Neural differentiation of the human neuroblastoma cell line IMR32 induces production of a thyrotropin-releasing hormone-like peptide

The human neuroblastoma cell line IMR32 produces and secretes substantial amounts of TRH-immunoreactivity (TRH-IR) as measured with radioimmunoassay (RIA) using the nonspecific antiserum 4319. It was found that synthesis of TRH-IR is dependent on neural differentiation: under serum-free conditions these cells exhibit neural characteristics as defined by morphological and biochemical standards. After culture for 2–5 days in serum-free medium cells grew large neural processes and expressed neuron-specific markers whereas glial-specific markers were absent. TRH-IR became detectable after 4–8 days serum-free conditions. Northern blot and chromatographic analysis, however, failed to detect proTRH mRNA and authentic TRH in these cells. Moreover, TRH-IR was undetectable in the RIA using TRH-specific antiserum 8880. TRH-IR produced by differentiated cells was retained on a QAE Sephadex A-25 anion-exchange column and thus negatively charged. HPLC analysis showed coelution with the synthetic peptide pGlu-Glu-ProNH2. Study of the mechanisms regulating production of this novel peptide in these cells should further elucidate the role differentation plays in the synthesis of neuropeptides

Hepatic lipase gene expression is transiently induced by gonadotropic hormones in rat ovaries

Hepatic lipase (HL) gene expression was studied in rat ovaries. A transcript lacking exons 1 and 2 could be detected by reverse transcription-polymerase chain reaction (RT-PCR) in the ovaries of mature cyclic females and of immature rats treated with pregnant mare serum followed by human chorionic gonadotropin (hCG) to induce superovulation. By competitive RT-PCR the HL transcript was quantified. Low levels of HL mRNA were detected in ovaries of mature cyclic females and of immature rats. During superovulation HL mRNA was several fold higher than in mature cyclic rats and transiently increased to a maximum at 2 days after hCG treatment. Pulse-labelling of ovarian cells and ovarian slices with [35S]methionine followed by immunoprecipitation with polyclonal anti-HL IgGs showed de novo synthesis of a 47 kDa HL-related protein. Expression of the protein was transiently induced by gonadotropins with a peak at 2 days after hCG treatment. Induction of liver-type lipase activity occurred only after HL mRNA and synthesis of the HL-related protein had returned to pre-stimulatory levels. We conclude that in rat ovaries the HL gene is expressed into a variant mRNA and a 47 kDa protein. The expression of the HL gene in ovaries is inducible and precedes the expression of the mature, enzymatically active liver-type lipase

Effects of thyroid status and thyrostatic drugs on hepatic glucuronidation of lodothyronines and other substrates in rats - Induction of phenol UDP-glucuronyltransferase by methimazole

Glucuronidation of iodothyronines in rat liver is catalyzed by at least three UDP-glucuronyltransferases (UGTs): bilirubin UGT, phenol UGT, and androsterone UGT. Bilirubin and phenol UGT activities are regulated by thyroid hormone, but the effect of thyroid status on hepatic glucuronidation of iodothyronines is unknown. We examined the effects of hypothyroidism induced by treatment of rats with propylthiouracil (PTU) or methimazole (MMI) or by thyroidectomy as well as the effects of T4-induced hyperthyroidism on the hepatic UGT activities for T4, T3, bilirubin, p-nitrophenol (PNP), and androsterone. Bilirubin UGT activity was increased in MMI- or PTU-induced hypothyroid and thyroidectomized rats, and decreased in hyperthyroid animals. T4 and, to a lesser extent, T3 UGT activities were increased in MMI- or PTU-induced hypothyroid rats, and T4 but not T3 glucuronidation also showed a significant increase in thyroidectomized rats. T4 but not T3 UGT activity was slightly decreased in hyperthyroid rats. While PNP UGT activity was decreased in thyroidectomized rats and increased in hyperthyroid animals, it was also markedly increased by MMI and slightly increased by PTU-induced hypothyroidism. In T4-substituted rats, MMI did not affect T4, T3, bilirubin and androsterone UGT activities but again strongly induced PNP UGT activity, indicating that this represented a direct induction of PNP UGT by the drug independent of its thyrostatic action. Androsterone UGT activity was hardly affected by thyroid status. Our results suggest a modest, negative control of the hepatic glucuronidation of thyroid hormone by thyroid status, which may be mediated by changes in bilirubin UGT activity. To our knowledge, this is the first report of the marked induction of a hepatic enzyme by MMI, which is not mediated by its thyroid hormone-lowering effect

Evidence that the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in human serum may not be secreted by the pituitary gland

Recent studies have revealed that TRH-like immunoreactivity (TRH-LI) in human serum is predominantly pGlu-Glu-ProNH2 (< EEP-NH2), a peptide previously found in, among others tissues, the pituitary gland of various mammalian species. In the rat pituitary, < EEP-NH2 is present in gonadotrophs and its pituitary content is regulated by gonadal steroids and gonadotrophin-releasing hormone (GnRH). Hence, we reasoned that < EEP-NH2 in human serum may also arise, at least in part, from the pituitary, and that its secretion may correlate with that of gonadotrophins. Therefore, blood was simultaneously sampled from both inferior petrosal sinuses, which are major sites of the venous drainage of the pituitary gland, and a peripheral vein from seven patients with suspected adrenocorticotrophin-secreting pituitary tumours. In addition, in six postmenopausal and six cyclic women, peripheral vein blood was collected at 10-min intervals for 6 h, then a standard 100 micrograms GnRH test was performed. In the sera, TRH-LI was estimated by RIA with antiserum 4319, which binds most tripeptides that share the N- and C-terminal amino acids with TRH (pGlu-His-ProNH2). In addition, LH and FSH were measured in these sera b

Renal clearance of the thyrotropin-releasing hormone-like peptide pyroglutamyl-glutamyl-prolineamide in humans

TRH-like peptides have been identified that differ from TRH (pGlu-His-ProNH2) in the middle amino acid. We have estimated TRH-like immunoreactivity (TRH-LI) in human serum and urine by RIA with TRH-specific antiserum 8880 or with antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2. TRH was undetectable in serum (< 25 pg/mL), but TRH-LI was detected with antiserum 4319 in serum of 27 normal subjects, 21 control patients, and 12 patients with carcinoid tumors (range 17-45, 5-79, and 18-16,600 pg/mL, respectively). Because serum was kept for at least 2 h at room temperature, which causes degradation of TRH, pGlu-Phe-ProNH2, and pGlu-Tyr-ProNH2, serum TRH-LI is not caused by these peptides. On high-performance liquid chromatography, serum TRH-LI coeluted with pGlu-Glu-ProNH2 (< EEP-NH2), a peptide produced in, among others, the prostate. Urine of normals and control patients also contained TRH-LI (range 1.14-4.97 and 0.24-5.51 ng/mL, respectively), with similar levels in males and females. TRH represented only 2% of urinary TRH-LI, and anion-exchange chromatography and high-performance liquid chromatography revealed that most TRH-LI in urine was < EEP-NH2. In patients with carcinoid tumors, increased urinary TRH-LI levels were noted (range 1.35-962.4 ng/mL). Urinary TRH-LI correlated positively with urinary creatinine, and the urinary clearance rate of TRH-LI was similar to the glomerular filtration rate. In addition, serum TRH-LI was increased in 17 hemodialysis patients (43-373 pg/mL). This suggests that serum < EEP-NH2 is cleared by glomerular filtration wit

Renal clearance of the thyrotropin-releasing hormone-like peptide pyroglutamyl-glutamyl-prolineamide in humans

TRH-like peptides have been identified that differ from TRH (pGlu-His-ProNH2) in the middle amino acid. We have estimated TRH-like immunoreactivity (TRH-LI) in human serum and urine by RIA with TRH-specific antiserum 8880 or with antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2. TRH was undetectable in serum (< 25 pg/mL), but TRH-LI was detected with antiserum 4319 in serum of 27 normal subjects, 21 control patients, and 12 patients with carcinoid tumors (range 17-45, 5-79, and 18-16,600 pg/mL, respectively). Because serum was kept for at least 2 h at room temperature, which causes degradation of TRH, pGlu-Phe-ProNH2, and pGlu-Tyr-ProNH2, serum TRH-LI is not caused by these peptides. On high-performance liquid chromatography, serum TRH-LI coeluted with pGlu-Glu-ProNH2 (< EEP-NH2), a peptide produced in, among others, the prostate. Urine of normals and control patients also contained TRH-LI (range 1.14-4.97 and 0.24-5.51 ng/mL, respectively), with similar levels in males and females. TRH represented only 2% of urinary TRH-LI, and anion-exchange chromatography and high-performance liquid chromatography revealed that most TRH-LI in urine was < EEP-NH2. In patients with carcinoid tumors, increased urinary TRH-LI levels were noted (range 1.35-962.4 ng/mL). Urinary TRH-LI correlated positively with urinary creatinine, and the urinary clearance rate of TRH-LI was similar to the glomerular filtration rate. In addition, serum TRH-LI was increased in 17 hemodialysis patients (43-373 pg/mL). This suggests that serum < EEP-NH2 is cleared by glomerular filtration with little tubular resorption. The possible role of the prostate as a source of urinary TRH-LI was evaluated in 11 men with prostate cancer, showing a 25% decrease in urinary TRH-LI excretion after prostatectomy (0.19 +/- 0.02 vs. 0.15 +/- 0.01 ng/mumol creatinine, mean +/- SEM). However, TRH-LI was similar in spontaneously voided urine and in urine obtained through a nephrostomy cannula from 16 patients with unilateral urinary tract obstruction (0.15 +/- 0.01 vs. 0.14 +/- 0.01 ng/mumol creatinine). These data indicate that: 1) TRH-LI in human serum represents largely < EEP-NH2, which is cleared by renal excretion; 2) part of urinary < EEP-NH2 is derived from prostatic secretion into the blood and not directly into urine; and 3) urinary < EEP-NH2 can be used as marker for carcinoid tumors

Different effects of continuous infusion of interleukin-1 and interleukin-6 on the hypothalamic-hypophysial-thyroid axis

The cytokines interleukin-1 (IL-1) and IL-6 are thought to be important mediators in the suppression of thyroid function during nonthyroidal illness. In this study we compared the effects of IL-1 and IL-6 infusion on the hypothalamus-pituitary-thyroid axis in rats. Cytokines were administered by continuous ip infusion of 4 micrograms IL-1 alpha/day for 1, 2, or 7 days or of 15 micrograms IL-6/day for 7 days. Body weight and temperature, food and water intake, and plasma TSH, T4, free T4 (FT4), T3, and corticosterone levels were measured daily, and hypothalamic pro-TRH messenger RNA (mRNA) and hypophysial TSH beta mRNA were determined after termination of the experiments. Compared with saline-treated controls, infusion of IL-1, but not of IL-6, produced a transient decrease in food and water intake, a transient increase in body temperature, and a prolonged decrease in body weight. Both cytokines caused transient decreases in plasma TSH and T4, which were greater and more prolonged with IL-1 than with IL-6, whereas they effected similar transient increases in the plasma FT4 fraction. Infusion with IL-1, but not IL-6, also induced transient decreases in plasma FT4 and T3 and a transient increase in plasma corticosterone. Hypothalamic pro-TRH mRNA was significantly decreased (-73%) after 7 days, but not after 1 or 2 days, of IL-1 infusion and was unaffected by IL-6 infusion. Hypophysial TSH beta mRNA was significantly decreased after 2 (-62%) and 7 (-62%) days, but not after 1 day, of IL-1 infusion and was unaffected by IL-6 infusion. These results are in agreement with previous findings that IL-1, more so than IL-6, directly inhibits thyroid hormone production. They also indicate that IL-1 and IL-6 both decrease plasma T4 binding. Furthermore, both cytokines induce an acute and dramatic decrease in plasma TSH before (IL-1) or even without (IL-6) a decrease in hypothalamic pro-TRH mRNA or hypophysial TSH beta mRNA, suggesting that the acute decrease in TSH secretion is not caused by decreased pro-TRH and TSH beta gene expression. The TSH-suppressive effect of IL-6, either administered as such or induced by IL-1 infusion, may be due to a direct effect on the thyrotroph, whereas additional effects of IL-1 may involve changes in the hypothalamic release of somatostatin or TRH.(ABSTRACT TRUNCATED AT 400 WORDS

Determinants of lenalidomide response with or without erythropoiesis-stimulating agents in myelodysplastic syndromes:the HOVON89 trial

A randomized phase-II study was performed in low/int-1 risk MDS (IPSS) to study efficacy and safety of lenalidomide without (arm A) or with (arm B) ESA/G-CSF. In arm B, patients without erythroid response (HI-E) after 4 cycles received ESA; G-CSF was added if no HI-E was obtained by cycle 9. HI-E served as primary endpoint. Flow cytometry and next-generation sequencing were performed to identify predictors of response. The final evaluation comprised 184 patients; 84% non-del(5q), 16% isolated del(5q); median follow-up: 70.7 months. In arm A and B, 39 and 41% of patients achieved HI-E; median time-to-HI-E: 3.2 months for both arms, median duration of-HI-E: 9.8 months. HI-E was significantly lower in non-del(5q) vs. del(5q): 32% vs. 80%. The same accounted for transfusion independency-at-week 24 (16% vs. 67%), but similar in both arms. Apart from presence of del(5q), high percentages of bone marrow lymphocytes and progenitor B-cells, a low number of mutations, absence of ring sideroblasts, and SF3B1 mutations predicted HI-E. In conclusion, lenalidomide induced HI-E in patients with non-del(5q) and del(5q) MDS without additional effect of ESA/G-CSF. The identified predictors of response may guide application of lenalidomide in lower-risk MDS in the era of precision medicine. (EudraCT 2008-002195-10).</p