Endotoxin at the Maternal-Fetal Interface in a Resource-Constrained Setting: Risk Factors and Associated Birth Outcomes.

Low- and middle-income countries (LMICs) carry a high burden of infectious diseases associated with impaired gut integrity, leading to microbial translocation. Pregnancies in this setting are at high risk of fetal growth restriction (FGR). We examined the association among specific risk factors for impaired gut integrity (schistosomiasis, hookworm infection, and alcohol consumption), blood endotoxin levels, and FGR. Endotoxins, lipopolysaccharide-binding proteins (LBPs), and cytokines were measured in blood from women at 32 weeks gestation, the maternal-fetal interface (MFI) at delivery, and cord blood at delivery. Resolution of schistosomiasis had no impact on endotoxin levels; however, maternal hookworm infection and alcohol consumption were associated with modest increases in endotoxin at the MFI. Cytokines responses within the maternal peripheral blood and blood from the MFI were positively associated with endotoxins, but many cord blood cytokines were negatively associated with endotoxins. Newborns with FGR also had higher levels of endotoxins at the MFI. Risk factors for microbial translocation may lead to increased levels of endotoxins at the MFI, which may contribute to poor growth in utero.

Reduced melanocortin production causes sexual dysfunction in male mice with POMC neuronal insulin and leptin insensitivity.

Proopiomelanocortin (POMC)-derived peptides like α-melanocyte-stimulating hormone (MSH) substantially improve hepatic insulin sensitivity and regulate energy expenditure. Melanocortinergic agents are also powerful inducers of sexual arousal that are being investigated for a possible therapeutic role in erectile dysfunction. It is currently unclear whether reduced melanocortin (MC) activity may contribute to the sexual dysfunction accompanying obesity and type 2 diabetes. Male rodents with leptin and insulin resistance targeted to POMC neurons (leptin receptor [LepR]/insulin receptor [IR]POMC mice) exhibit obesity, hyperinsulinemia, hyperglycemia, and systemic insulin resistance. In this study, we demonstrate that LepR/IRPOMC males are also subfertile due to dramatic alterations in sexual behavior. Remarkably, these reproductive changes are accompanied by decreased α-MSH production not present when a single receptor type is deleted. Unexpectedly, behavioral sensitivity to α-MSH and MC receptor expression are also reduced in LepR/IRPOMC males, a potential adaptation of the MC system to altered α-MSH production. Together, these results suggest that concurrent insulin and leptin resistance in POMC neurons in individuals with obesity or type 2 diabetes can reduce endogenous α-MSH levels and impair sexual function.

Central Sirt1 regulates body weight and energy expenditure along with the POMC-derived peptide α-MSH and the processing enzyme CPE production in diet-induced obese male rats.

In the periphery, the nutrient-sensing enzyme Sirtuin 1 (silent mating type information regulation 2 homolog 1 [Sirt1]) reduces body weight in diet-induced obese (DIO) rodents. However, the role of hypothalamic Sirt1 in body weight and energy balance regulation is debated. The first studies to reveal that central Sirt1 regulates body weight came from experiments in our laboratory using Sprague-Dawley rats. Central inhibition of Sirt1 decreased body weight and food intake as a result of a forkhead box protein O1 (FoxO1)-mediated increase in the anorexigenic proopiomelanocortin (POMC) and decrease in the orexigenic Agouti-related peptide in the hypothalamic arcuate nucleus. Here, we demonstrate that central inhibition of Sirt1 in DIO decreased body weight and increased energy expenditure at higher levels as compared with the lean counterpart. Brain Sirt1 inhibition in DIO increased acetylated FoxO1, which in turn increased phosphorylated FoxO1 via improved insulin/phosphorylated AKT signaling. Elevated acetylated FoxO1 and phosphorylated FoxO1 increased POMC along with the α-melanocyte-stimulating hormone (α-MSH) maturation enzyme carboxypeptidase E, which resulted in more of the bioactive POMC product α-MSH released into the paraventricular nucleus. Increased in α-MSH led to augmented TRH levels and circulating T3 levels (triiodothyronine, thyroid hormone). These results indicate that inhibiting hypothalamic Sirt1 in DIO enhances the activity of the hypothalamic-pituitary-thyroid axis, which stimulates energy expenditure. Because we show that blocking central Sirt1 causes physiological changes that promote a negative energy balance in an obese individual, our results support brain Sirt1 as a significant target for weight loss therapeutics.

Central Sirt1 regulates body weight and energy expenditure along with the POMC-derived peptide α-MSH and the processing enzyme CPE production in diet-induced obese male rats.

In the periphery, the nutrient-sensing enzyme Sirtuin 1 (silent mating type information regulation 2 homolog 1 [Sirt1]) reduces body weight in diet-induced obese (DIO) rodents. However, the role of Sirt1 in the brain, particularly the hypothalamus, in body weight and energy balance regulation is debated. Among the first studies to reveal that central Sirt1 regulates body weight came from experiments in our laboratory using Sprague Dawley rats. In that study, central inhibition of Sirt1 decreased body weight and food intake as a result of a Forkhead box protein O1 (FoxO1)-mediated increase in the anorexigenic proopiomelanocortin (POMC) and decrease in the orexigenic Agouti-related peptide in the hypothalamic arcuate nucleus. Here, we demonstrate that central inhibition of Sirt1 in DIO decreased body weight and increased energy expenditure at higher levels as compared with the lean counterpart. Brain Sirt1 inhibition in DIO increased acetylated FoxO1, which, in turn, increased phosphorylated FoxO1 via improved insulin/pAKT signaling. Elevated acetylated FoxO1 and phosphorylated FoxO1 increased POMC along with the α-MSH maturation enzyme carboxypeptidase E, which resulted in more of the bioactive POMC product α-MSH released into the paraventricular nucleus. Increased in α-MSH led to augmented TRH levels and circulating T3 levels (thyroid hormone). These results indicate that inhibiting hypothalamic Sirt1 in DIO enhances the activity of the hypothalamic-pituitary-thyroid axis, which stimulates energy expenditure. Because we show that blocking central Sirt1 causes physiological changes that promote a negative energy balance in an obese individual, our results support brain Sirt1 as a significant target for weight loss therapeutics.

Temporal changes in nutritional state affect hypothalamic POMC peptide levels independently of leptin in adult male mice.

Hypothalamic proopiomelanocortin (POMC) neurons constitute a critical anorexigenic node in the central nervous system (CNS) for maintaining energy balance. These neurons directly affect energy expenditure and feeding behavior by releasing bioactive neuropeptides but are also subject to signals directly related to nutritional state such as the adipokine leptin. To further investigate the interaction of diet and leptin on hypothalamic POMC peptide levels, we exposed 8- to 10-wk-old male POMC-Discosoma red fluorescent protein (DsRed) transgenic reporter mice to either 24-48 h (acute) or 2 wk (chronic) food restriction, high-fat diet (HFD), or leptin treatment. Using semiquantitative immunofluorescence and radioimmunoassays, we discovered that acute fasting and chronic food restriction decreased the levels of adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), and ß-endorphin in the hypothalamus, together with decreased DsRed fluorescence, compared with control ad libitum-fed mice. Furthermore, acute but not chronic HFD or leptin administration selectively increased α-MSH levels in POMC fibers and increased DsRed fluorescence in POMC cell bodies. HFD and leptin treatments comparably increased circulating leptin levels at both time points, suggesting that transcription of Pomc and synthesis of POMC peptide products are not modified in direct relation to the concentration of plasma leptin. Our findings indicate that negative energy balance persistently downregulated POMC peptide levels, and this phenomenon may be partially explained by decreased leptin levels, since these changes were blocked in fasted mice treated with leptin. In contrast, sustained elevation of plasma leptin by HFD or hormone supplementation did not significantly alter POMC peptide levels, indicating that enhanced leptin signaling does not chronically increase Pomc transcription and peptide synthesis.

Morphine treatment selectively regulates expression of rat pituitary POMC and the prohormone convertases PC1/3 and PC2.

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, ß-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.

Obesity induces hypothalamic endoplasmic reticulum stress and impairs proopiomelanocortin (POMC) post-translational processing.

It was shown previously that abnormal prohormone processing or inactive proconverting enzymes that are responsible for this processing cause profound obesity. Our laboratory demonstrated earlier that in the diet-induced obesity (DIO) state, the appetite-suppressing neuropeptide α-melanocyte-stimulating hormone (α-MSH) is reduced, yet the mRNA of its precursor protein proopiomelanocortin (POMC) remained unaltered. It was also shown that the DIO condition promotes the development of endoplasmic reticulum (ER) stress and leptin resistance. In the current study, using an in vivo model combined with in vitro experiments, we demonstrate that obesity-induced ER stress obstructs the post-translational processing of POMC by decreasing proconverting enzyme 2, which catalyzes the conversion of adrenocorticotropin to α-MSH, thereby decreasing α-MSH peptide production. This novel mechanism of ER stress affecting POMC processing in DIO highlights the importance of ER stress in regulating central energy balance in obesity.

Mechanisms by which the orexigen NPY regulates anorexigenic α-MSH and TRH.

Protein posttranslational processing is a cellular mechanism fundamental to the generation of bioactive peptides, including the anorectic α-melanocyte-stimulating hormone (α-MSH) and thyrotropin-releasing hormone (TRH) peptides produced in the hypothalamic arcuate (ARC) and paraventricular (PVN) nuclei, respectively. Neuropeptide Y (NPY) promotes positive energy balance in part by suppressing α-MSH and TRH. The mechanism by which NPY regulates α-MSH output, however, is not well understood. Our results reveal that NPY inhibited the posttranslational processing of α-MSH's inactive precursor proopiomelanocortin (POMC) by decreasing the prohormone convertase-2 (PC2). We also found that early growth response protein-1 (Egr-1) and NPY-Y1 receptors mediated the NPY-induced decrease in PC2. NPY given intra-PVN also decreased PC2 in PVN samples, suggesting a reduction in PC2-mediated pro-TRH processing. In addition, NPY attenuated the α-MSH-induced increase in TRH production by two mechanisms. First, NPY decreased α-MSH-induced CREB phosphorylation, which normally enhances TRH transcription. Second, NPY decreased the amount of α-MSH in the PVN. Collectively, these results underscore the significance of the interaction between NPY and α-MSH in the central regulation of energy balance and indicate that posttranslational processing is a mechanism that plays a specific role in this interaction.

Family members CREB and CREM control thyrotropin-releasing hormone (TRH) expression in the hypothalamus.

Thyrotropin-releasing hormone (TRH) in the paraventricular nucleus (PVN) of the hypothalamus is regulated by thyroid hormone (TH). cAMP response element binding protein (CREB) has also been postulated to regulate TRH expression but its interaction with TH signaling in vivo is not known. To evaluate the role of CREB in TRH regulation in vivo, we deleted CREB from PVN neurons to generate the CREB1(ΔSIM1) mouse. As previously shown, loss of CREB was compensated for by an up-regulation of CREM in euthyroid CREB1(ΔSIM1) mice but TSH, T4 and T3 levels were normal, even though TRH mRNA levels were elevated. Interestingly, TRH mRNA expression was also increased in the PVN of CREB1(ΔSIM1) mice in the hypothyroid state but became normal when made hyperthyroid. Importantly, CREM levels were similar in CREB1(ΔSIM1) mice regardless of thyroid status, demonstrating that the regulation of TRH by T3 in vivo likely occurs independently of the CREB/CREM family.

Biosynthesis of proTRH-derived peptides in prohormone convertase 1 and 2 knockout mice.

Prohormone convertases (PCs) 1 and 2 are the primary endoproteases involved in the post-translational processing of proThyrotropin Releasing Hormone (proTRH) to give rise to TRH and other proposed biologically active non-TRH peptides. Previous evidence suggests that PC1 is responsible for most proTRH cleavage events. Here, we used the PC1 and PC2 knockout (KO) mouse models to examine the effects of PC1 or PC2 loss on proTRH processing. The PC1KO mouse presented a decrease in five proTRH-derived peptides, whereas the PC2KO mouse showed only lesser reduction in three TRH (Gln-His-Pro), TRH-Gly (Gln-His-Pro-Gly), and the short forms preproTRH(178-184) (pFQ(7)) and preproTRH(186-199) (pSE(14)) of pFE(22) (preproTRH(178-199)). Also, PC1KO and not PC2KO showed a decrease in pEH(24) indicating that PC1 is more important in generating this peptide in the mouse, which differs from previous studies using rat proTRH. Furthermore, downstream effects on thyroid hormone levels were evident in PC1KO mice, but not PC2KO mice suggesting that PC1 plays the more critical role in producing bioactive hypophysiotropic TRH. Yet loss of PC1 did not abolish TRH entirely indicating a complementary action for both enzymes in the normal processing of proTRH. We also show that PC2 alone is responsible for catalyzing the conversion of pFE(22) to pFQ(7) and pSE(14), all peptides implicated in regulation of suckling-induced prolactin release. Collectively, results characterize the specific roles of PC1 and PC2 in proTRH processing in vivo.

Maintenance of the thyroid axis during diet-induced obesity in rodents is controlled at the central level.

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 T(4/3) seen in obese animals is due mostly to central leptin action. We also found that T(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(3) and pSTAT3 signaling appears essential to maintain the HPT axis at normal levels in conditions such as obesity.

SIRT1 deacetylase in POMC neurons is required for homeostatic defenses against diet-induced obesity.

Feeding on high-calorie (HC) diets induces serious metabolic imbalances, including obesity. Understanding the mechanisms against excessive body weight gain is critical for developing effective antiobesity strategies. Here we show that lack of nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1 in pro-opiomelanocortin (POMC) neurons causes hypersensitivity to diet-induced obesity due to reduced energy expenditure. The ability of leptin to properly engage the phosphoinositide 3-kinase (PI3K) signaling in POMC neurons and elicit remodeling of perigonadal white adipose tissue (WAT) is severely compromised in mutant mice. Also, electrophysiological and histomorphomolecular analyses indicate a selective reduction in sympathetic nerve activity and brown-fat-like characteristics in perigonadal WAT of mutant mice, suggesting a physiologically important role for POMC neurons in controlling this visceral fat depot. In summary, our results provide direct genetic evidence that SIRT1 in POMC neurons is required for normal autonomic adaptations against diet-induced obesity.

Role of a pro-sequence in the secretory pathway of prothyrotropin-releasing hormone.

The biogenesis of rat thyrotropin releasing hormone (TRH) involves the processing of its precursor (proTRH) into five biologically active TRH peptides and several non-TRH peptides where two of them had been attributed potential biological functions. This process implicates 1) proper folding of proTRH in the endoplasmic reticulum after its biosynthesis and exit to the Golgi apparatus and beyond, 2) initial processing of proTRH in the trans Golgi network and, 3) sorting of proTRH-derived peptides to the regulated secretory pathway. Previous studies have focused on elucidating the processing and sorting determinants of proTRH. However, the role of protein folding in the sorting of proTRH remains unexplored. Here we have investigated the role in the secretion of proTRH of a sequence comprising 22 amino acid residues, located at the N-terminal region of proTRH, residues 31-52. Complete deletion of these 22 amino acids dramatically compromised the biosynthesis of proTRH, manifested as a severe reduction in the steady state level of proTRH in the endoplasmic reticulum. This effect was largely reproduced by the deletion of only three amino acid residues, 40PGL42, within the proTRH31-52 sequence. The decreased steady state level of the mutant DeltaPGL was due to enhanced endoplasmic reticulum-associated protein degradation. However, the remnant of DeltaPGL that escaped degradation was properly processed and sorted to secretory granules. Thus, these results suggest that the N-terminal domain within the prohormone sequence does not act as "sorting signal" in late secretion; instead, it seems to play a key role determining the proper folding pathway of the precursor and, thus, its stability.

Prothyrotropin-releasing hormone targets its processing products to different vesicles of the secretory pathway.

Prothyrotropin-releasing hormone (pro-TRH) is initially cleaved by the prohormone convertase-1/3 (PC1/3) in the trans-Golgi network generating N- and C-terminal intermediate forms that are then packed into secretory vesicles. However, it is not known whether these peptides are differentially sorted within the secretory pathway. This is of key importance because the processing products of several prohormones fulfill different biological functions. Using AtT20 cells stably transfected with prepro-TRH cDNA, we found that two specific N- and C-terminal peptides were located in different vesicles. Furthermore, the C-terminal pro-TRH-derived peptides were more efficiently released in response to KCl and norepinephrine, a natural secretagogue of TRH. Similar sorting and secretion of N- and C-terminal peptides occurs in vivo. When we blocked the initial proteolytic processing by a mutagenic approach, the differential sorting and secretion of these peptides were prevented. In summary, our data show that pro-TRH-derived peptides are differentially sorted within the secretory pathway and that the initial cleavage in the trans-Golgi network is key to this process. This could be a common mechanism used by neuroendocrine cells to regulate independently the secretion of different bioactive peptides derived from the same gene product.

Cold exposure increases the biosynthesis and proteolytic processing of prothyrotropin-releasing hormone in the hypothalamic paraventricular nucleus via beta-adrenoreceptors.

Different physiological conditions affect the biosynthesis and processing of hypophysiotropic proTRH in the hypothalamic paraventricular nucleus, and consequently the output of TRH. Early studies suggest that norepinephrine (NE) mediates the cold-induced activation of the hypothalamic-pituitary-thyroid axis at a central level. However, the specific role of NE on the biosynthesis and processing of proTRH has not been fully investigated. In this study, we found that NE affects gene transcription, protein biosynthesis, and secretion in TRH neurons in vitro; these changes were coupled with an up-regulation of prohormone convertase enzymes (PC) 1/3 and PC2. In vivo, NE is the main mediator of the cold-induced activation of the hypothalamic-pituitary-thyroid axis at the hypothalamic level, in which it potently stimulates the biosynthesis and proteolytic processing of proTRH through a coordinated up-regulation of the PCs. This activation occurs via beta-adrenoreceptors and phosphorylated cAMP response element binding signaling. In contrast, alpha-adrenoreceptors regulate TRH secretion but not proTRH biosynthesis and processing. Therefore, this study provides novel information on the molecular mechanisms of control of hypophysiotropic TRH biosynthesis.

Differential effects of fasting and leptin on proopiomelanocortin peptides in the arcuate nucleus and in the nucleus of the solitary tract.

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.

Role of the central melanocortin circuitry in adaptive thermogenesis of brown adipose tissue.

The central melanocortin 4 receptor (MC4R) plays a critical role in energy homeostasis, although little is known regarding its role in the regulation of adaptive thermogenesis of brown adipose tissue (BAT). Here we show using retrograde transsynaptic tracing with attenuated pseudorabies virus coupled with dual-label immunohistochemistry that specific subsets of MC4R-expressing neurons in multiple nuclei of the central nervous system known to regulate sympathetic outflow polysynaptically connect with interscapular BAT (IBAT). Furthermore, we show that MC4R-/- and agouti-related peptide-treated mice are defective in HF diet-induced up-regulation of uncoupling protein 1 in IBAT. Additionally, MC4R-/- mice exposed to 4 C for 4 h exhibit a defect in up-regulation of uncoupling protein 1 levels in IBAT. Our results provide a neuroanatomic substrate for MC4R regulating sympathetically mediated IBAT thermogenesis and demonstrate that the MC4R is critically required for acute high-fat- and cold-induced IBAT thermogenesis.

The role of intracerebroventricular administration of leptin in the stimulation of prothyrotropin releasing hormone neurons in the hypothalamic paraventricular nucleus.

We previously have shown that leptin regulates proTRH in the paraventricular nucleus (PVN) of the hypothalamus through two pathways. The first one acts directly on proTRH neurons, and the second one (indirect) acts through the melanocortin system (arcuate nucleus). However, it is unknown whether the direct or the indirect pathways of leptin action on proTRH neurons occurs on separated or on the same subsets of neurons within the PVN region. We used immunostaining for the phosphorylated signal transducer and activator of transcription 3 to localize direct leptin signaling, and the phosphorylated cAMP response element binding protein to localize indirect signaling on proTRH neurons in animals intracerebroventricularly injected with leptin. With this approach we were able to identify two subsets of neuronal populations responsive to leptin, which are distributed in different regions within the PVN. ProTRH neurons directly responsive to leptin were located mainly in the medial and posterior part of the PVN, and they were not primarily related to the hypothalamic pituitary thyroid axis. Whereas, proTRH neurons indirectly responsive (through alpha-MSH) to leptin were located mainly in the anterior, medial, and periventricular part of the PVN, and related to the hypothalamic pituitary thyroid axis. In addition, alpha-MSH showed to affect the processing of proTRH and up-regulated the prohormone convertase 1/3. In this study, we show evidence supporting the hypothesis that in the PVN there are subpopulations of proTRH neurons responding to leptin, which is dependent upon the way leptin reaches its primary target(s) in the hypothalamus. These findings are critical to a better understanding of leptin-mediated actions in energy expenditure.

Thyroid hormones selectively regulate the posttranslational processing of prothyrotropin-releasing hormone in the paraventricular nucleus of the hypothalamus.

Over the last few years, our laboratory has demonstrated that different physiological conditions or stressors affect the posttranslational processing of hypophysiotropic and nonhypophysiotropic proTRH and, consequently, the output of TRH and other proTRH-derived peptides. These alterations in proTRH processing are generally associated with parallel changes in the levels of two members of the family of prohormone convertases 1/3 and 2 (PC1/3 and PC2). An important regulator of proTRH is thyroid hormone, which is the peripheral end product of the hypothalamic (TRH)-pituitary (TSH)-thyroid (T3/4) (HPT) axis. In this study we investigated the effect of thyroid status on the processing of proTRH inside and outside the HPT axis. Our data showed that high levels of thyroid hormone down-regulated PC1/3 and PC2 and TRH synthesis, which led to an accumulation of intermediate forms of proTRH processing. Conversely, low levels of thyroid hormone up-regulated proTRH synthesis and PC1/3 and PC2 levels. Control of the activity of PCs and proTRH processing occurred specifically in the paraventricular nucleus, whereas no change due to thyroid status was found in the lateral hypothalamus or preoptic area. The posttranslational regulation of proTRH processing in the paraventricular nucleus by thyroid status is a novel aspect of the regulation of the HPT axis, which may have important implications for the pathophysiology of hypo- and hyperthyroidism.

Regulation of hypothalamic prohormone convertases 1 and 2 and effects on processing of prothyrotropin-releasing hormone.

Regulation of energy balance by leptin involves regulation of several neuropeptides, including thyrotropin-releasing hormone (TRH). Synthesized from a larger inactive precursor, its maturation requires proteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). Since this maturation in response to leptin requires prohormone processing, we hypothesized that leptin might regulate hypothalamic PC1 and PC2 expression, ultimately leading to coordinated processing of prohormones into mature peptides. Using hypothalamic neurons, we found that leptin stimulated PC1 and PC2 mRNA and protein expression and also increased PC1 and PC2 promoter activities in transfected 293T cells. Starvation of rats, leading to low serum leptin levels, decreased PC1 and PC2 gene and protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Exogenous administration of leptin to fasted animals restored PC1 levels in the median eminence (ME) and the PVN to approximately the level found in fed control animals. Consistent with this regulation of PCs in the PVN, concentrations of TRH in the PVN and ME were substantially reduced in the fasted animals relative to the fed animals, and leptin reversed this decrease. Further analysis showed that proteolytic cleavage of pro-thyrotropin-releasing hormone (proTRH) at known PC cleavage sites was reduced by fasting and increased in animals given leptin. Combined, these findings suggest that leptin-dependent stimulation of hypothalamic TRH expression involves both activation of trh transcription and stimulation of PC1 and PC2 expression, which lead to enhanced processing of proTRH into mature TRH.