Effect of thyrotropin-releasing hormone stimulation testing on the oral sugar test in horses when performed as a combined protocol.

BACKGROUND: The use of parallel dynamic tests to identify insulin dysregulation (ID) and pituitary pars intermedia dysfunction (PPID) in horses could have better diagnostic utility than measuring baseline hormone concentrations, if the tests do not alter diagnostic interpretation of one another. HYPOTHESIS: Performing a thyrotropin-releasing hormone (TRH) stimulation test before an oral sugar test (OST) would not affect results of OST. ANIMALS: Twenty-six healthy university-owned horses. METHODS: A prospective randomized placebo-controlled, crossover design was used to evaluate 3 OST protocols: OST alone, TRH followed by OST (TRH + OST), and placebo followed by OST (placebo + OST). Agreement for plasma insulin concentrations and diagnostic interpretation were assessed with Bland-Altman and logistic regression analyses, respectively. RESULTS: Bland-Altman analysis of TRH + OST versus OST alone showed good agreement between testing protocols, with bias ± SD for insulin concentrations at baseline 0.4 ± 4.7 µIU/mL (95% limits of agreement [LOA], -8.8 to 9.7), 60 minute -0.5 ± 22.6 µIU/mL (95% LOA, -44.7 to 43.8), and 90 minute 1.9 ± 20.6 µIU/mL (95% LOA, -38.5 to 42.4) after OST, similar to placebo + OST versus OST alone. Diagnostic interpretation (positive/negative) was not different between protocols (TRH + OST versus OST alone [P = .78], placebo + OST versus OST alone [P = .77], or TRH + OST versus placebo + OST [P = .57]). CONCLUSIONS AND CLINICAL IMPORTANCE: Concurrent testing for PPID and ID with a TRH stimulation test before an OST is an acceptable diagnostic tool for investigation of endocrinopathies in horses and allows accurate testing to be performed efficiently in 1 visit.

Effect of dietary carbohydrates and time of year on ACTH and cortisol concentrations in adult and aged horses.

Diagnosis of equine pituitary pars intermedia dysfunction (PPID) remains a challenge as multiple factors (stress, exercise, and time of year) influence ACTH and cortisol concentrations. To assess endocrine status in a study designed to evaluate the effects of age and diet on glucose and insulin dynamics, we performed thyrotropin-releasing hormone (TRH) stimulation tests and overnight dexamethasone suppression tests in March, May, August, and October on 16 healthy Thoroughbred and Standardbred mares and geldings. Horses were grouped by age: adult (mean ± SD; 8.8 ± 2.9 yr; n = 8) and aged (20.6 ± 2.1 yr; n = 8). None of the horses showed clinical signs (hypertrichosis, regional adiposity, skeletal muscle atrophy, lethargy) of pituitary pars intermedia dysfunction. Horses were randomly assigned to groups of 4, blocked for age, and fed grass hay plus 4 isocaloric concentrate diets (control, starch-rich, fiber-rich, and sugar-rich) using a balanced Latin square design. Data were analyzed using a multivariable linear mixed regression model. Baseline ACTH was significantly higher in aged horses (mean ± standard error of the mean; 60.0 ± 10.7 pg/mL) adapted to the starch-rich diet compared to adult horses (15.7 ± 12.0 pg/mL) on the same diet (P = 0.017). After controlling for age and diet, baseline ACTH concentrations were significantly increased in October (57.7 ± 7.1 pg/mL) compared to March (13.2 ± 7.1 pg/mL; P < 0.001), May (12.4 ± 7.1 pg/mL; P < 0.001), and August (24.2 ± 7.1 pg/mL; P < 0.001), whereas post-TRH ACTH was higher in August (376.6 ± 57.6 pg/mL) and October (370.9 ± 57.5 pg/mL) compared to March (101.9 ± 57.3 pg/mL; P < 0.001) and May (74.5 ± 57.1 pg/mL; P < 0.001). Aged horses had significantly higher post-dexamethasone cortisol on the starch-rich diet (0.6 ± 0.1 µg/dL) compared to the sugar-rich diet (0.2 ± 0.1 µg/dL; P = 0.021). Post-dexamethasone cortisol was significantly higher in October (0.6 ± 0.1 µg/dL) compared to March (0.3 ± 0.1 µg/dL; P = 0.005), May (0.2 ± 0.1 µg/dL; P < 0.001), and August (0.3 ± 0.1 µg/dL; P = 0.004). Breed did not influence ACTH or cortisol measurements. In conclusion, in addition to age and time of year, diet is a potential confounder as animals on a starch-rich diet may be incorrectly diagnosed with pituitary pars intermedia dysfunction.

Pituitary pars intermedia dysfunction does not necessarily impair insulin sensitivity in old horses.

Equine pituitary pars intermedia dysfunction (PPID) has been associated with reduced insulin sensitivity in comparison with younger adult horses; however, the difference in insulin sensitivity between horses with PPID and aged-matched controls has not been well characterized. The objective of the study was to determine if aged horses with PPID had reduced insulin sensitivity and alterations in the insulin-mediated signaling pathways in the skeletal muscle when compared with healthy aged horses. Isoglycemic hyperinsulinemic clamp procedures were conducted in 12 horses that were classified as either PPID (n = 6; age: 25.0 ± 2.5 yr; mean ± standard deviation) or non-PPID, aged-matched controls (control) (n = 6; age: 25.7 ± 2.0 yr). Blood samples were taken before and during the clamp procedures to measure plasma glucose, insulin, and amino acid concentrations, and 2 muscle biopsies were collected from the gluteus medius muscle, one in the basal state and the second at the end of the clamp procedure (insulin-stimulated state). Plasma insulin concentrations increased ∼9-fold during the clamp compared with basal conditions (P < 0.001) in both groups. During the last 30 min of the clamp, the rate of glucose infusion required to maintain isoglycemia in horses with PPID was similar to that in the control horses (P = 0.67). The plasma concentrations of most indispensible amino acids were lower in the insulin-stimulated state than the basal state (P < 0.05). PPID status did not have an effect on the activation of factors associated with protein synthesis and breakdown; however, factors associated with protein synthesis had increased phosphorylation in the insulin-stimulated state, compared with basal. The results from this study provide evidence that PPID is not always associated with impairments in insulin sensitivity.

Tailless and Atrophin control Drosophila aggression by regulating neuropeptide signalling in the pars intercerebralis.

Aggressive behaviour is widespread throughout the animal kingdom. However, its mechanisms are poorly understood, and the degree of molecular conservation between distantly related species is unknown. Here we show that knockdown of tailless (tll) increases aggression in Drosophila, similar to the effect of its mouse orthologue Nr2e1. Tll localizes to the adult pars intercerebralis (PI), which shows similarity to the mammalian hypothalamus. Knockdown of tll in the PI is sufficient to increase aggression and is rescued by co-expressing human NR2E1. Knockdown of Atrophin, a Tll co-repressor, also increases aggression, and both proteins physically interact in the PI. tll knockdown-induced aggression is fully suppressed by blocking neuropeptide processing or release from the PI. In addition, genetically activating PI neurons increases aggression, mimicking the aggression-inducing effect of hypothalamic stimulation. Together, our results suggest that a transcriptional control module regulates neuropeptide signalling from the neurosecretory cells of the brain to control aggressive behaviour.

Angiogenesis in the intermediate lobe of the pituitary gland alters its structure and function.

The pars distalis (PD) and the pars intermedia (PI) have the same embryonic origin, but their morphological and functional characteristics diverge during development. The PD is highly vascularized, whereas the highly innervated PI is essentially non-vascularized. Based on our previous finding that vascular endothelial growth factor-A (VEGF-A) is involved in vascularization of the rat PD, attempt was made to generate transgenic Xenopus expressing VEGF-A specifically in the melanotrope cells of the PI as a model system for studying the significance of vascularization or avascularization for the functional differentiation of the pituitary. The PI of the transgenic frogs, examined after metamorphosis, were distinctly vascularized but poorly innervated. The experimentally induced vascularization in the PI resulted in a marked increase in tissue volume and a decrease in the expression of both alpha-melanophore-stimulating hormone (α-MSH) and prohormone convertase 2, a cleavage enzyme essential for generating α-MSH. The transgenic animals had low plasma α-MSH concentrations and displayed incomplete adaptation to a black background. To our knowledge, this is the first report indicating that experimentally induced angiogenesis in the PI may bring about functional as well as structural alterations in this tissue.

Seasonal variation in results of diagnostic tests for pituitary pars intermedia dysfunction in older, clinically normal geldings.

OBJECTIVE: To determine whether seasonal variations exist in endogenous plasma ACTH, plasma α-melanocyte-stimulating hormone (α-MSH), serum cortisol, and serum insulin concentrations and in the results of a dexamethasone suppression test for older, clinically normal geldings in Alabama. DESIGN: Cohort study. ANIMALS: 15 healthy mixed-breed geldings (median age, 14 years). PROCEDURES: Sample collection was repeated monthly for 12 months. Dexamethasone (0.04 mg/kg [0.02 mg/lb], IM) was administered and cortisol concentrations were determined at 15 and 19 hours. Radioimmunoassays were used to measure ACTH, α-MSH, cortisol, and insulin concentrations at each testing time. Hormone concentrations were compared between months via repeated-measures ANOVA and correlated with age within each month. RESULTS: A significant time effect was found between months for α-MSH and insulin concentrations. Endogenous cortisol and ACTH concentrations remained within existing reference ranges. Significant correlations were detected between age and ACTH concentration for several fall and winter months and between age and insulin concentration for September. CONCLUSIONS AND CLINICAL RELEVANCE: Older horses have higher ACTH concentrations in several fall and winter months and higher insulin concentrations in September than do younger horses. Seasonally specific reference ranges are required for α-MSH and insulin concentrations, with significantly higher concentrations detected in the fall. Practitioners should be advised to submit samples only to local laboratories that can provide such reference ranges for their local geographic region.

Long-lasting intrinsic optical changes observed in the neurointermediate lobe of the mouse pituitary reflect volume changes in cells of the pars intermedia.

BACKGROUND/AIMS: Complex intrinsic optical changes (light scattering) are readily observed in the neurointermediate lobe of the mouse pituitary gland following electrical stimulation of the infundibular stalk. Our laboratory has previously identified three distinct phases within the light scattering signal: two rapid responses to action potential stimulation and a long duration recovery. The rapid light scattering signals, restricted to the neurohypophysial portion (posterior pituitary) of the neurointermediate lobe, consist of an E-wave and an S-wave that reflect excitation and secretion, respectively. The E-wave has the approximate shape of the action potential and includes voltage- and current-related components and is independent of Ca(2+) entry. The S-wave is related to Ca(2+) entry and exocytosis. The slow recovery phase of the light scattering signal, which we designated the R-wave, is less well characterized. METHODS: Using high temporal resolution light scattering measurements, we monitored intrinsic optical changes in the neurointermediate lobe of the mouse pituitary gland. Pharmacological interventions during the measurements were employed. RESULTS: The data presented here provide optical and pharmacological evidence suggesting that the R-wave, which comprises signals from the posterior pituitary as well as from the pars intermedia, mirrors volume changes in pars intermedia cells following a train of stimuli applied to the infundibular stalk. These volume changes were blocked by the GABA-receptor antagonists bicuculline and picrotoxin, and were mimicked by direct application of GABA in the absence of electrical stimulation. CONCLUSIONS: These results emphasize the importance of central GABAergic projections into the neurointermediate lobe, and the potential role of GABA in effecting hormone release from the pars intermedia.

Plasticity in the melanotrope neuroendocrine interface of Xenopus laevis.

Melanotrope cells of the amphibian pituitary pars intermedia produce alpha-melanophore-stimulating hormone (alpha-MSH), a peptide which causes skin darkening during adaptation to a dark background. The secretory activity of the melanotrope of the South African clawed toad Xenopus laevis is regulated by multiple factors, both classical neurotransmitters and neuropeptides from the brain. This review concerns the plasticity displayed in this intermediate lobe neuroendocrine interface during physiological adaptation to the environment. The plasticity includes dramatic morphological plasticity in both pre- and post-synaptic elements of the interface. Inhibitory neurons in the suprachiasmatic nucleus, designated suprachiasmatic melanotrope-inhibiting neurons (SMINs), possess more and larger synapses on the melanotrope cells in white than in black-background adapted animals; in the latter animals the melanotropes are larger and produce more proopiomelanocortin (POMC), the precursor of alpha-MSH. On a white background, pre-synaptic SMIN plasticity is reflected by a higher expression of inhibitory neuropeptide Y (NPY) and is closely associated with postsynaptic melanotrope plasticity, namely a higher expression of the NPY Y1 receptor. Interestingly, melanotrope cells in such animals also display higher expression of the receptors for thyrotropin-releasing hormone (TRH) and urocortin 1, two neuropeptides that stimulate alpha-MSH secretion. Possibly, in white-adapted animals melanotropes are sensitized to neuropeptide stimulation so that, when the toad moves to a black background, they can immediately initiate alpha-MSH secretion to achieve rapid adaptation to the new background condition. The melanotrope cell also produces brain-derived neurotrophic factor (BDNF), which is co-sequestered with alpha-MSH in secretory granules within the cells. The neurotrophin seems to control melanotrope cell plasticity in an autocrine way and we speculate that it may also control presynaptic SMIN plasticity.

Possible involvement of brain-derived neurotrophic factor (BDNF) in the innervation of dopaminergic neurons from the rat periventricular nucleus to the pars intermedia.

Developing neurons are guided to their appropriate targets by specific guidance substances that have neurotrophic actions. The aim of the present study was to elucidate the mechanism by which hypothalamic neurons reach the pars intermedia (PI) by correlating the development of dopaminergic (DA) neurons arising in the periventricular nucleus (PeV) of fetal rats with the expression of brain-derived neurotrophic factor (BDNF) in the rat pituitary. The differentiation of DA neurons was observed by immunohistochemistry using an antibody against tyrosine hydroxylase (TH), whereas the ontogenesis of BDNF mRNA in the PI was examined by in situ hybridization and RT-PCR. Immunoreactive TH-neurons were first observed in the PeV at embryonic day (E) 16.5, following which time their axons elongated toward the pituitary. TH-positive reactions were observed in the connective tissue between the PI and the pars nervosa at E20.5. Innervation of the PI by TH-positive neurons was determined at postnatal day (P) 1.5; however, BDNF mRNA was first detected in the PI cells at E17.5, with an increase in its expression clearly visible at E21.5 and continuing high expression levels in the PI thereafter. These results suggest that BDNF is a specific guidance cue for DA neurons elongating from the PeV to the PI.