in DA turnover in diabetics and nondiabetics, but attenuated increases in 5-HT turnover in diabetic rats. CORT concentration of diabetics and nondiabetics measured in complete quiet did not differ. Relative to these measures, only diabetics had elevated CORT when either restrained or kept in the same room with restrained rats with food and water removed. Open-field exploration was suppressed by restraint in diabetics only. All diabetic rats showed decreased locomotion in a novel environment which was normalized during a second exposure to the apparatus. Together, these results suggest that diabetes-induced disruptions in open-field activity are related to anxiety rather than to motor or energy deficits, and may be related to impaired 5-HT and CORT systems.

Diabetes mellitus: Stress, neurochemistry and behavior

Neurochemical alterations in several rodent models of insulin-dependent diabetes are compared and their relevance to behavioral and physiological pathology in the clinical disorder is discussed. In the majority of rodent models, reductions in metabolism of norepinephrine (NE), dopamine (DA) and serotonin (5HT) in the central nervous system (CNS) have been reported. While there are two reports of increased 5HT turnover in CSF or post-mortem brains of diabetic humans experiencing severe ketoacidosis, these patients were receiving insulin therapy. Insulin appears to have effects on monoamines opposite to that of chronic hyperglycemia. Both in rodent models and in clinical populations, there is widespread evidence of enhanced hormonal and behavioral responsiveness to stress. There are findings in rodent models indicating that hormonal responses to stress are related to CNS monoamine activity. The mechanisms responsible for both hormonal and CNS alterations in diabetes, as well as their involvement in behavioral pathology, can best be investigated further using animal models.

Streptozotocin-Induced Decreases in Serotonin Turnover Are Prevented by Thyroidectomy

Although characterized as hypothyroid, streptozotocin-diabetic rats have reduced serotonin turnover (5-hydroxyindoleacetic acid/serotonin, 5-HIAA/5-HT) in brain stem, while hypothyroid rats have increased 5-HIAA/5-HT. In the present study the two treatments were combined to determine if they affected 5-HIAA/5-HT through the same mechanism. In addition, an alternative method was used to assess 5-HT activity in thyroidectomized (TX) rats, i.e. measurement of 5-HT disappearance after inhibition of tryptophan hydroxylase with p-chlorophenylalanine (PCPA). Adult male rats were first TX (experiment 1) or given methimazole (METH; experiment 3). Two weeks later, diabetes (DB) was induced with streptozotocin in hypothyroid rats and euthyroid controls. Two weeks later, functional measurements were taken. Rats were then killed, and spinal cord and brain stem serotonin turnover (5-HIAA/5-HT), as well as plasma T3, T4 and corticosterone (CORT) concentrations were measured. TX attenuated diabetic hyperphagia and weight loss. DB alone led to moderate reductions in T3 and T4, but the hormones were barely detectable in plasma of TX and METH rats. CORT was elevated in DB but was not affected by TX. Open field activity was not affected by DB or TX. TX and METH significantly increased 5-HIAA/5-HT in both spinal cord and brain stem. TX also led to enhanced disappearance of 5-HT after PCPA. DB significantly reduced 5-HIAA/5-HT, suggesting independent effects of the treatments. However, DB-TX rats still had significantly higher 5-HIAA/5-HT than control-sham surgery rats, while DB-METH rats had 5-HIAA/5-HT indistinguishable from controls. In both cases, prior induction of primary hypothyroidism interfered with the expected diabetes-induced reduction in 5-HT turnover.

Altered responses to environmental stress in streptozotocin-diabetic rats ☆

Metabolic and biochemical adaptations were compared in streptozotocin-diabetic and nondiabetic control rats exposed for 24 hours to a cold environment (4 degrees C) or hypobaric hypoxia (simulated altitude = 12,000 ft). In the cold, diabetic rats had greater reductions in adrenal norepinephrine (NE) and greater elevations in urinary NE and epinephrine excretion. However, diabetics did not increase food intake, whereas cold-exposed nondiabetic rats did. 5-HT turnover was reduced in hypothalamus and elevated in brain stem in both diabetics and nondiabetics. Responses to hypoxia were different. Both diabetics and nondiabetics reduced food and water intake and had elevated plasma glucose concentrations. Diabetics had elevated urinary NE excretion. Hypothalamic NE concentration and dopamine turnover were significantly reduced by hypoxia. Brain stem 5-HT turnover was also reduced in nondiabetics but not in diabetics. Thus, diabetics had a different response profile to the environmental stressors than nondiabetics. In addition, the two stressors elicited different responses. Some stressors may be more debilitating in diabetics. The greater reactivity of the sympathetic nervous system in diabetics suggests a mechanism by which stress leads to increased risk of metabolic complications in diabetes mellitus.

Reemergence of spontaneous hypertension in hypoxia-protected rats returned to normoxia as adults

Five-week-old male spontaneously hypertensive rats (SHR) were either exposed to hypoxia or maintained in normoxia. Groups of rats were returned to normoxia after 8 or 12 weeks exposure to hypoxia while others remained in hypoxia or normoxia throughout the study. Subdivisions of the groups were sacrificed 2 or 6 weeks after return to normoxia at the same time as were rats continuously exposed to either normoxia or hypoxia. Hypoxia attenuated the development of systemic hypertension (P less than 0.05); however, this protection dissipated partially when rats were returned to normoxia. Norepinephrine concentration was significantly elevated and serotonin turnover (5-hydroxyindoleacetic acid/serotonin 5HIAA/5HT) was significantly decreased in caudal brainstem of hypoxic SHR and both were gradually normalized upon return to normoxia. Similarly, left ventricular hypertrophy was attenuated and adrenal catecholamine contents were increased with hypoxic exposure. Both gradually normalized upon return to normoxia. Mechanisms associated with the development of spontaneous hypertension reemerge when adult, previously hypoxic SHR are returned to a normoxic environment. These findings implicate long-term changes in central noradrenergic and serotonergic function as components of the cardiovascular adaptation to hypoxia which includes hypoxic moderation of spontaneous hypertension.

Central catecholaminergic responses in hypoxie moderation of spontaneous hypertension

Exposure to hypobaric hypoxia (H; simulated altitude = 3658 m) was initiated in 5-week-old, male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKy) normotensive rats while normoxic controls (N) for both groups were maintained under laboratory conditions. Significant attenuation of systolic arterial blood pressure was evident in SHR-H relative to SHR-N (125 +/- 6 vs. 145 +/- 5, mmHg; p less than 0.05) while blood pressure in the normotensive, Wistar-Kyoto rat was not affected by 20 days of exposure to hypoxia (WKy-H, 116 +/- 2 vs. WKy-N, 117 +/- 5, mmHg). Increased contents of norepinephrine and dopamine in brain stem, striatum, hypothalamus, and frontal cortex in SHR versus WKy indicated a possible involvement of central catecholaminergic mechanisms with spontaneous hypertension. Hypoxia significantly decreased neuronal contents of both neurotransmitters, typically on both days studied (days 4 and 21 of altitude treatment). In striatum and hypothalamus, dihydroxyphenylacetic acid to dopamine ratios indicated that dopamine turnover was decreased with hypoxia. Hypoxia elicits catecholaminergic responses consistent with profiles found following ICV administration of 6-hydroxydopamine, a sympatholytic agent that also prevents the development of spontaneous hypertension. Hypoxic mitigation of spontaneous hypertension may occur via mechanisms initiated at the level of the CNS.

Time-Dependent Changes in Catecholamine Turnover in Spontaneously Hypertensive Rats Exposed to Hypoxia

Abstract In three separate experiments, 4 to 5-week-old spontaneously hypertensive rats (SHR) and normotensive controls (Wistar-Kyoto [WKy]) were exposed to hypobaric hypoxia (simulated altitude = 3658 m) for 3 hr, 3 days, or 3 weeks. Comparable groups were maintained in ambient laboratory conditions (normoxia). Hypoxia prevented the increase in blood pressure noted in 8-week-old normoxic SHR. Right ventricular hypertrophy first occurred after 3 days of hypoxia, and was found in both SHR and WKy. Catecholamine turnover was measured using the tyrosine hydroxylase inhibitor, α-methyl-p-tyrosine. In myocardium, both strains evidenced hypoxia-induced changes in norepinephrine (NE) turnover, which was increased at 3 hr, normalized at 3 days, and increased again at 3 weeks. Reduced basal NE concentration at 3 days indicated a temporary deficit in synthetic capacity, which would allow maintenance of a heightened neuronal output. Catecholamine turnover in right and left ventricles differed little in response to hypoxia, in spite of differential hemodynamic demands on SHR versus WKy or on right versus left ventricle. In contrast to findings in myocardium, significant interactive effects between strain and altitude exposure were noted for adrenal catecholamine turnover. Specifically, hypoxia exerted a suppressive influence in SHR that was not evident in WKy, and this may represent an important component of hypoxia-induced protection against the development of spontaneous hypertension.

Sympathetic and metabolic correlates of hypoxic moderation of spontaneous hypertension in the rat

Abstract Exposure to hypobaric hypoxia (H: simulated altitude = 3658 m) was initiated in 5-week-old, male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKy) normotensive rats while normoxic controls (N) for both groups were maintained under laboratory conditions. Significant attenuation of systolic arterial blood pressure was evident in SHR-H relative to SHR-N (125 ± 6 vs 145 ± 5 mm Hg; P < 0.05) but not in WKy-H relative to WKy-N (WKy-H, 116 ± 2 vs WKy-N, 117 ± 5 mm Hg). Hypoxia significantly decreased metabolic efficiency in both normotensive and hypertensive rats, although being both more severe and accompanied by significantly impaired growth rate in SHR-H. Urinary excretion of norepinephrine in the SHR was elevated relative to WKy, irrespective of altitude treatment, while hypoxia elicited similar increases in urinary excretion of norepinephrine in both SHR and WKy. Myocardial and adrenal contents of norepinephrine were significantly reduced following 3 days of simulated altitude exposure in both strains of rats. Tissue contents of norepinephrine in hypoxic rats returned to normoxic levels by 21 days of simulated altitude. Both urine and tissue indices provided consistent indirect evidence that changes in sympathetic neuronal activity in response to hypoxia were similar in normotensive and hypertensive rats. These findings suggest that prior reports of reduced α-adrenergic responsiveness in vasculature from hypoxia-exposed SHR reflect a postsynaptic event that is regulated independently of norepinephrine release from sympathetic nerve terminals.