Robert J. Kemppainen

Effect of endotoxin on pituitary hormone secretion in sheep.

Endotoxin, a potent stimulator of the immune system and an important mediator in the pathophysiology of septic shock, has been shown to alter the release of certain hormones following its systemic administration. The purpose of this study was to determine the effects of endotoxin on pituitary hormone secretion both in vivo and in vitro in sheep, with emphasis placed on its effects on growth hormone (GH) release. Endotoxin (400 ng/kg i.v.) increased plasma GH, adrenocorticotropic hormone (ACTH), cortisol and prolactin, while it decreased luteinizing hormone (LH) pulse frequency (p < 0.05). Plasma levels of tumor necrosis factor, a major mediator of endotoxin effects, also increased following endotoxin administration. Endotoxin did not affect the GH response to human GH-releasing hormone. In vitro studies evaluated the effect of endotoxin to alter GH secretion from dispersed sheep anterior pituitary cells at dosages of 1, 10 and 50 micrograms/ml, with samples collected at 4, 8 and 24 h. Endotoxin increased pituitary GH secretion at 24 h for 1 microgram/ml (p < 0.05) and at all time periods for 10 and 50 micrograms/ml (p < 0.05). It also led to an increased release of ACTH and LH in vitro. The results of this study demonstrate the ability of endotoxin to alter pituitary hormone secretion both in vivo and in vitro in sheep, suggesting a direct effect of endotoxin on the pituitary gland.

Modulation of Growth Hormone-Releasing Factor Stimulated Growth Hormone Secretion by Plasma Glucose and Free Fatty Acid Concentrations in Sheep

Effects of plasma glucose and free fatty acid (FFA) concentrations on bovine growth hormone-releasing factor (bGRF)-induced release of growth hormone (GH) were examined in ovariohysterectomized sheep. In experiment 1, the effects of an infusion of insulin (0.025 U/kg BW.h-1), glucose (40 mg/kg BW.h-1), insulin plus glucose or saline on the subsequent effects of bGRF on plasma GH concentrations were determined. Insulin-induced hypoglycemia inhibited GRF effects on plasma GH concentrations while glucose infusion enhanced bGRF actions. Infusing a higher glucose dose (120 mg/kg BW.h-1) had no effect on GRF actions. Subsequently, infusion of FFA (0.25 g/kg/.h-1), nicotinic acid (50 mg/kg BW) or saline for 1 h prior to bGRF injection demonstrated that FFA inhibited GRF actions but FFA depletion by nicotinic acid infusion had no effect on GRF actions. Nicotinic acid (40 mg/kg BW.h-1) infused for 2 h prior to bGRF injection significantly enhanced bGRF-stimulated GH secretion. Finally, to determine whether central nervous system glucopenia produced similar effects to insulin-induced hypoglycemia, 2-deoxyglucose (500 mg) was injected into the lateral ventricle followed in 1 by the i.v. injection of bGRF. The central glucopenia produced by 2-DG inhibited GRF-stimulated GH release. These data demonstrate that decreased peripheral or central nervous system glucose availability and exogenous administration of FFA antagonized GRF-induced release of GH. And, pharmacologic depletion of circulating FFA for at least 2 h facilitated GRF-induced release of GH.

Regulation of Dexras1 Expression by Endogenous Steroids

Dexras1, a newly identified member of the Ras superfamily of proteins, was discovered in AtT-20 corticotrope cells because its expression was induced in response to glucocorticoids (dexamethasone; Dex). As yet, the function of Dexras1 is unknown, but its rapid induction in response to glucocorticoids suggests the possibility that it may be involved in negative feedback regulation of corticotropin secretion. To better understand the control of Dexras1 expression, possible effects of other steroid hormones on its expression were studied in both AtT-20 cells and in mouse pituitaries. AtT-20 cells were treated with each of 6 steroids [aldosterone, corticosterone (Cort), Dex, β-estradiol (E2), progesterone and testosterone] for 2 h. Dexras1 expression was assessed using both reverse transcription polymerase chain reaction (RT-PCR) and Northern analysis. Expression of the gene was only induced in response to glucocorticoid treatment (Dex or Cort). The 6 steroids were also injected into mice, pituitaries were harvested and total RNA was obtained for RT-PCR analysis. Surprisingly, treatment with E2, not only injection of glucocorticoids, induced Dexras1 expression in mouse pituitary. Other steroids were without effect. The results suggest that in AtT-20 corticotropes, Dexras1 expression is only induced by glucocorticoid-type steroids. In pituitary glands of mice, the gene’s expression is also responsive to E2. We conclude that either Dexras1 expression in corticotropes from normal mice is regulated differently from that in AtT-20 cells, or that Dexras1 is also expressed in other pituitary cells than corticotropes.

Neurotransmitter Receptor Agonists Regulate Growth Hormone Gene Expression in Cultured Ovine Pituitary Cells

Abstract The regulation of growth hormone (GH) secretion and GH mRNA content by the dopaminergic agonist, bromocriptine (BRO); the β-adrenergic agonist; isoproterenol (ISO); the α1-adrenergic agonist, methoxamine (MET); the α2-adrenergic agonist, clonidine (CLON); the serotonergic agonist, quipazine (QUIP); somatostatin (SS) and GH-releasing hormone (GHRH) were studied using cultured ovine anterior pituitary cells. Clonidine and BRO (10-6 M) inhibited basal and GHRH (10-10 M)-stimulated GH release. Bromocriptine enhanced GH mRNA content and potentiated the GHRH (10-8 M)-stimulated content of GH mRNA, while CLON had no effect on GH mRNA. Quipazine had little effect on GH secretion and no effect on GH mRNA content. Methoxamine and ISO (10-6 M) increased basal secretion of GH and both enhanced GHRH-stimulated GH secretion. Both MET and ISO increased GH mRNA content of cultured ovine pituitary cells. Somatostatin (10-7 M) inhibited GHRH-stimulated GH secretion and GH mRNA accumulation. These results support the hypothesis that neurotransmitters may regulate or interact to further modulate pituitary hormone release. Moreover, the data indicate that neurotransmitters may not only regulate secretion but also regulate GH mRNA content and thus affect hormone synthesis.

Use of Compounded Adrenocorticotropic Hormone (ACTH) for Adrenal Function Testing in Dogs

Serum cortisol concentrations were measured in five healthy dogs in response to five adrenocorticotropic hormone (ACTH) preparations. Cortisol concentrations were similar at time 0 (pre-ACTH) and at 30 and 60 minutes after injection of all forms of ACTH. However, at 90 and 120 minutes post-ACTH, serum cortisol concentrations were significantly lower following injection of two compounded forms of ACTH. The data showed that injection of four compounded forms of ACTH caused elevations in serum cortisol concentrations of a similar magnitude as cosyntropin in samples collected 60 minutes after administration; but concentrations at later times varied, depending on the type of ACTH used.

Alterations in Insulin and Glucagon Secretion by Monosodium Glutamate Lesions of the Hypothalamic Arcuate Nucleus

This study was performed to determine the effects of monosodium glutamate (MSG) induced lesions of the hypothalamic arcuate nucleus (ARC) on glucose tolerance and insulin and glucagon secretion in male golden hamsters. Eight day old hamsters were given a single s.c. injection of 5.8 mg/g BW MSG or hypertonic saline (controls). Studies were initiated when the hamsters were 3 months of age. At this age there were no body weight differences. Glucose (180 mg/100 g BW) was administered via stomach tube to 18 control and 18 MSG-treated hamsters. Animals were anesthetized with ether and a single blood sample from the portal vein was taken either before or at 30 or 60 min after glucose administration (n = 6/group). Glucose concentrations were similar in both groups at all time periods. Insulin concentrations in the MSG group were significantly (P less than 0.05) elevated in MSG-treated hamsters compared to controls at the 60 min time point. Glucose suppressed glucagon (P less than 0.05) in control but not in MSG-treated hamsters. The MSG group had significantly more glucagon (P less than 0.05) in portal vein blood at 30 min after glucose administration than did the control hamsters. Molar insulin/glucagon ratios did not differ between the 2 groups which likely accounts for the lack of differences in blood glucose levels. These results suggest a role for the ARC in regulating pancreatic function.

Autoantibodies to triiodothyronine and thyroxine in a golden retriever.

A golden retriever presented with signs of hypothyroidism occurring in conjunction with autoantibodies to both triiodothyronine (T3) and thyroxine (T4). The autoantibodies caused the apparent concentrations of total T3, total T4, and free T4 by analog assay to be high. However, free T4 concentration was nondetectable when measured using a dialysis assay. The dogs clinical condition markedly improved in response to L-thyroxine therapy, and the free T4 concentration by dialysis assay increased into the normal range. Thyroid hormone autoantibodies can confuse the diagnostic evaluation for suspected hypothyroidism. In dogs with autoantibodies to T4, measurement of free T4 by dialysis assay is useful for both diagnostic and therapeutic monitoring purposes.

Characterization of Canine Triiodothyronine (T3) Autoantibodies and Their Effect on Total T3 in Canine Serum

Abstract A study of 3,5,3′-L-triiodothyronine autoantibody (T3 AA) in 18 dogs revealed an average apparent affinity constant for T3 of 2.24 ± 1.78 × 1010 M -1, an average T3 binding capacity of 639.3 ± 666.5 ng/dl and a low thyroxine (T4) cross-reactivity (<1%) in all samples tested. A valid radioimmunoassay (RIA) procedure which involved heat treatment of samples for 1 hr at 70°C and assay on Sephadex minicolumns was developed for measuring T3 in the presence of T3 AA. Total T3 was elevated (x = 374.8 ± 158.4 ng/dl) in samples in which T4 was in the normal canine range, but T3 was lower (x = 96.1 ± 63.3 ng/dl) in samples with T4 values in the hypothyroid range. For each sample the concentration of T3 not bound by T3 AA was calculated from the total T3 concentration, the affinity constant, and the binding capacity. In dogs with normal total T4 concentrations the average calculated T3 not bound by T3 AA was 147.2 ± 144.4 ng/dl while in dogs with low total T4 the value was 15.7 ± 26.3 ng/dl (normal canine range is 45-150 ng/dl). Canine samples containing T3 AA were compared to serum from three rabbits actively immunized against T3 to provide anti-T3 for commercial RIA. The rabbit T3-antisera had an average T3 affinity constant similar to those of the canine samples (1.57 × 1010 M -1), but had average titer, T3 binding capacity, and total T3 values more than 10-fold higher. Our findings indicate that, in dogs with serum containing T3 AA and normal total T4 concentrations, a compensatory mechanism appears to exist to maintain non-T3 AA bound T3 within the range of normal total T3. This compensatory mechanism does not operate in those dogs with insufficient thyroid activity to maintain normal total T4 values.

Exogenous thyrotoxicosis in dogs attributable to consumption of all-meat commercial dog food or treats containing excessive thyroid hormone: 14 cases (2008-2013)

OBJECTIVEnTo describe findings in dogs with exogenous thyrotoxicosis attributable to consumption of commercially available dog foods or treats containing high concentrations of thyroid hormone.nnnDESIGNnRetrospective and prospective case series.nnnANIMALSn14 dogs.nnnPROCEDURESnMedical records were retrospectively searched to identify dogs with exogenous thyrotoxicosis attributable to dietary intake. One case was found, and subsequent cases were identified prospectively. Serum thyroid hormone concentrations were evaluated before and after feeding meat-based products suspected to contain excessive thyroid hormone was discontinued. Scintigraphy was performed to evaluate thyroid tissue in 13 of 14 dogs before and 1 of 13 dogs after discontinuation of suspect foods or treats. Seven samples of 5 commercially available products fed to 6 affected dogs were analyzed for thyroxine concentration; results were subjectively compared with findings for 10 other commercial foods and 6 beef muscle or liver samples.nnnRESULTSnTotal serum thyroxine concentrations were high (median, 8.8 μg/dL; range, 4.65 to 17.4 μg/dL) in all dogs at initial evaluation; scintigraphy revealed subjectively decreased thyroid gland radionuclide in 13 of 13 dogs examined. At ≥ 4 weeks after feeding of suspect food or treats was discontinued, total thyroxine concentrations were within the reference range for all dogs and signs associated with thyrotoxicosis, if present, had resolved. Analysis of tested food or treat samples revealed a median thyroxine concentration for suspect products of 1.52 μg of thyroxine/g, whereas that of unrelated commercial foods was 0.38 μg of thyroxine/g.nnnCONCLUSIONS AND CLINICAL RELEVANCEnResults indicated that thyrotoxicosis can occur secondary to consumption of meat-based products presumably contaminated by thyroid tissue, and can be reversed by identification and elimination of suspect products from the diet.

Effect of glucocorticoid administration on serum aldosterone concentration in clinically normal dogs

OBJECTIVEnTo evaluate the effects of oral administration of anti-inflammatory dosages of prednisone for 28 days on serum aldosterone, cortisol, and electrolyte concentrations in clinically normal dogs.nnnANIMALSn10 dogs.nnnPROCEDURESnOn days 1 through 28, 5 dogs received prednisone (0.55 mg/kg, PO, q 12 h) and 5 dogs received similar treatments with a placebo (empty capsules). Serum cortisol and aldosterone concentrations before and after ACTH stimulation testing and serum electrolyte concentrations were measured before (day 0 [baseline]), during (days 7, 14, 21, and 28), and after (days 35 and 42) treatment.nnnRESULTSnAt baseline, variables did not differ between the 2 groups. Serum cortisol concentrations before and after ACTH stimulation testing did not change from baseline values in placebo-treated dogs. In prednisone-treated dogs, serum chloride and corrected chloride concentrations were significantly lower on days 7, 14, 21, and 28 and serum bicarbonate concentrations were significantly higher on days 14, 21, and 28, compared with baseline values. Serum cortisol concentrations before and after ACTH stimulation testing were significantly lower than baseline values during prednisone treatment. Serum aldosterone concentration after ACTH stimulation testing was significantly lower than baseline on day 35 (ie, 1 week after discontinuation of prednisone treatment) but returned to baseline by day 42 in prednisone-treated dogs.nnnCONCLUSIONS AND CLINICAL RELEVANCEnAdministration of anti-inflammatory dosages of prednisone caused significant changes in serum chloride, bicarbonate, and cortisol concentrations in clinically normal dogs. Although ACTH-stimulated serum aldosterone concentrations were unchanged from baseline during glucocorticoid administration, values decreased after treatment cessation but quickly returned to baseline values.