Effect of hypothalamic preparations on human omental adipose tissue in vitro

The lipolytic responses of rat and human adipose tissue to several pituitary hormones, catecholamines, and purified preparations from human and porcine hypothalami have been compared. Fragments of human omental adipose tissue, obtained at surgery, or rat epididymal adipose tissue were incubated in vitro for 2 hr in 2 ml of Krebs-Ringer bicarbonate medium containing 3% bovine albumin. Acetic acid extracts of human and porcine stalk median eminence tissue were purified by gel filtration on Sephadex G-25. Lipid-mobilizing factor (LMF) activity emerged in an area occupied by peptides with a molecular weight of 3000–5000. This lipolytic fraction was found to be free of thyroid-stimulating hormone (TSH) and catecholamines. The ACTH content of this fraction was found to be less than 10 mU/mg. Addition of 20–60 μg/ml of porcine or human LMF to the medium significantly increased lipolysis, as measured by glycerol release into the medium, from human and rat adipose tissue. Human LMF elicited a greated lipolytic response in human omental adipose tissue than did the procine LMF, but porcine LMF was more potent in rat epididymal adipose tissue. This suggests that a species specificity may exist for the lipolytic activity of hypothalamic lipid mobilizers. Responsiveness of human omental adipose tissue to adrenalin was lower than that of rat epididymal adipose tissue. Addition of corticotropin A (10–20 μg/ml), human growth hormone (10–30 μg/ml), and alpha or beta melanocyte-stimulating hormone (MSH) (10–30 μg/ml) had no effect on lipolysis when incubated with human omental adipose tissue. However, the addition of 1.5–3.0 mU/ml of human TSH significantly increased the release of glycerol from human omental adipose tissue. These data suggest that human hypothalamic extracts, like those of porcine hypothalamic extracts, contain a lipid-mobilizing factor or factors that may differ chemically from corticotropin, TSH, growth hormone, and the catecholamines

Studies on the Inactivation of Thyrotropin-Releasing Hormone (TRH)

Summary Inactivation of porcine thyrotropin-releasing hormone (TRH) by plasma fractions in vitro was determined using mice maintained on a low iodine diet and pre-treated with 5 μCi of 125I and 0.085 μg of triiodothyronine. Incubation of TRH with porcine, bovine, or human serum caused a complete inactivation in 30 min. The optimum pH for the inactivation was about 7 and the optimal temperature was between 30 and 40°. The rate of inactivation of TRH was proportional to the enzyme concentration and time. Preheating rat plasma to 56° for 30 min greatly reduced this inactivation. When plasma fractions of porcine, bovine, and human origin were incubated with TRH in Krebs-Ringer bicarbonate, pH 7.4, at 37° for 30 min, alpha, beta, and gamma globulin fractions caused an 80-90% inactivation of added TRH. Albumin and fibrinogen caused a 40-50% reduction in TRH activity while the beta-lipoprotein fraction only induced a slight inactivation of TRH. Incubation of TRH with slices of rat liver, kidney, brain cortex, and skeletal muscle tissue also abolished TRF activity. Prior boiling of these tissues reduced the inactivation of TRH. These results are best explained by the presence of an enzyme in the plasma and various tissues which is capable of inactivating TRH

A Study on the Mode of Administration of Thyrotropin Releasing Hormone (TRH) in Mice

Administration of purified porcine TRH to mice by intravenous subcutaneous, intraperitoneal, or intramuscular routes produced a significant release of TSH from the anterior pituitary gland. Subcutaneous administration of TRH in dilute acetic acid or 5% carboxymethylcellulose produced a prompt but somewhat smaller response at 2 h that increased between 2 and 4 h. Subcutaneous injections of TRH emulsified in glycerol and in sesame oil were associated with an approximately 30% decrease in response at 2 h as compared with intravenously administered TRH. TRH emulsified in Freund’s adjuvant and injected subcutaneously was associated with a 62% decrease in response at 2 h followed by marked increase at 4 h. It is suggested that Freund’s adjuvant, glycerol, and sesame oil slow the rate of absorption of TRH from the injection site. Plasma radioactivity levels, after the injection of tritiated TRH, correlate with the patterns of response seen when TRH is followed by bioassay using the same vehicle and mode of administration

Studies on the Thyrotropin-Releasing Hormone (TRH)∗ Activity in Peripheral Blood

Summary Significant TRH-like activity was detected in the blood of thyroidectomized-hypophysectomized rats after (but not before) exposure to mild cold. Thus mild cold is capable of acting as a specific stimulus for the release of TRH in rats. This provides the first evidence for the possible physiologic importance of TRH. A study was made of the conditions under which TRH can be detected in blood. The presence of TRH in the peripheral circulation may be dependent upon the levels of the enzyme(s) responsible for its destruction. TRH is not detectable in the peripheral circulation unless the endogenous level of thyroid hormones is sufficiently reduced. Thyroid hormones are able to influence the levels or activity of the enzyme(s) responsible for the inactivation of TRH

The distribution, half-life, and excretion of tritiated luteinizing hormone-releasing hormone (LH-RH) in rats

Tritiated LH-RH was prepared by the Wilzbach method and repurified. After the injection of 1.0μCi of tritiated LH-RH into normal female rats, radioactivity disappeared rapidly from the circulation. The half-life of tritiated LH-RH, calculated from the disappearance curves, was about 7 min. Tritiated LH-RH was distributed in a volume of 38.4 ml or about 16% of the body weight. After 60 min, approximately 15% of the radioactivity was found in the urine. A significant accumulation of radioactivity, indicated by tissue to plasma ratio (T/P) greater than 1.0, occurred in the pineal gland, anterior and posterior pituitaries, the liver and kidney, but not in the cerebral cortex, hypothalamus, muscle tissue or adrenal glands. Concentration of the radioactivity by the liver and kidney indicates that these tissues are major sites for the inactivation and excretion of LH-RH

In vitro Studies with Thyrotropin Releasing Factor.

Summary Porcine TRF stimulates the release of TSH from rat anterior pituitaries in vitro at doses as small as 0.01 nanog. By increasing the doses of TRF, greater amounts of TSH are released into the incubation media. The pituitary response to TRF is inhibited by small amounts of T3 and T4. Act D does not abolish the response to TRF, indicating that de novo synthesis of TSH is not required for TRF to exert its effect, a- and β-MSH do not stimulate the release of TSH in vitro and did not reverse the inhibitory effect of T4 on TSH release in vitro. Preincubation with Act D reverses the inhibition of TSH release induced by T3 and T4. This may indicate that Act D interferes with the formation of inhibitory substances, induced by T3 and T4, which suppress the release of TSH after TRF

Inhibition of growth of pancreatic carcinomas in animal models by analogs of hypothalamic hormones (tumor weight and volume reduction/somatostatin analogs/luteinizing hormone-releasing hormone analogs)

Using animal models of acinar and ductal pancreatic cancer, we investigated the effect of analogs of hy- pothalamic hormones on tumor growth. In Wistar/Lewis rats bearing the acinar pancreatic tumor DNCP-322, chronic ad- ministration of (L-5-Br-Trp8)somatostatin-14 significantly de- creased tumor weights and volume. Somatostatin-28 and the cyclic hexapeptide analog of somatostatin cyclo(Pro-Phe-D- Trp-Lys-Thr-Phe) failed to influence the growth of this tumor. The agonistic analog of luteinizing hormone-releasing hor- mone (D-Trp6)LH-RH also significantly decreased tumor weight and volume in this model and reduced testosterone lev- els and the weights of the ventral prostate and testes. In Syrian hamsters bearing ductal type of pancreatic carcinoma, chronic administration of (L-5-Br-Trp8)somatostatin diminished tu- mor weights and volume. The percentage change in tumor vol- ume was significantly decreased when compared to control an- imals. In one experiment, cyclic hexapeptide of somatostatin also inhibited growth of this tumor. (D-Trp6)LH-RH, given twice daily or injected in the form of microcapsules for con- stant controlled release, significantly decreased tumor weight and volume and suppressed serum testosterone levels. Ham- sters castrated 4 days after transplantation of the pancreatic tumors showed a significant decrease in weight and volume of these tumors. This suggests that pancreatic cancers may, at least in part, be sex hormone sensitive. (D-Trp6)LH-RH may decrease the growth of pancreatic carcinomas by suppressing androgens. Somatostatin analogs reduce the growth of pancre- atic ductal and acinar cancers, probably by inhibiting the re- lease or stimulatory action of gastrointestinal hormones on tu- mor cells (or both). Inhibition of animal models of pancreatic tumors by chronic administration of somatostatin analogs and (D-Trp6)LH-RH suggests that these compounds should be con- sidered for the development of a new hormonal therapy for cancer of the pancreas