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The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, β-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert.

Pro-opiomelanocortin (POMC) neurons function as key regulators of metabolism and physiology by releasing prohormone-derived neuropeptides with distinct biological activities. However, our understanding of early events in prohormone maturation in the ER remains incomplete. Highlighting the significance of this gap in knowledge, a single POMC cysteine-to-phenylalanine mutation at position 28 (POMC-C28F) is defective for ER processing and causes early onset obesity in a dominant-negative manner in humans through an unclear mechanism. Here, we report a pathologically important role of Sel1L-Hrd1, the protein complex of ER-associated degradation (ERAD), within POMC neurons. Mice with POMC neuron-specific Sel1L deficiency developed age-associated obesity due, at least in part, to the ER retention of POMC that led to hyperphagia. The Sel1L-Hrd1 complex targets a fraction of nascent POMC molecules for ubiquitination and proteasomal degradation, preventing accumulation of misfolded and aggregated POMC, thereby ensuring that another fraction of POMC can undergo normal posttranslational processing and trafficking for secretion. Moreover, we found that the disease-associated POMC-C28F mutant evades ERAD and becomes aggregated due to the presence of a highly reactive unpaired cysteine thiol at position 50. Thus, this study not only identifies ERAD as an important mechanism regulating POMC maturation within the ER, but also provides insights into the pathogenesis of monogenic obesity associated with defective prohormone folding., Introduction Hypothalamic pro-opiomelanocortin (POMC) neurons critically mediate leptin signaling and regulate feeding behavior and systemic metabolic homeostasis via the secretion of several bioactive neuropeptides derived from the POMC protein precursor [...]

The thyrotropin- (TRH) releasing hormone precursor (26 kDa) undergoes proteolytic cleavage at either of two sites, generating N-terminal 15 kDa/9.5 kDa or C-terminal 16.5/10 kDa intermediate forms that are processed further to yield five copies of TRH-Gly and seven non-TRH peptides. Glucocorticoids (Gcc) have been shown to enhance TRH gene expression in three different cell systems in vitro, an effect that occurs, at least in part, through transcriptional activation. Although this implies that an increase of TRH prohormone biosynthesis would take place, this had not been demonstrated as yet. We report here that the synthetic glucocorticoid dexamethasone (Dex) substantially elevated the de novo biosynthesis of the intact 26-kDa TRH prohormone and its intermediate products of processing in cultured anterior pituitary cells, an observation that is consistent with an overall upregulation of both the biosynthesis and degradation of the TRH precursor. We reasoned that Gcc may act not only at the transcriptional, but also at the translational/posttranslational level. To address this question we chose a different cell system, AtT20 cells transfected with a cDNA encoding preproTRH. Since TRH gene expression in these cells is driven by the CMV-IE promoter and not by an endogenous "physiological" promoter, these cells provide an ideal model to study selectively the effects of Gcc on the translation and posttranslational processing of proTRH without interference from a direct transcriptional activation of the TRH gene. Dex caused a significant 75.7% increase in newly synthesized 26-kDa TRH prohormone, suggesting that the glucocorticoid raised the translation rate. We then demonstrated that Dex treatment accelerated TRH precursor processing. Of interest, processing of the N- vs the C-terminal intermediate was influenced differentially by the glucocorticoid. Although the N-terminal intermediate product of processing accumulated, the C-terminal intermediate was degraded more rapidly. Consistent with these observations was the finding that the intracellular accumulation of the N-terminally derived peptide preproTRH 25-50 was enhanced, but levels of the C-terminally derived peptide preproTRH208-255 were reduced. Accumulation of TRH itself, whose five copies are N- and C-terminally derived, was also enhanced. We conclude that Gcc induce changes in the biosynthesis and processing of proTRH by increasing the translation rate and by differentially influencing the processing of N- vs C-terminal intermediates of the precursor molecule. These effects of Gcc at the translational and posttranslational levels result in an increase in TRH production accompanied by differential effects on the accumulation of N- and C-terminal non-TRH peptides.

The hypothalamic-pituitary-thyroid (HPT) axis is a major contributor in maintaining energy expenditure and body weight, and the adipocyte hormone leptin regulates this axis by increasing TRH levels in the fed state. Leptin stimulates TRH directly in the hypothalamic paraventricular nucleus (PVN; direct pathway) and indirectly by regulating proopiomelnocortin neurons in the hypothalamic arcuate nucleus (ARC; indirect pathway). Whereas the indirect pathway is fully functional in lean animals, it is inactive during diet-induced obesity (DIO) because of the establishment of leptin resistance. Despite this, the HPT axis activity in obese humans and rodents remains within the normal levels or slightly higher. Therefore, in this study, we aimed to determine the mechanism(s) by which the HPT axis is still active despite leptin resistance. With a combination of using the Sprague-Dawley rat physiological model and the Zuker rat that bears a mutation in the leptin receptor, we were able to demonstrate that under DIO conditions the HPT axis is regulated at the central level, but only through the direct pathway of leptin action on TRH neurons. Deiodinase enzymes, which are present in many tissues and responsible for converting thyroid hormones, were not statistically different between lean and DIO animals. These data suggest that the increase in T4/3 seen in obese animals is due mostly to central leptin action. We also found that [T.sub.3] feedback inhibition on the prepro-TRH gene is controlled partially by leptin-induced pSTAT3 signaling via the TRH promoter. This interactive relationship between [T.sub.3] and pSTAT3 signaling appears essential to maintain the HPT axis at normal levels in conditions such as obesity. doi: 10.1152/ajpendo.00448.2010.

The [alpha]-melanocyte-stimulating hormone ([alpha]-MSH), derived from proopiomelanocortin (POMC), is generated by a posttranslational processing mechanism involving the prohormone convertases (PCs) PC1/3 and PC2. In the brain, [alpha]-MSH is produced in the arcuate nucleus (ARC) of the hypothalamus and in the nucleus of the solitary tract (NTS) of the medulla. This peptide is key in controlling energy balance, as judged by changes observed at transcriptional level. However, little information is available regarding the biosynthesis of the precursor POMC and the production of its processed peptides during feeding, fasting, and fasting plus leptin in the ARC compared with the NTS in conjunction with the PC activity. In this study we found that, in the ARC, pomc mRNA, POMC-derived peptides, and PC1/3 all decreased during fasting, and administration of leptin reversed these effects. In contrast, in the NTS, where there is a large amount of a 28.1-kDa peptide similar in size to POMC, the 28.1-kDa peptide and other POMC-derived peptides, including [alpha]-MSH, were further accumulated in fasting conditions, whereas pomc mRNA decreased. These changes were not reversed by leptin. We also observed that, during fasting, PC2 levels decreased in the NTS. These data suggest that, in the NTS, fasting induced changes in POMC biosynthesis, and processing is independent of leptin. These observations indicate that changes in energy status affect POMC in the brain in a tissue-specific manner. This represents a novel aspect in the regulation of energy balance and may have implications in the pathophysiology of obesity. processing; adrenocorticotropin hormone; [alpha]-melanocyte-stimulating hormone doi:10.1152/ajpendo.00466.2006

The prohormone convertases (PCs), PC1/3 and PC2, are involved in the tissuespecific endoproteolytic posttranslational processing of many hormonal precursors within the secretory pathway. One important prohormone, pro-thyrotropin-releasing hormone (TRH), is expressed in both hypophysiotropic (where it regulates the secretion of thyroid-stimulating hormone) and nonhypophysiotropic regions of the brain. Pro-TRH is processed at specific sites in the secretory pathway, primarily by PC1/3 followed by PC2. We hypothesized that thyroid hormone status in specific nuclei of the brain would alter pro-TRH processing by inducing changes in PC1/3 and PC2 expression. Therefore, we examined pro-TRH, PC1/3, and PC2 coexpression and coregulation in the paraventricular nucleus (PVN), lateral hypothalamus (LH), and ventromedial nucleus (VMN) of hypothyroid and euthyroid rats. Our results show that 6-n-propyl-2-thiouracil (PTU) treatment producing hypothyroidism induced a significant increase in the expression of PC1/3, PC2, and pro-TRH in the PVN and LH, but not VMN. When confocal studies were performed, an increase in colocalization of PC1/3 or PC2 in pro-TRH was observed only in PVN, a response that was especially prominent in the ventral and medial areas of the PVN. PTU did not regulate colocalization in the VMH or LH. Regulation of colocalization of processing enzyme and prohormone expression is a novel mechanism to alter hormonal biosynthesis. posttranslational processing; hyperthyroidism; hypothyroidism; prohormone convertase; hypothalamus; pro-thyrotropin-releasing hormone; peptide

The central melanocortin system is critical in the regulation of appetite and body weight, and leptin exerts its anorexigenic actions partly by increasing hypothalamic proopiomelanocortin (POMC) expression. The POMC-derived peptide [alpha]-melanocyte-stimulating hormone ([alpha]MSH) is a melanocortin 4 receptor agonist, and its potency in reducing energy intake is strongly increased by N-acetylation. The reason for the higher biological activity of N-acetylated [alpha]MSH (Act-[alpha]MSH) compared with that of N-desacetylated [alpha]MSH (Des-[alpha]MSH) is unclear, and regulation of acetylation by leptin has not been investigated. We show here that total hypothalamic [alpha]MSH levels are decreased in leptin-deficient ob/ob mice and increased in leptin-treated ob/ob and C57BL/6J mice. The increase in total [alpha]MSH occurred as soon as 3 h after leptin injection and was entirely due to an increase in Act-[alpha]MSH. Consistent with this observation, leptin rapidly induced the enzymatic activity of a N-acetyltransferase in the hypothalamus of mice. In 293T cells expressing the melanocortin 4 receptor, Act-[alpha]MSH is far more potent than Des-[alpha]MSH in stimulating cAMP accumulation, an effect caused by a dramatically increased stability of Act-[alpha]MSH. Moreover, Des-[alpha]MSH is rapidly degraded in the hypothalamus after intracerebroventricular injection in rats and was less potent in inhibiting energy intake. The results suggest that leptin activates a N-acetyltransferase in POMC neurons, leading to increased hypothalamic levels of Act-[alpha]MSH. Due to its increased stability, this posttranslational modification of [alpha]MSH may play a critical role in leptin action via the central melanocortin pathway.