Involvement of human peroxisomes in biosynthesis and signaling of steroid and peptide hormones.

Although peroxisomes exert essential biological functions, cell type-specific features of this important organelle are still only superficially characterized. An intriguing new aspect of peroxisomal function was recently uncovered by the observation that the peptide hormones ß-lipotropin (ß-LPH) and ß-endorphin are localized to peroxisomes in various human tissues. This suggests a functional link between peptide hormone metabolism and peroxisomes. In addition, because endocrine manifestations that affect steroid hormones are often found in patients suffering from inherited peroxisomal disorders, the question has been raised whether peroxisomes are also involved in steroidogenesis. With this chapter, we will review several crucial aspects concerning peroxisomes and hormone metabolism.

Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry.

The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d(10)-Leu) and used mass spectrometry to measure the biosynthetic rate of gamma-lipotropin in the AtT-20 cell line and insulin in the INS-1 cell line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the cells and media. The relative levels of the d-labeled forms are greater in the media than in the cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 cells were stimulated with KCl or forskolin, the ratio of d- to H-labeled gamma-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 cells reduced the ratio of d- to H-labeled gamma-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 cells, 30 microm chloroquine for 3 or 6 h significantly reduced the rate of formation of gamma-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 microm chloroquine for 6 h showed a significant decrease in the rate of formation of insulin in both cells and media. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d10-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines.

Altered processing of pro-orphanin FQ/nociceptin and pro-opiomelanocortin-derived peptides in the brains of mice expressing defective prohormone convertase 2.

The bioactivity of neuropeptides can be regulated by a variety of post-translational modifications, including proteolytic processing. Here, gene-targeted mice producing defective prohormone convertase 2 (PC2) were used to examine the post-translational processing of two neuroendocrine prohormones, pro-opiomelanocortin (POMC) and pro-orphanin FQ (pOFQ)/nociceptin (N), in the brain. Reversed-phase HPLC and gel-exclusion chromatography were combined with specific radioimmunoassays to analyze the processing patterns of these two prohormones in the hypothalamus and the amygdala. In the case of POMC, the lack of PC2 activity completely prevented carboxy-shortening of beta-endorphins and greatly diminished conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin. Although conversion of beta-lipotropin to beta-endorphin decreased, the lack of PC2 activity caused an increase in beta-lipotropin and beta-endorphin levels in the mutant animals, but no increases in POMC or biosynthetic intermediates were seen. The extent of OFQ/N production was significantly lower in PC2-deficient mice and there was an accumulation of relatively large amounts of pOFQ/N and biosynthetic intermediates. These results demonstrate that PC2 is directly involved in the biogenesis of two brain neuropeptides in vivo and suggest that the specific prohormone and cellular context influences neuropeptide processing by PCs.

Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor.

Chromogranin B (CgB, secretogranin I) is a widespread constituent of neuroendocrine secretory granules whose function is unknown. To determine whether CgB affects the sorting of peptide hormone and neuropeptide precursors to secretory granules, we overexpressed CgB in AtT-20 cells, which exhibit an only moderate capacity to sort proopiomelanocortin and proteolytic fragments derived therefrom. In mock-transfected AtT-20 cells, a substantial proportion of newly synthesized proopiomelanocortin and its two primary proteolytic products generated in the trans-Golgi network, the N-terminal 23-kDa fragment containing adrenocorticotropin and the C-terminal beta-lipotropin fragment, was secreted via the constitutive pathway. Two- to three-fold overexpression of CgB markedly reduced the constitutive secretion of the 23-kDa fragment, but not beta-lipotropin and tripled the amount of adrenocorticotropin generated and stored in secretory granules. Our results indicate the existence of neuroendocrine-specific helper proteins which promote the sorting from the trans-Golgi network to secretory granules of certain processing intermediates derived from peptide hormone and neuropeptide precursors and demonstrate that CgB functions as such.

Altered ratios of beta-endorphin: beta-lipotropin released from anterior lobe corticotropes with increased secretory drive. I. Effects of diminished glucocorticoid secretion.

Previous studies have demonstrated that acute stress or ovine corticotropin-releasing hormone (oCRH) in vivo, or oCRH in vitro, stimulates release of beta-endorphin over beta-lipotropin from anterior pituitary corticotropes. This occurs despite the predominance of beta-lipotropin in corticotrope peptide stores. In vitro studies with primary anterior pituitary cultures suggested that chronic exposure to oCRH results in a shift towards more beta-lipotropin secretion into the media than with short-term exposure. The current studies explored whether increased secretory drive in vivo results in a similar shift towards more beta-lipotropin. We used removal of glucocorticoids by adrenalectomy or metyrapone blockade of corticosterone synthesis, to stimulate endogenous secretion of CRH and vasopressin. Both treatments resulted in shifts of the ratio of beta-endorphin: beta-lipotropin in plasma of experimental animals in comparison to the sham-treated control rats. In vitro testing with oCRH of anterior lobe cultures from adrenalectomized or metyrapone-treated rats demonstrated similar effects of these treatments on the ratio of beta-endorphin:beta-lipotropin. These changes occurred despite similar ratios of beta-endorphin:beta-lipotropin in anterior pituitary peptide stores.

[In vitro processing of the artificial analog of beta-lipotropin]. / Issledovanie protsessinga in vitro iskusstvennogo analoga beta-lipotropina.

The processing of the recombinant analogue of beta-lipotropin (beta-LHP) having 11 additional N-terminal amino acid residues and separated from the hormone by the processing signal, was investigated using rat adrenal secretory granule lysate as a test system of processing "in vitro". It was found that incubation of the beta-LPH analogue with secretory granule enzymes leads to its limited specific degradation with a release of native beta-endorphin. It is concluded that the additional N-terminal amino acids induced no qualitative changes in beta-LPH processing.

The ontogeny of immunoreactive beta-endorphin and beta-lipotropin in the rat ovary.

We have investigated the ontogeny of, and the effect of hypophysectomy on, immunoreactive beta-endorphin in rat ovaries. Total levels rose with ovarian weight from nondetectable levels at 5 days of age to approximately 0.15 pmol/ovary at 80 days; thereafter, the levels remained constant through 201 days of age. Hypophysectomy decreased both ovarian weight and the total content of immunoreactive beta-endorphin, but the concentration per weight was not significantly altered. Most of the immunoreactive beta-endorphin before puberty chromatographed like authentic beta-endorphin, but after puberty most chromatographed like beta-lipotropin. Hypophysectomy did not alter this chromatographic pattern.

Posttranslational processing of opioid pro-opiomelanocortin products in piglets.

We have measured levels of beta-lipotropin, beta-endorphin, and N-acetyl-beta-endorphin in the plasma, cerebrospinal fluid (CSF), and caudal medullary brain containing the respiratory-related portion of the nucleus tractus solitarius (NTS) of 2.5 +/- 1.0- (SD) and 38.2 +/- 3.7-day-old naive uninstrumented piglets. Time of day, ambient atmosphere, temperature, handling, sound, light, and nutritional status were kept constant. Experimental procedure included decapitation and rapid collection, processing, and freezing of tissues until analysis by radioimmunoassay. Young, compared with older piglets, have higher measured levels of beta-lipotropin in the plasma and CSF and of N-acetyl-beta-endorphin in all three body compartments. Although measured levels of beta-endorphin-like immunoreactivity are also higher in the plasma and CSF of the young group, the calculated level of beta-endorphin is higher only in the CSF. In the NTS, both the measured and calculated active endorphin appear higher in the older group, but this difference is not significant. Excess beta-endorphin in the CSF of neonates may explain the relative immaturity of their respiratory functions at birth.

Expression of mouse proopiomelanocortin in an insulinoma cell line. Requirements for beta-endorphin processing.

Proopiomelanocortin (POMC) is a neuroendocrine precursor protein which is processed at paired basic amino acids in a tissue-specific manner. To study this phenomenon, a vaccinia virus recombinant, which directs the synthesis of mouse POMC (VV:mPOMC) was constructed and used to infect epithelial (BSC-40) and endocrine (Rin m5F) cell lines. Bona fide mPOMC was produced in both cell types and beta-endorphin immunoreactivity was secreted in a nonregulated manner from BSC-40 cells and in a regulated manner from Rin m5F cells. Although the precursor was not cleaved to smaller beta-MSH or beta-endorphin immunoreactive peptides in BSC-40 cell extracts, Rin m5F cells produced primarily authentic gamma-lipotropin and des-acetyl beta-endorphin. Furthermore, production of these peptides was restricted to the regulated secretory pathway in Rin m5F cells. Site-directed mutagenesis was then used to change the inefficiently recognized Lys-Lys potential cleavage site near the carboxyl terminus of beta-endorphin to Lys-Arg. Expression of the mutant precursor in Rin m5F cells resulted in the synthesis of both des-acetyl beta-endorphin and beta-endorphin.

Proadrenocorticotropin/endorphin production and messenger ribonucleic acid levels in primary intermediate pituitary cultures: effects of serum, isoproterenol, and dibutyryl adenosine 3',5'-monophosphate.

Long term primary rat intermediate pituitary cells cultured in complete serum-free medium (CSFM) were previously shown to exhibit a low basal secretory rate and maintain a stable cellular level of immunoactive pro-ACTH/endorphin (PAE)-related peptides. The present studies used biosynthetic labeling techniques to demonstrate that the biosynthesis of PAE-derived peptides declined as a function of the time the cultures were maintained in CSFM. Compared to hormone biosynthesis in freshly isolated cells, the ability of melanotropes maintained in CSFM to synthesize hormone declined approximately 3-fold after 5 days and 7-fold after 10 days. The endoproteolytic processing of PAE was not changed. This diminution in biosynthetic rate was not observed in serum-supplemented cultures; the rates of hormone secretion and hormone biosynthesis were both substantially higher in cultures maintained in serum-containing medium, suggesting that stimulatory factors were present in serum supplements. Chronic treatment of intermediate pituitary cultures in CSFM with 100 microM (Bu)2cAMP or 100 nM isoproterenol mimicked the effects of serum on total PAE production, hormone biosynthesis, and PAE mRNA levels. The diminished biosynthetic ability of cultures maintained in CSFM for several days could be restored by subsequent chronic (B mu)2cAMP or isoproterenol treatment. Taken together with previous information, these results suggest that melanotropes maintained in CSFM exist in a functionally basal state and that stimulatory agents, in addition to inhibitory inputs such as dopamine, exert significant regulatory control over intermediate pituitary lobe function in vivo.

Comparison of acute and chronic secretagogue regulation of proadrenocorticotropin/endorphin synthesis, secretion, and messenger ribonucleic acid production in primary cultures of rat anterior pituitary.

The acute and chronic effects of secretagogues activating cAMP-dependent pathways (CRH and cAMP) and activating cAMP-independent pathways [phenylephrine and phorbol 12-myristate 13-acetate (PMA)] on anterior pituitary function were examined in serum-free cultures. Applied acutely, PMA produced a greater stimulation of ACTH/endorphin secretion than CRH or cAMP. However, the effects of CRH and cAMP on secretion were maintained for up to 12 days, while those of PMA and phenylephrine diminished rapidly. Secretagogue effects on pro-ACTH/endorphin biosynthesis were determined by immunoprecipitation of biosynthetically labeled beta-endorphin-related peptides. Cultures exposed to CRH or cAMP and [3H]tyrosine for 12 h produced 1.7 +/- 0.2- and 1.6 +/- 0.1-fold more newly synthesized beta-endorphin-related material than control cells. Cultures exposed to phenylephrine or PMA synthesized 1.3 +/- 0.1- and 1.4 +/- 0.1-fold more peptide than control cells. Exposure of cells to CRH or cAMP for 12 days increased pro-ACTH/endorphin biosynthesis to a greater extent than the 12-h treatment (3.0 +/- 0.1- and 2.5 +/- 0.3-fold over control value, respectively). Exposure to phenylephrine or PMA for 12 days had the same effect on pro-ACTH/endorphin biosynthesis as exposure for 12 h. After acute or chronic secretagogue exposure, the cells secreted relatively more newly synthesized beta-lipotropin than beta-endorphin. Levels of pro-ACTH/endorphin mRNA in cultures treated acutely (12 h) or chronically (12 days) with CRH, cAMP, or phenylephrine changed in parallel with rates of pro-ACTH/endorphin biosynthesis. In contrast, chronic exposure to PMA stimulated biosynthesis while reducing pro-ACTH/endorphin mRNA levels. In summary, these results suggest that factors that activate cAMP-dependent pathways are more powerful stimulators of pro-ACTH/endorphin biosynthesis than factors that activate cAMP-independent pathways; the cAMP-dependent pathway may be primarily responsible for regenerating depleted hormone reserves.

Effects of ethanol treatment and withdrawal on biosynthesis and processing of proopiomelanocortin by the rat neurointermediate lobe.

Male Sprague Dawley rats were chronically pair-fed with liquid diets containing 6.5% (vol/vol) ethanol, or equicaloric sucrose. After 21 days the ethanol-containing diet was discontinued and both groups were fed the sucrose diet. Groups of animals were killed on day 22 (0 day of ethanol withdrawal) and 1, 3, 8, and 15 days after ethanol withdrawal and the neurointermediate lobes (NILs) were removed and incubated with [3H]phenylalanine for 3 h. Chronic ethanol treatment induced an increase in the biosynthesis and release of beta-endorphin-like peptides by the rat NIL. After ethanol withdrawal the beta-endorphin-like immunoreactivity content in the NIL and the in vitro release of immunoreactive beta-endorphin (beta EP) by the NIL were significantly lower than in the controls on the first day, whereas no significant difference was found on days 3, 8, and 15 after ethanol withdrawal. The in vitro incorporation of [3H]phenylalanine into POMC, beta-lipotropin and beta EP was found to be higher in the ethanol-treated animals than in the controls on days 0, 1, and 3 after ethanol withdrawal, with no significant difference on days 8 and 15 after ethanol withdrawal. Furthermore, in both the ethanol-treated animals and their pair-fed controls the rate of incorporation of [3H]phenylalanine into total proteins, POMC, beta-lipotropin, and beta EP was significantly higher on days 8 and 15 after ethanol withdrawal than on the day of ethanol withdrawal (day 0), suggesting the implication of a nutritional factor. HPLC analysis of the beta EP peptides indicated that the percentage of acetylated forms of beta EP was higher in the NIL of the alcohol-treated animals, especially on days 8 and 15 after ethanol withdrawal. This observation suggests that though the rates of biosynthesis and release of beta EP-related peptides have returned to normal at 15 days after ethanol treatment, the activity of the enzyme responsible for the acetylation of beta EP remained elevated.

Neurointermediate lobe transplanted under the kidney capsule modifies the activity of the neurointermediate lobe in situ, but does not respond to opiate treatment.

To investigate the possibility of a direct effect of morphine on the pars intermedia cells of the pituitary gland, rat neurointermediate lobes (NIL) were transplanted under the kidney capsule. At 2 and 8 days posttransplantation the NIL transplant had maintained its morphological integrity. However, at 15 days posttransplantation the morphological integrity of the NIL transplant had started to deteriorate. The NIL transplant contained, synthesized, and released beta-endorphin-like peptides. It was noticed that there was very little beta-endorphin in the radiolabelled biosynthesized products, suggesting that either the maturation processing of proopiomelanocortin was modified, or that beta-endorphin was released immediately as soon as it was formed and did not accumulate in the tissue. In support of the latter possibility was the elevated content of beta-endorphin-like immunoreactivity in the sera of rats with a NIL under the kidney capsule. Furthermore, the NIL transplant seemed to produce a substance or substances which could decrease the content, the biosynthesis and the release of beta-endorphin-like peptides by the NIL in situ. Treatment with either morphine or naloxone for 5 days did not change the beta-endorphin-like immunoreactivity content in the NIL transplanted under the kidney capsule. However, a distinct decrease in the beta-endorphin-like immunoreactivity in the NIL in situ of animals with or without a NIL transplant was observed following the morphine treatment. Naloxone treatment induced a decrease in the beta-endorphin-like immunoreactivity content in the hypothalamus, but had no effect on the beta-endorphin-like immunoreactivity content in the anterior lobe and NIL of the pituitary gland in situ or in the NIL transplant.

Effects of adrenalectomy and dexamethasone administration on the level of prepro-corticotropin-releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin/beta-lipotropin precursor mRNA in the pituitary in rats.

RNA blot hybridization analysis with cloned rat CRF precursor (prepro-CRF) cDNA as a probe showed that prepro-CRF mRNA existed in rat hypothalamic and extrahypothalamic brain tissue, whereas it was undetectable in the pituitary and adrenal. To study the effect of glucocorticoid on the level of prepro-CRF mRNA in the hypothalmus and that of ACTH/beta-lipotropin (beta LPH) precursor mRNA in the pituitary, effects of adrenalectomy and dexamethasone administration were studied in rats. Adrenalectomy markedly raised mRNA coding for ACTH/beta LPH precursor in the anterior pituitary, but not in the neurointermediate pituitary lobe. Hypothalamic pre-pro-CRF mRNA increased only to 152% of the control value, 7 days after adrenalectomy. The administration of dexamethasone (200 micrograms/day for 7 days) started immediately after adrenalectomy lowered the ACTH/beta LPH precursor mRNA level in the anterior pituitary to 19% of the intact control value, whereas the level of prepro-CRF mRNA in the hypothalamus decreased only to 102%. These results suggest that glucocorticoids exert their feedback effect at the level of gene expression on both hypothalamic CRF neurons and pituitary corticotropes. Although the possibility that CRF neurons insensitive to glucocorticoid in the hypothalamus might blunt the change in the prepro-CRF mRNA could not be ruled out, it is also possible that the effect of glucocorticoids on the pituitary is dominant.

[Genetic engineering of peptide hormones]. / Geneticheskaia inzheneriia peptidnykh gormonov.

The application of different approaches for preparing DNAs coding for peptide hormones was demonstrated. The libraries of human, bovine and porcine pituitaries cDNA were obtained starting from their total mRNAs. Screening of these libraries revealed clones containing human, bovine and porcine growth hormone sequences, cDNAs for bovine ACTH-beta-lipotropin precursor and for bovine and porcine prolactin. The gene of human calcitonin was created by combination of chemical and enzymatic synthesis. This synthetic gene was further cloned in pBR322. The expression of cloned human growth hormone cDNA under control of different Escherichia coli promoters was studied and physico-chemical and biological properties of the growth hormone produced by E. coli were tested.

Differentiation of cells producing polypeptide hormones (ACTH, MSH, LPH, alpha- and beta-endorphin, GH and PRL) in the fetal porcine anterior pituitary.

The aim of the present study on the fetal porcine pituitary was (1) to detect by means of the immunoperoxidase technique the earliest stages of cells producing polypeptide hormones: beta-MSH, ACTH, beta-LPH, alpha- and beta-endorphin, growth hormone (GH) and prolactin (PRL), (2) to study the development of the synthesis and the storage of these hormones during fetal life, and (3) to detect whether several hormones can be located in one and the same cell. The corticotropic cells were revealed as the earliest functional elements of the fetal anterior pituitary. Our results indicate clearly that ACTH, beta-MSH, beta-LPH, alpha- and beta-endorphin appear at 34 days in the same regular, round or ovoid cells; no differences in the time of their appearance could be observed. The ACTH-cells, irregular or angular in shape and endowed with cytoplasmic processes such as described in the adult pituitary, were not seen until day 50. The first GH-cells were detected between 40 to 45 days of fetal life. From day 45 to 90, the GH-cells greatly increased in number and in staining intensity of their progressively extending cytoplasmic area, but they displayed the same regular and round shape. The PRL-cells were the last cell type to appear in the fetal pituitary. The first PRL-cells, small in size and round or ovoid in shape with a high nucleus/cytoplasm ratio, were detected at day 70. At day 80, the PRL-cells increased in size and staining intensity. They displayed an irregular elongated or stellated shape and cytoplasmic processes resembling those characteristic of the adult pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)