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Recent preclinical research exploring how neuropeptide transmitter systems regulate motivated behavior reveal the increasing importance of sex as a critical biological variable. Neuropeptide systems and their central circuits both contribute to sex differences in a range of motivated behaviors and regulate sex-specific behaviors. In this short review, we explore the current research of how sex as a biological variable influences several distinct motivated behaviors that are modulated by the melanin-concentrating hormone (MCH) neuropeptide system. First, we review how MCH regulates feeding behavior within the context of energy homeostasis differently between male and female rodents. Then, we focus on MCH's role in lactation as a sex-specific process within the context of energy homeostasis. Next, we discuss the sex-specific effects of MCH on maternal behavior. Finally, we summarize the role of MCH in drug-motivated behaviors. While these topics are traditionally investigated from different scientific perspectives, in this short review we discuss how these behaviors share commonalities within the larger context of motivated behaviors, and that sex differences discovered in one area of research may impact our understanding in another. Overall, our review highlights the need for further research into how sex differences in energy regulation associated with reproduction and parental care contribute to regulating motivated behaviors., (© 2024. The Author(s).)

Memory storage in the brain is one of the most extensively studied subjects in neuroscience. However, due to the highly complex structure of the memory-related systems in the brain, the mystery remains unsolved. Consolidation is one of the most important parts of the memory process, and one that can be affected by numerous neurodegenerative diseases. Hypothalamic melanin-concentrating hormone (MCH) neuronal activity has been of particular interest to researchers in terms of the association between sleep, neurodegenerative diseases, and memory consolidation. We used Pmch-Cre animals to investigate the role of MCH neuronal activity in memory consolidation. In order to observe the differences in memory consolidation, we chemogenetically inhibited MCH neurons using the DREADD method and measured hippocampus-dependent memory performance with a novel object recognition test applicable to early memory impairment in Alzheimer's disease. Our results revealed no significant improvement or worsening with MCH inhibition, suggesting that the role of MCH should now be evaluated in a wider setting., Competing Interests: Conflict of interest statement None., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Over the past years, pituitary hormones and their receptors have been shown to have non-traditional actions that allow them to bypass the hypothalamus-pituitary-effector glands axis. Bone cells-osteoblasts and osteoclasts-express receptors for growth hormone, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), adrenocorticotrophic hormone (ACTH), prolactin, oxytocin, and vasopressin. Independent skeletal actions of pituitary hormones on bone have been studied using genetically modified mice with haploinsufficiency and by activating or inactivating the receptors pharmacologically, without altering systemic effector hormone levels. On another front, the discovery of a TSH variant (TSH-βv) in immune cells in the bone marrow and skeletal action of FSHβ through tumor necrosis factor α provides new insights underscoring the integrated physiology of bone-immune-endocrine axis. Here we discuss the interaction of each pituitary hormone with bone and the potential it holds in understanding bone physiology and as a therapeutic target.

Parental behaviour is a comprehensive set of neural responses to social cues. The neural circuits that govern parental behaviour reside in several putative nuclei in the brain. Melanin concentrating hormone (MCH), a neuromodulator that integrates physiological functions, has been confirmed to be involved in parental behaviour, particularly in crouching behaviour during nursing. Abolishing MCH neurons in innate MCH knockout males promotes infanticide in virgin male mice. To understand the mechanism and function of neural networks underlying parental care and aggression against pups, it is essential to understand the basic organisation and function of the involved nuclei. This review presents newly discovered aspects of neural circuits within the hypothalamus that regulate parental behaviours.

Melanin-concentrating hormone (MCH)-expressing neurons are key regulators of energy and glucose homeostasis. Here, we demonstrate that they provide dense projections to the median eminence (ME) in close proximity to tanycytes and fenestrated vessels. Chemogenetic activation of MCH neurons as well as optogenetic stimulation of their projections in the ME enhance permeability of the ME by increasing fenestrated vascular loops and enhance leptin action in the arcuate nucleus of the hypothalamus (ARC). Unbiased phosphoRiboTrap-based assessment of cell activation upon chemogenetic MCH neuron activation reveals MCH-neuron-dependent regulation of endothelial cells. MCH neurons express the vascular endothelial growth factor A (VEGFA), and blocking VEGF-R signaling attenuates the leptin-sensitizing effect of MCH neuron activation. Our experiments reveal that MCH neurons directly regulate permeability of the ME barrier, linking the activity of energy state and sleep regulatory neurons to the regulation of hormone accessibility to the ARC., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Glutamate transporter 1 (GLT1) is the main astrocytic transporter that shapes glutamatergic transmission in the brain. However, whether this transporter modulates sleep-wake regulatory neurons is unknown. Using quantitative immunohistochemical analysis, we assessed perisomatic GLT1 apposition with sleep-wake neurons in the male rat following 6 h sleep deprivation (SD) or following 6 h undisturbed conditions when animals were mostly asleep (Rest). We found that SD decreased perisomatic GLT1 apposition with wake-promoting orexin neurons in the lateral hypothalamus compared with Rest. Reduced GLT1 apposition was associated with tonic presynaptic inhibition of excitatory transmission to these neurons due to the activation of Group III metabotropic glutamate receptors, an effect mimicked by a GLT1 inhibitor in the Rest condition. In contrast, SD resulted in increased GLT1 apposition with sleep-promoting melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus. Functionally, this decreased the postsynaptic response of MCH neurons to high-frequency synaptic activation without changing presynaptic glutamate release. The changes in GLT1 apposition with orexin and MCH neurons were reversed after 3 h of sleep opportunity following 6 h SD. These SD effects were specific to orexin and MCH neurons, as no change in GLT1 apposition was seen in basal forebrain cholinergic or parvalbumin-positive GABA neurons. Thus, within a single hypothalamic area, GLT1 differentially regulates excitatory transmission to wake- and sleep-promoting neurons depending on sleep history. These processes may constitute novel astrocyte-mediated homeostatic mechanisms controlling sleep-wake behavior. SIGNIFICANCE STATEMENT Sleep-wake cycles are regulated by the alternate activation of sleep- and wake-promoting neurons. Whether and how astrocytes can regulate this reciprocal neuronal activity are unclear. Here we report that, within the lateral hypothalamus, where functionally opposite wake-promoting orexin neurons and sleep-promoting melanin-concentrating hormone neurons codistribute, the glutamate transporter GLT1, mainly present on astrocytes, distinctly modulates excitatory transmission in a cell-type-specific manner and according to sleep history. Specifically, GLT1 is reduced around the somata of orexin neurons while increased around melanin-concentrating hormone neurons following sleep deprivation, resulting in different forms of synaptic plasticity. Thus, astrocytes can fine-tune the excitability of functionally discrete neurons via glutamate transport, which may represent novel regulatory mechanisms for sleep., (Copyright © 2018 the authors 0270-6474/18/382506-14$15.00/0.)

Electrical signals generated by molecularly-distinct classes of lateral hypothalamus (LH) neurons have distinct physiological consequences. For example, LH orexin neurons promote net body energy expenditure, while LH non-orexin neurons [VGAT, melanin-concentrating hormone (MCH)] drive net energy conservation. Appropriate switching between such physiologically-opposing LH outputs is traditionally thought to require cell-type-specific chemical modulation of LH firing. However, it was recently found that, in vivo, the LH neurons are also physiologically exposed to electrical oscillations of different frequency bands. The role of the different physiological oscillation frequencies in firing of orexin vs non-orexin LH neurons remains unknown. Here, we used brain-slice whole-cell patch-clamp technology to target precisely-defined oscillation waveforms to individual molecularly-defined classes LH cells (orexin, VGAT, MCH, GAD65), while measuring the action potential output of the cells. By modulating the frequency of sinusoidal oscillatory input, we found that high-frequency oscillations (γ, ≈30-200 Hz) preferentially silenced the action potential output orexin LH cells. In contrast, low frequencies (δ-θ, ≈0.5-7 Hz) similarly permitted outputs from different LH cell types. This differential control of orexin and non-orexin cells by oscillation frequency was mediated by cell-specific, impedance-unrelated resonance mechanisms. These results substantiate electrical oscillations as a novel input modality for cell-type-specific control of LH firing, which offers an unforeseen way to control specific cell ensembles within this highly heterogeneous neuronal cluster.

Study Objectives: Sleep reduction after stroke is linked to poor recovery in patients. Conversely, a neuroprotective effect is observed in animals subjected to acute sleep deprivation (SD) before ischemia. This neuroprotection is associated with an increase of the sleep, melanin concentrating hormone (MCH) and orexin/hypocretin (OX) systems. This study aims to 1) assess the relationship between sleep and recovery; 2) test the association between MCH and OX systems with the pathological mechanisms of stroke., Methods: Sprague-Dawley rats were assigned to four experimental groups: (i) SD_IS: SD performed before ischemia; (ii) IS: ischemia; (iii) SD_Sham: SD performed before sham surgery; (iv) Sham: sham surgery. EEG and EMG were recorded. The time-course of the MCH and OX gene expression was measured at 4, 12, 24 hours and 3, 4, 7 days following ischemic surgery by qRT-PCR., Results: A reduction of infarct volume was observed in the SD_IS group, which correlated with an increase of REM sleep observed during the acute phase of stroke. Conversely, the IS group showed a reduction of REM sleep. Furthermore, ischemia induces an increase of MCH and OX systems during the acute phase of stroke, although, both systems were still increased for a long period of time only in the SD_IS group., Conclusions: Our data indicates that REM sleep may be involved in the neuroprotective effect of SD pre-ischemia, and that both MCH and OX systems were increased during the acute phase of stroke. Future studies should assess the role of REM sleep as a prognostic marker, and test MCH and OXA agonists as new treatment options in the acute phase of stroke., Competing Interests: The authors have declared that no competing interests exist.

Melanin-concentrating hormone (MCH) is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area. Recent studies have reported that MCH neurons are active during rapid eye movement (REM) sleep, but their physiological role in the regulation of sleep/wakefulness is not fully understood. To determine the physiological role of MCH neurons, newly developed transgenic mouse strains that enable manipulation of the activity and fate of MCH neurons in vivo were generated using the recently developed knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction system. The activity of these cells was controlled by optogenetics by expressing channelrhodopsin2 (E123T/T159C) or archaerhodopsin-T in MCH neurons. Acute optogenetic activation of MCH neurons at 10 Hz induced transitions from non-REM (NREM) to REM sleep and increased REM sleep time in conjunction with decreased NREM sleep. Activation of MCH neurons while mice were in NREM sleep induced REM sleep, but activation during wakefulness was ineffective. Acute optogenetic silencing of MCH neurons using archaerhodopsin-T had no effect on any vigilance states. Temporally controlled ablation of MCH neurons by cell-specific expression of diphtheria toxin A increased wakefulness and decreased NREM sleep duration without affecting REM sleep. Together, these results indicate that acute activation of MCH neurons is sufficient, but not necessary, to trigger the transition from NREM to REM sleep and that MCH neurons also play a role in the initiation and maintenance of NREM sleep., (Copyright © 2014 the authors 0270-6474/14/336896-14$15.00/0.)

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice. Three weeks later MCH neurons were stimulated for 1 min every 5 min for 24 h. A 10 Hz stimulation at the start of the night hastened sleep onset, reduced length of wake bouts by 50%, increased total time in non-REM and REM sleep at night, and increased sleep intensity during the day cycle. Sleep induction at a circadian time when all of the arousal neurons are active indicates that MCH stimulation can powerfully counteract the combined wake-promoting signal of the arousal neurons. This could be potentially useful in treatment of insomnia.

Background & Aims: Specific neuronal circuits modulate autonomic outflow to liver and white adipose tissue. Melanin-concentrating hormone (MCH)-deficient mice are hypophagic, lean, and do not develop hepatosteatosis when fed a high-fat diet. Herein, we sought to investigate the role of MCH, an orexigenic neuropeptide specifically expressed in the lateral hypothalamic area, on hepatic and adipocyte metabolism., Methods: Chronic central administration of MCH and adenoviral vectors increasing MCH signaling were performed in rats and mice. Vagal denervation was performed to assess its effect on liver metabolism. The peripheral effects on lipid metabolism were assessed by real-time polymerase chain reaction and Western blot., Results: We showed that the activation of MCH receptors promotes nonalcoholic fatty liver disease through the parasympathetic nervous system, whereas it increases fat deposition in white adipose tissue via the suppression of sympathetic traffic. These metabolic actions are independent of parallel changes in food intake and energy expenditure. In the liver, MCH triggers lipid accumulation and lipid uptake, with c-Jun N-terminal kinase being an essential player, whereas in adipocytes MCH induces metabolic pathways that promote lipid storage and decreases lipid mobilization. Genetic activation of MCH receptors or infusion of MCH specifically in the lateral hypothalamic area modulated hepatic lipid metabolism, whereas the specific activation of this receptor in the arcuate nucleus affected adipocyte metabolism., Conclusions: Our findings show that central MCH directly controls hepatic and adipocyte metabolism through different pathways., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

GABAergic neurons are vital for brain function. Their neurochemical and electrical features have been classically characterized in the cortex, but in the lateral hypothalamic area (LHA), such knowledge is lacking, despite the emerging roles of LHA GABAergic cells in feeding and sleep. We used GAD65-GFP transgenic mice, developed for studies of cortical GABAergic cells, to determine fundamental properties of LHA GAD65 neurons, and compare them to 'classical' GABAergic cell types of the cortex, and to previously described classes of LHA cells. Whole-cell patch-clamp recordings in acute brain slices revealed that, unlike cortical GABAergic interneurons, LHA GAD65 neurons were intrinsically depolarized and fired action potentials spontaneously. Similar to cortical GABAergic cells, LHA GAD65 cells fell into four major subtypes based on evoked firing: fast spiking, late spiking, low threshold spiking and regular spiking. Three-dimensional reconstructions of biocytin-filled neurons, performed after the patch-clamp analysis, did not reveal striking morphological differences between these electrophysiological subtypes. Peptide transmitters expressed in known classes of LHA projection neurons, namely melanin-concentrating hormone (MCH) and hypocretin/orexin (hcrt/orx), were not detected in LHA GAD65 cells. Approximately 40% of LHA GAD65 cells were directly inhibited by physiological increases in extracellular glucose concentration. Glucose inhibition was most prevalent in the fast spiking subpopulation, although some glucose-responsive neurons were found in each electrophysiological subpopulation. These results suggest that LHA GAD65 neurons are electrically different from 'classical' GABAergic neurons of the cortex, are neurochemically distinct from LHA hcrt/orx and MCH cells, but partly resemble hcrt/orx cells in their glucose responses.

Neurons that synthesize melanin-concentrating hormone (MCH) colocalize GABA, regulate energy homeostasis, modulate water intake, and influence anxiety, stress, and social interaction. Similarly, vasopressin and oxytocin can influence the same behaviors and states, suggesting that these neuropeptides may exert part of their effect by modulating MCH neurons. Using whole cell recording in MCH-green fluorescent protein (GFP) transgenic mouse hypothalamic brain slices, we found that both vasopressin and oxytocin evoked a substantial excitatory effect. Both peptides reversibly increased spike frequency and depolarized the membrane potential in a concentration-dependent and tetrodotoxin-resistant manner, indicating a direct effect. Substitution of lithium for extracellular sodium, Na(+)/Ca(2+) exchanger blockers KB-R7943 and SN-6, and intracellular calcium chelator BAPTA, all substantially reduced the vasopressin-mediated depolarization, suggesting activation of the Na(+)/Ca(2+) exchanger. Vasopressin reduced input resistance, and the vasopressin-mediated depolarization was attenuated by SKF-96265, suggesting a second mechanism based on opening nonselective cation channels. Neither vasopressin nor oxytocin showed substantial excitatory actions on lateral hypothalamic inhibitory neurons identified in a glutamate decarboxylase 67 (GAD67)-GFP mouse. The primary vasopressin receptor was vasopressin receptor 1a (V1aR), as suggested by the excitation by V1aR agonist [Arg(8)]vasotocin, the selective V1aR agonist [Phe(2)]OVT and by the presence of V1aR mRNA in MCH cells, but not in other nearby GABA cells, as detected with single-cell RT-PCR. Oxytocin receptor mRNA was also detected in MCH neurons. Together, these data suggest that vasopressin or oxytocin exert a minimal effect on most GABA neurons in the lateral hypothalamus but exert a robust excitatory effect on presumptive GABA cells that contain MCH. Thus, some of the central actions of vasopressin and oxytocin may be mediated through MCH cells.

Thyrotropin-releasing hormone (TRH) increases activity and decreases food intake, body weight, and sleep, in part through hypothalamic actions. The mechanism of this action is unknown. Melanin-concentrating hormone (MCH) and hypocretin/orexin neurons in the lateral hypothalamus (LH) together with neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the arcuate nucleus play central roles in energy homeostasis. Here, we provide electrophysiological evidence from GFP-reporter transgenic mouse brain slices that shows TRH modulates the activity of MCH neurons. Using whole-cell current-clamp recording, we unexpectedly found that TRH and its agonist, montrelin, dose-dependently inhibited MCH neurons. Consistent with previous reports, TRH excited hypocretin/orexin neurons. No effect was observed on arcuate nucleus POMC or NPY neurons. The TRH inhibition of MCH neurons was eliminated by bicuculline and tetrodotoxin, suggesting that the effect was mediated indirectly through synaptic mechanisms. TRH increased spontaneous IPSC frequency without affecting amplitude and had no effect on miniature IPSCs or EPSCs. Immunocytochemistry revealed little interaction between TRH axons and MCH neurons, but showed TRH axons terminating on or near GABA neurons. TRH inhibition of MCH neurons was attenuated by Na(+)-Ca(2+) exchanger (NCX) inhibitors, TRPC channel blockers and the phospholipase C inhibitor U-73122. TRH excited LH GABA neurons, and this was also reduced by NCX inhibitors. Finally, TRH attenuated the excitation of MCH neurons by hypocretin. Together, our data suggest that TRH inhibits MCH neurons by increasing synaptic inhibition from local GABA neurons. Inhibition of MCH neurons may contribute to the TRH-mediated reduction in food intake and sleep.

Background: There are many types of ovarian activity that occur in women. This review provides information on the relationship between the hormone values and the degree of biological response to the hormones including the frequency and degree of uterine bleeding. The continuous process is termed the 'Continuum' and is thus similar to other processes in the body., Methods: This review draws on information already published from monitoring ovarian activity by urinary oestrogen and pregnanediol measurements using timed 24-h specimens of urine. Much of the rationalization was derived from 5 to 6 year studies of girls progressing from childhood to adulthood, women progressing through menopause, and the return of fertility post-partum. During these times, all the reported types of ovarian activity were encountered., Results: All cycle types can be understood in terms of steps in the normal maturation of fertility at the beginning of reproductive life, its return post-partum and its demise at menopause. Each step merges into the next and therefore the sequence is termed the 'Continuum'. Unpredictable movement from fertile to infertile types and back can occur at any time during reproductive life. Stress is a major causative factor. Hormonal definitions for each step, the relevance of the various cycle types in determining fertility and in the initiation of uterine bleeding and the roles of the pituitary hormones in causing them, are presented., Conclusions: The findings explain the erratic fertility of women and why ovulation is not always associated with fertility. They provide an understanding of the various types of ovarian activity and their relation to pituitary function, fertility and uterine bleeding.

Although the neuronal pathways within the hypothalamus critical in controlling feeding and energy expenditure and projecting to brown adipose tissue (BAT) have been identified and their peptidergic content characterized, endogenous action of such peptides in the control of BAT activity has not been elucidated. Here male Sprague Dawley rats received infusions of either melanin-concentrating hormone antagonist (SNAP-7941) (1 microg/microl x h), orexin A receptor antagonist (SB-334867-A; 1 microg/microl x h), combined SB-334867-A (1 microg/microl x h), and SNAP-7941 (1 microg/microl x h), or melanocortin-3/4 receptor antagonist (SHU9119) (1 microg/microl x h) via an indwelling cannula in the lateral ventricle attached to s.c. implanted osmotic minipump. BAT temperature, physical activity, body weight, food intake, and changes in uncoupling protein (UCP)-1 were measured. SB-334867-A and SNAP-7941 significantly increased BAT temperature and UCP1 expression and reduced food intake and body weight. Combined infusion of SB-334867-A and SNAP-7941 produced a pronounced response that was greater than the addition of the individual effects in all parameters measured. SHU9119 significantly decreased BAT temperature and UCP1 expression and increased feeding and body weight. In a second series of experiments, the effect of SB-334867-A and SNAP-7941 alone or combination on the expression of the Fos protein was determined. SB-334867-A and SNAP-7941 increased Fos expression in key hypothalamic and brainstem feeding-related regions. In combination, these antagonists produced a greater than additive elevation of Fos expression in most of the regions evaluated. These findings support a role for endogenous orexigenic and anorexigenic hypothalamic peptides acting in concert to create a thermogenic tone via BAT activity.

Two lamprey glycoprotein hormone receptors (lGpH-R I and II) highly similar with gnathostome GpH-Rs were cloned from sea lamprey testes and thyroid, respectively. Vertebrate glycoprotein protein receptors have a large extracellular domain (ED) containing a leu rich domain (LRD) linked to a rhodopsin-like transmembrane domain (TMD) through a highly divergent linker region (signal specificity domain, SSD or 'hinge' region) and a third major segment, the intracellular domain. To determine the potential roles of the different domains in the activation of the receptor following ligand-receptor binding, functional assays were performed on lGpH-R I/rat luteinizing hormone (LH)-R domain swapped chimeric receptors. These results show that the functional roles of the lamprey glycoprotein-receptor I (lGpH-R I) domains are conserved compared with its Gnathostome homologs. The ability of different glycoprotein hormones to activate chimeric lamprey/rat receptors suggests that the selectivity of the GpH-Rs in respect to their ligands is not controlled exclusively by a single domain but is the result of specific interactions between domains. We hypothesize that these interactions were refined during millions of years of co-evolution of the receptors with their cognate ligands under particular intramolecular, intermolecular and physiological constraints., (© The Author(s) 2010. Published by Oxford University Press.)

Normal hypothalamopituitary development is closely related to that of the forebrain and is dependent upon a complex genetic cascade of transcription factors and signaling molecules that may be either intrinsic or extrinsic to the developing Rathke's pouch. These factors dictate organ commitment, cell differentiation, and cell proliferation within the anterior pituitary. Abnormalities in these processes are associated with congenital hypopituitarism, a spectrum of disorders that includes syndromic disorders such as septo-optic dysplasia, combined pituitary hormone deficiencies, and isolated hormone deficiencies, of which the commonest is GH deficiency. The highly variable clinical phenotypes can now in part be explained due to research performed over the last 20 yr, based mainly on naturally occurring and transgenic animal models. Mutations in genes encoding both signaling molecules and transcription factors have been implicated in the etiology of hypopituitarism, with or without other syndromic features, in mice and humans. To date, mutations in known genes account for a small proportion of cases of hypopituitarism in humans. However, these mutations have led to a greater understanding of the genetic interactions that lead to normal pituitary development. This review attempts to describe the complexity of pituitary development in the rodent, with particular emphasis on those factors that, when mutated, are associated with hypopituitarism in humans.

Somatolactin alpha (SLa) is a fish-specific peptide hormone secreted from the pituitary. Its functions have been investigated for the last decade but are still under debate. We previously reported a frame-shift mutation on SLa in a medaka mutant, color interfere (ci), which shows defects in body-color regulation, lipid storage, and cortisol secretion. In this study, we examined the effects of introducing a DNA fragment which constitutively expresses wild-type SLa under regulation of the beta-actin (Actb) promoter into the ci genome. We successfully visualized the transgene expression by taking advantage of an internal ribosome entry site (IRES) and the green fluorescent protein (GFP). The transgenic medaka, Actb-SLa:GFP, exhibited a reversed body-color phenotype of ci; more orange xanthophores and less white leucophores. We also detected more black melanophores and less silver iridophores, which indicates SLa's comprehensive role in regulating all types of pigment cells in the skin. Unexpectedly, the defects in lipid/cortisol contents remained in Actb-SLa:GFP. Therefore, the causal relationship between SLa and lipid/cortisol metabolisms relapses to an open question which needs to be reassessed by other types of experiments. Both the Actb-SLa:GFP and ci fish grow and mature similar to wild type, indicating SLa contributes little to growth regulation in spite of the fact that it binds to a teleost-specific paralog of growth-hormone receptor in vitro. The present study provides definitive evidence for SLa's principal and indispensable role in body-color regulation in medaka.

Melanin-concentrating hormone (MCH) is expressed primarily in the hypothalamus and has a positive impact on feeding behavior and energy balance. Although MCH is expressed in the gastrointestinal tract, its role in this system remains elusive. We demonstrate that, compared to wild type, mice genetically deficient in MCH had substantially reduced local inflammatory responses in a mouse model of experimental colitis induced by intracolonic administration of 2,4,6 trinitrobenzene sulfonic acid (TNBS). Likewise, mice receiving treatments with an anti-MCH antibody, either prophylactically or after the establishment of colitis, developed attenuated TNBS-associated colonic inflammation and survived longer. Consistent with a potential role of MCH in intestinal pathology, we detected increased colonic expression of MCH and its receptor in patients with inflammatory bowel disease. Moreover, we found that human colonic epithelial cells express functional MCH receptors, the activation of which induces IL-8 expression. Taken together, these results clearly implicate MCH in inflammatory processes in the intestine and perhaps elsewhere.

The lateral hypothalamus (LH) has long been known as a homeostasis center of the brain that modulates feeding behavior, arousal and reward. The hypocretins (Hcrts, also called orexins) and melanin-concentrating hormone (MCH) are neuropeptides produced in two intermingled populations of a few thousand neurons in the LH. The Hcrts have a prominent role in regulating the stability of arousal, since Hcrt system deficiency leads to narcolepsy. MCH is an important modulator of energy balance, as MCH system deficiency in mice leads to leanness and increased metabolism. Recently, MCH has been proposed to modulate rapid eye movement sleep in rodents. In this review, we propose a working model of the cross-talk between Hcrt and MCH circuits that may provide an arousal balance system to regulate complex goal-oriented behaviors.

The LHX3 LIM-homeodomain transcription factor is required for correct development of the mammalian pituitary gland and spinal motoneurons. Mutations in the LHX3 gene underlie complex diseases featuring combined anterior pituitary hormone deficiency and, in specific cases, loss of neck rotation considered to result from nervous system abnormalities. The molecular basis for LHX3 protein actions in both normal and aberrant pituitary and nervous system development is poorly understood. In this study, the gene regulatory abilities of mutant LHX3 proteins associated with distinct types of diseases (LHX3a A210V, LHX3a E173Ter, and LHX3a W224Ter) were investigated. The capacity of these proteins to activate pituitary hormone and transcription factor gene promoters, nervous system target genes, and to localize to the nucleus of pituitary cells was measured. Consistent with the symptoms of patients with these mutations, the abnormal proteins displayed diminished capacities to activate the promoters of genes expressed in the pituitary gland. On nervous system promoters, several mutant proteins retained some activity. The ability of the mutant proteins to concentrate in the nucleus of pituitary cells was correlated with the retention of defined nuclear localization signals in the protein sequence, except for the E173Ter protein which unexpectedly localizes to the nucleus, likely due to the insertion of cryptic nuclear localization signals by a frame shift caused by the mutation. This study extends the molecular characterization of the severe neuroendocrine diseases associated with LHX3 gene mutations.

Pituitary hormones are essential for normal growth and metabolic responsiveness after birth, but their role before birth remains unclear. This study examined the effects of hypophysectomizing fetal sheep on their growth and glucose metabolism during the late normal and extended periods of gestation, and on their metabolic response to maternal fasting for 48 h near term. Fetal hypophysectomy reduced crown rump length (CRL), limb lengths, and body weight but increased ponderal index relative to controls near normal term. It also lowered the daily rate of crown rump length increment uniformly from 35 d before, to 20 d after normal term. Hypophysectomized (HX) fetuses had normal weight-specific rates of umbilical uptake, utilization, and oxidation of glucose but lower rates of umbilical oxygen uptake than controls near term. All these metabolic rates were significantly less in HX fetuses during the extended period of gestation than in HX and intact fetuses near normal term. In contrast to controls, glucogenesis was negligible in HX fetuses during maternal fasting. Consequently, the rate of glucose utilization decreased significantly in fasted HX but not intact fetuses. Conversely, the rate of CO(2) production from glucose carbon decreased in fasted intact but not HX fetuses. Fetal hypophysectomy also prevented the fasting-induced increases in plasma cortisol and norepinephrine concentrations seen in controls. These findings demonstrate that the pituitary hormones are important in regulating the growth rate and adaptive responses of glucose metabolism to undernutrition in fetal sheep. They also suggest that fetal metabolism is altered when gestational length is extended.

Cannabinoids modulate energy homeostasis and decrease cognitive arousal, possibly by acting on hypothalamic neurons including those that synthesize melanin-concentrating hormone (MCH) or hypocretin/orexin. Using patch-clamp recordings, we compared the actions of cannabinoid agonists and antagonists on identified MCH or hypocretin neurons in green fluorescent protein-expressing transgenic mice. The cannabinoid type-1 receptor (CB1R) agonist R-(+)-[2,3-dihydro-5-methyl-3-(4-morpho linylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212,2) depolarized MCH cells and increased spike frequency; in contrast, WIN55,212,2 hyperpolarized and reduced spontaneous firing of the neighboring hypocretin cells, both results consistent with reduced activity seen with intracerebral cannabinoid infusions. These effects were prevented by AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a CB1R antagonist, and by tetrodotoxin, suggesting no postsynaptic effect on either neuron type. In MCH cells, depolarizing WIN55,212,2 actions were abolished by the GABA(A) receptor antagonist bicuculline, suggesting that the CB1R-mediated depolarization was attributable to reduced synaptic GABA release. WIN55,212,2 decreased spontaneous IPSCs, reduced the frequency but not amplitude of miniature IPSCs, and reduced electrically evoked synaptic currents in MCH cells. Glutamate microdrop experiments suggest that WIN55,212,2 acted on axons arising from lateral hypothalamus local inhibitory cells that innervate MCH neurons. In hypocretin neurons, the reduced spike frequency induced by WIN55,212,2 was attributable to presynaptic attenuation of glutamate release; CB1R agonists depressed spontaneous and evoked glutamatergic currents and reduced the frequency of miniature EPSCs. Cannabinoid actions on hypocretin neurons were abolished by ionotropic glutamate receptor antagonists. Together, these results show that cannabinoids have opposite effects on MCH and hypocretin neurons. These opposing actions could help explain the increase in feeding and reduction in arousal induced by cannabinoids.

Melanin-concentrating hormone (MCH) is a cyclic peptide originally identified as a 17-amino-acid circulating hormone in teleost fish, where it is secreted by the pituitary in response to stress and environmental stimuli. In fish, MCH lightens skin color by stimulating aggregation of melanosomes, pigment-containing granules in melanophores, cells of neuroectodermal origin found in fish scales. Although the peptide structure between fish and mammals is highly conserved, in mammals, MCH has no demonstrable effects on pigmentation; instead, based on a series of pharmacological and genetic experiments, MCH has emerged as a critical hypothalamic regulator of energy homeostasis, having effects on both feeding behavior and energy expenditure.

The level of somatotropin and thyreotropin hormones was measured in blood plasma of patients with non-insulin-dependent diabetes mellitus with and without diabetic retinopathy (DR). Fifty eight patients (26 males and 32 females) aged from 35 to 75 were examined. The patients were divided into two groups. First group included 29 patients (13 males and 16 females) with non-insulin-dependent diabetes mellitus without DR. Second group included the same number of patients with non-insulin-dependent diabetes mellitus complicated by DR. The concentration of somatotropin hormones was increased and thyreotropin was decreased in patients with non-insulin-dependent diabetes mellitus with DR. The results suggest that somatotropin and thyreotropin contributes to the development of diabetic retinopathy in humans.

A number of different neuropeptides exert powerful concerted controls on feeding behavior and energy balance, most of them being produced in hypothalamic neuronal networks under stimulation by anabolic and catabolic peripheral hormones such as ghrelin and leptin, respectively. These peptide-expressing neurons interconnect extensively to integrate the multiple opposing signals that mediate changes in energy expenditure. In the present review I have summarized our current knowledge about two key peptidic systems involved in regulating appetite and energy homeostasis, the melanocortin system (alpha-MSH, agouti and Agouti-related peptides, MC receptors and mahogany protein) and the melanin-concentrating hormone system (proMCH-derived peptides and MCH receptors) that contribute to satiety and feeding-initiation, respectively, with concurrent effects on energy expenditure. I have focused particularly on recent data concerning transgenic mice and the ongoing development of MC/MCH receptor antagonists/agonists that may represent promising drugs to treat human eating disorders on both sides of the energy balance (anorexia, obesity).

The level of thyrotropin (TSH) secretion is determined by the balance of TSH-releasing hormone (TRH) and thyroid hormones. However, neuromedin B (NB), a bombesin-like peptide, highly concentrated in the pituitary, has been postulated to be a tonic inhibitor of TSH secretion. We studied the pituitary-thyroid axis in adult male mice lacking NB receptor (NBR-KO) and their wild-type (WT) littermates. At basal state, NBR-KO mice presented serum TSH slightly higher than WT (18%, P< 0.05), normal intra-pituitary TSH content, and no significant changes in alpha and beta TSH mRNA levels. Serum thyroxine was normal but serum triiodothyronine (T3) was reduced by 24% (P< 0.01) in NBR-KO mice. Pituitaries of NBR-KO mice exhibited no alteration in prolactin mRNA expression but type I and II deiodinase mRNA levels were reduced by 53 and 42% respectively (P< 0.05), while TRH receptor mRNA levels were importantly increased (78%, P< 0.05). The TSH-releasing effect of TRH was significantly higher in NBR-KO than in WT mice (7.1-and 4.0-fold respectively), but, while WT mice presented a 27% increase in serum T3 (P< 0.05) after TRH, NBR-KO mice showed no change in serum T3 after TRH. NBR-KO mice did not respond to exogenous NB, while WT showed a 30% reduction in serum TSH. No compensatory changes in mRNA expression of NB or other bombesin-related peptides and receptors (gastrin-releasing peptide (GRP), GRP-receptor and bombesin receptor subtype-3) were found in the pituitary of NBR-KO mice. Therefore, the data suggest that NB receptor pathways are importantly involved in thyrotroph gene regulation and function, leading to a state where TSH release is facilitated especially in response to TRH, but probably with a less-bioactive TSH. Therefore, the study highlights the important role of NB as a physiological regulator of pituitary-thyroid axis function and gene expression.

The observation that loss of orexin (hypocretin) neurons causes human narcolepsy raises the possibility that other acquired disorders might also result from loss of hypothalamic neurons. To test this possibility for body weight, mice with selective loss of melanin concentrating hormone (MCH) neurons were generated. MCH was chosen to test because induced mutations of the MCH gene in mice cause hypophagia and leanness. Mice with ablation of MCH neurons were generated using toxin (ataxin-3)-mediated ablation strategy. The mice appeared normal but, after 7 weeks, developed reduced body weight, body length, fat mass, lean mass, and leptin levels. Leanness was characterized by hypophagia and increased energy expenditure. To study the role of MCH neurons on obesity secondary to leptin deficiency, we generated mice deficient in both ob gene product (leptin) and MCH neurons. Absence of MCH neurons in ob/ob mice improved obesity, diabetes, and hepatic steatosis, suggesting that MCH neurons are important mediators of the response to leptin deficiency. These data show that loss of MCH neurons can lead to an acquired leanness. This has implications for the pathogenesis of acquired changes of body weight and might be considered in clinical settings characterized by substantial weight changes later in life.

Because there are sex differences in feeding behavior in rats, we looked for a possible sex difference in the response to glucose of melanin-concentrating hormone (MCH) neurons in the lateral hypothalamic area using phosphorylated cAMP response element-binding protein (pCREB) as a marker of neural activity. Intact male rats and female rats at diestrus 2, proestrus, or estrus were fed normally or fasted for 48 h and injected with saline or glucose (400 mg/kg). Thereafter, preparations were subjected to immunohistochemical processing for the double staining of MCH and pCREB. Fasting increased the ratio of MCH neurons with pCREB (double-stained cells) in both male and female rats. In fasted rats, glucose injection decreased the ratio of double-stained cells more promptly in females than in males. The magnitude of decrease caused by glucose was greater at proestrus and estrus than at diestrus 2. Gonadectomy in males enhanced and in females attenuated the response of MCH neurons to glucose. Testosterone and estrogen replacement in males and females, respectively, restored the response of MCH neurons to glucose. The demonstrated sex differences in the response of MCH neurons to glucose correlated well with the gonadal steroid milieu; thus, MCH neurons may play an important role in sex differences in feeding behavior.

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) is a class A G protein-coupled receptor. The MCH system has been linked to a variety of physiological functions, including the regulation of feeding and energy metabolism. We recently reported the importance of a dibasic motif in the membrane-proximal C-terminal region for MCH1R function. Here we reveal that an Arg residue in intracellular loop 2 of MCH1R plays a critical role in receptor function. We analyzed the roles of two distinct motifs, BBXXB and BXBB (in which B is a basic residue and X is a nonbasic residue), located in the three intracellular loops of MCH1R. Triple-substitution mutants of intracellular loops 1 and 3 could still activate calcium mobilization, albeit with lower efficacy or potency. However, mutations in intracellular loop 2 led to a complete loss of induction of signal transduction without changing the high affinity constant (Kd) value. By analyzing a series of single-substitution mutants, a point mutation of Arg155 in intracellular loop 2 was found to be responsible for the signaling pathway elicited by MCH. In addition, substitution at positions corresponding to Arg155 in human MCH receptor 2 and rat somatostatin receptor 2 also markedly abolished their ligand-induced signaling capacities, indicating that this Arg is a recognition determinant in several G protein-coupled receptors.

The perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the regulation of behavioural arousal. The PF-LHA contains several cell types including neurones expressing the peptides, hypocretin (HCRT; also called orexin) and melanin-concentrating hormone (MCH). Evidence suggests that most of the PF-LHA neurones, including HCRT neurones, are active during waking and quiescent during non-rapid eye movement (non-NREM) sleep. The PF-LHA contains local GABAergic interneurones and also receives GABAergic inputs from sleep-promoting regions in the preoptic area of the hypothalamus. We hypothesized that increased GABA-mediated inhibition within PF-LHA contributes to the suppression of neuronal activity during non-REM sleep. EEG and EMG activity of rats were monitored for 2 h during microdialytic delivery of artificial cerebrospinal fluid (aCSF) or bicuculline, a GABAA receptor antagonist, into the PF-LHA in spontaneously sleeping rats during the lights-on period. At the end of aCSF or bicuculline perfusion, rats were killed and c-Fos immunoreactivity (Fos-IR) in HCRT, MCH and other PF-LHA neurones was quantified. In response to bicuculline perfusion into the PF-LHA, rats exhibited a dose-dependent decrease in non-REM and REM sleep time and an increase in time awake. The number of HCRT, MCH and non-HCRT/non-MCH neurones exhibiting Fos-IR adjacent to the microdialysis probe also increased dose-dependently in response to bicuculline. However, significantly fewer MCH neurones exhibited Fos-IR in response to bicuculline as compared to HCRT and other PF-LHA neurones. These results support the hypothesis that PF-LHA neurones, including HCRT neurones, are subject to increased endogenous GABAergic inhibition during sleep. In contrast, MCH neurones appear to be subject to weaker GABAergic control during sleep.

Objective: Treatment of male rodents with estradiol (E2) is associated with anorexia and weight loss by poorly understood mechanisms. We examined the role of the orexigenic hypothalamic peptide melanin-concentrating hormone (MCH) and the appetite-inhibiting, fat-derived hormone leptin in mediating E2-induced anorexia., Research Methods and Procedures: We studied the effect of E2 treatment (implantation of either E2 pellet or matching placebo) in male C57Bl/6J mice, as well as in a lean mouse model (MCH knockout mice) and an obese model (leptin-deficient ob/ob mice). We also studied the effect of E2 treatment in the context of high-fat diet., Results: We confirmed E2 dose-dependent anorexia in male wild type mice fed a normal chow diet. E2 treatment was associated with a significant decrease in body fat, serum leptin levels, and arcuate hypothalamic proopiomelanocortin expression. E2-implanted mice also showed increased hypothalamic neuropeptide Y and MCH expression. As MCH has been implicated in E2-induced hypophagia, we performed E2 pellet implantation in MCH knockout mice and observed hypophagia and weight loss, indicating that MCH is not an essential mediator of E2-induced anorexia. E2-implanted ob/ob mice also had hypophagia and weight loss, indicating that leptin is not essential for E2-induced anorexia. High-fat diet significantly exacerbated the effect of E2 treatment, leading to a 99.6% decrease in food intake at 48 hours and a 30% loss of body weight within 1 week., Discussion: The anorectic effects of E2 were independent of MCH and leptin. Our results suggested that E2 may have effects on nutrient preferences.

Mice lacking either bombesin receptor subtype (BRS)-3 or gastrin-releasing peptide receptor (GRP-R) exhibit feeding abnormalities. However, it is unclear how these receptors are associated with feeding regulation. In BRS-3-deficient mice, we found hyperphagia, subsequent hyperleptinemia, and brain leptin resistance that occurred after the onset of obesity. To explore the cause of this phenomenon, we examined changes in feeding responses to appetite-related neuropeptides in BRS-3-deficient, GRP-R-deficient, and wild-type littermate mice. Among orexigenic neuropeptides, the hyperphagic response to melanin-concentrating hormone (MCH) was significantly enhanced in BRS-3-deficient mice but not in GRP-R-deficient mice. In addition, the levels of MCH-R and prepro-MCH mRNAs in the hypothalamus of BRS-3-deficient mice were significantly more elevated than those of wild-type littermates. There was no significant difference in feeding between BRS-3-deficient and wild-type littermate mice after treatment with bombesin (BN), although the hypophagic response to low-dose BN was significantly suppressed in the GRP-R-deficient mice. These results suggest that upregulation of MCH-R and MCH triggers hyperphagia in BRS-3-deficient mice. From these results, we assume that the BRS-3 gene deletion upsets the mechanism by which leptin decreases the expression of MCH-R and that this effect may be mediated through neural networks independent of BN-related peptides such as GRP-R.

Prohormone convertases (PCs) are thought to represent the major proteinases involved in the biosynthetic processing of peptide hormone precursors to bioactive peptide products. The maturation of PC2 requires the aid of a helper protein, 7B2, in order for the zymogen to become an active enzyme species. The 7B2 and PC2 nulls should thus be functionally equivalent with regard to deficits in precursor processing. In this article, we have examined this proposition through the study of proopiomelanocortin (POMC) biosynthesis and granule content in both null models. RIA data indicate that both PC2 and 7B2 nulls lack pituitary alpha-MSH; interestingly, 7B2 nulls are still able to generate beta-endorphin from beta-lipotropin, whereas PC2 nulls contain little if any beta-endorphin. Labeling experiments demonstrate a build-up of POMC, high molecular weight intermediates, and intact ACTH, as well as the disappearance of alpha-MSH, in both null models. Electron microscopy of neurointermediate lobe melanotrophs reveals the presence of a significantly greater number of secretory granules in both 7B2 and PC2 nulls compared with wild-type controls. However, PC2 null melanotrophs contain twice as many granules as 7B2 null melanotrophs. Another difference between the two null models is a relatively enhanced accumulation of precursors in the PC2 null compared with the 7B2 null; these include not only PC2 substrates, but also presumed PC1 substrates. These data indicate that the two nulls are not phenotypically equivalent.

Recent studies have revealed the importance of fish-derived peptide hormones to human endocrinology. These peptides include melanin-concentrating hormone (MCH), urocortins (human urotensin-I), and urotensin-II. MCH, a hypothalamic peptide, is a potent stimulator on appetite. Urocortins, e.g. urocortin 1 and urocortin 3 (stresscopin), are endogenous ligands for the corticotropin-releasing factor (CRF) receptors, particularly CRF type 2 receptor, that mediates a vasodilator action, a positive inotropic action and a central appetite-inhibiting action. These actions mediated by CRF type 2 receptor may ameliorate the stress response. Human urotensin-II is a potent vasoconstrictor peptide, while it acts as a vasodilator on some arteries. Human urotensin-II is expressed in various types of cells and tissues, including cardiovascular tissues, as well as many types of tumor cells. Thus, these fish-derived peptides appear to play important roles in human physiology, such as appetite regulation, stress response and cardiovascular regulation, and also in diseases, for example, obesity, cardiovascular diseases and tumors. Development of antagonists/agonists against the receptors for these peptides may open new strategies for the treatment of various diseases, including obesity-related diseases, hypertension, heart failure and malignant tumors.

The worldwide prevalence of obesity is increasing at an alarming rate, with major adverse consequences for human health. This "obesity epidemic" is paralleled by a rapid and substantive increase in our understanding of molecular pathways and physiologic systems underlying the regulation of energy balance. While efforts to address the environmental factors that are responsible for the recent "epidemic" must continue, new molecular and physiologic insights into this system offer exciting possibilities for future development of successful therapies.

Short-term cold exposure of homeothermic animals leads to higher thermogenesis and food consumption accompanied by weight loss. An analysis of cDNA-macroarray was employed to identify candidate mRNA species that encode proteins involved in thermogenic adaptation to cold. A cDNA-macroarray analysis, confirmed by RT-PCR, immunoblot, and RIA, revealed that the hypothalamic expression of melanin-concentrating hormone (MCH) is enhanced by exposure of rats to cold environment. The blockade of hypothalamic MCH expression by antisense MCH oligonucleotide in cold-exposed rats promoted no changes in feeding behavior and body temperature. However, MCH blockade led to a significant drop in body weight, which was accompanied by decreased liver glycogen, increased relative body fat, increased absolute and relative interscapular brown adipose tissue mass, increased uncoupling protein 1 expression in brown adipose tissue, and increased consumption of lean body mass. Thus, increased hypothalamic MCH expression in rats exposed to cold may participate in the process that allows for efficient use of energy for heat production during thermogenic adaptation to cold.

Energy homeostasis is regulated by a complex network involving peripheral and central signals that determine food intake and energy expenditure. Melanin-concentrating hormone (MCH) plays an essential role in this process. Animals treated with MCH develop hyperphagia and obesity. Ablation of the prepro-MCH gene leads to a lean phenotype, as does ablation of the rodent MCH receptor, MCHR-1. MCH is overexpressed in the leptin-deficient ob/ob mouse, and we hypothesized that ablation of MCH in this animal would lead to attenuation of its obese phenotype. Compared with ob/ob animals, mice lacking both leptin and MCH (double null) had a dramatic reduction in body fat. Surprisingly, the hyperphagia of the ob/ob mouse was unaffected. Instead, leanness was secondary to a marked increase in energy expenditure resulting from both increased resting energy expenditure and locomotor activity. Furthermore, double-null mice showed improvements in other parameters impaired in ob/ob mice. Compared with ob/ob mice, double-null animals had increased basal body temperature, improved response to cold exposure, lower plasma glucocorticoid levels, improved glucose tolerance, and reduced expression of stearoyl-CoA desaturase 1 (SCD-1). These results highlight the importance of MCH in integration of energy homeostasis downstream of leptin and, in particular, the role of MCH in regulation of energy expenditure.

Peroxisome proliferator-activated receptor (PPAR) alpha is a nuclear receptor that is mainly expressed in tissues with a high degree of fatty acid oxidation such as liver, heart, and skeletal muscle. Unsaturated fatty acids, their derivatives, and fibrates activate PPARalpha. Male rats are more responsive to fibrates than female rats. We therefore wanted to investigate if there is a sex difference in PPARalpha expression. Male rats had higher levels of hepatic PPARalpha mRNA and protein than female rats. Fasting increased hepatic PPARalpha mRNA levels to a similar degree in both sexes. Gonadectomy of male rats decreased PPARalpha mRNA expression to similar levels as in intact and gonadectomized female rats. Hypophysectomy increased hepatic PPARalpha mRNA and protein levels. The increase in PPARalpha mRNA after hypophysectomy was more pronounced in females than in males. GH treatment decreased PPARalpha mRNA and protein levels, but the sex-differentiated secretory pattern of GH does not determine the sex-differentiated expression of PPARalpha. The expression of PPARalpha mRNA in heart or soleus muscle was not influenced by gender, gonadectomy, hypophysectomy, or GH treatment. In summary, pituitary-dependent hormones specifically regulate hepatic PPARalpha expression. Sex hormones regulate the sex difference in hepatic PPARalpha levels, but not via the sexually dimorphic GH secretory pattern.

We have studied male sexual differentiation of null mutant mice (-/-) for the thyroid-specific enhancer-binding protein (T/ebp or Nkx2.1) gene, a homeodomain transcription factor that plays a role in organogenesis of the thyroid, lung, ventral forebrain, and pituitary gland. Because the T/ebp/Nkx2.1 (-/-) mice do not develop the pituitary gland, their sexual differentiation, if any, must occur in the absence of action of gonadotropins and other pituitary hormones. The (-/-) mice survive only until birth (embryonic d 19-19.5 of pregnancy), and when their external and internal genitals were inspected at embryonic d 18.5, they were indistinguishable from the (+/-) and (+/+) control mice. The testis weights of (-/-) mice were 20% lower than in (+/+) and (+/-) mice. The testosterone content of the (-/-) testes (13.5 +/- 2.4 pg/gonad, mean +/- SEM, n = 11) was dramatically reduced, compared with (+/-) (165 +/- 22.5 pg, n = 14) and (+/+) (234 +/- 37.3 pg, n = 10) littermates. Light microscopy revealed no difference in seminiferous tubules, interstitial tissue, or relative proportions of the two-cell compartments between the (-/-) and (+/+) testes. However, electron microscopy confirmed that Leydig cells in the (-/-) testes were much smaller, with smaller mitochondria and proportion of smooth endoplasmic reticulum than found in the controls, which was in support of the low androgen content of the knockout testes. In conclusion, this study on T/ebp/Nkx2.1 knockout mice, devoid of the pituitary gland, demonstrates that pituitary hormone secretion is not needed for stimulation of sufficient fetal testicular androgen synthesis to induce male sexual differentiation. The endogenous testosterone level in the null mutant testes is 5-10% of the control level, which suggests that there is a considerable safety margin in the amount of testosterone that is needed for the male fetal masculinization.

Hepatic alcohol dehydrogenase (ADH) activity is higher in female than in male rats. Although sex steroids, thyroid, and growth hormone (GH) have been shown to regulate hepatic ADH, the mechanism(s) for sexual dimorphic expression is unclear. We tested the possibility that the GH secretory pattern determined differential expression of ADH. Gonadectomized and hypophysectomized male and female rats were examined. Hepatic ADH activity was 2.1-fold greater in females. Because protein and mRNA content were also 1.7- and 2.4-fold greater, results indicated that activity differences were due to pretranslational mechanisms. Estradiol increased ADH selectively in males, and testosterone selectively decreased activity and mRNA levels in females. Effect of sex steroids on ADH was lost after hypophysectomy; infusion of GH in males increased ADH to basal female levels, supporting a role of the pituitary-liver axis. However, GH and L-thyroxine (T4) replacements alone in hypophysectomized rats did not restore dimorphic differences for either ADH activity or mRNA levels. On the other hand, T4 in combination with intermittent administration of GH reduced ADH activity and mRNA to basal male values, whereas T4 plus GH infusion replicated female levels. These results indicate that the intermittent male pattern of GH secretion combined with T4 is the principal determinant of low ADH activity in male liver.

The hypothalamic neuropeptide melanin-concentrating hormone (MCH) has been implicated in a variety of physiological functions including the regulation of feeding and energy homeostasis. Two MCH receptors (MCHR1 and MCHR2) have been identified so far. To decipher the functional role of the MCH receptors, we have generated and phenotypically characterized mice rendered deficient in MCHR1 expression by homologous recombination. Inactivation of MCHR1 results in mice (MCHR1-/-) that are resistant to diet-induced obesity. With a high-fat diet, body fat mass is significantly lower in both male (4.7 +/- 0.6 g vs. 9.6 +/- 1.2 g) and female (3.9 +/- 0.2 vs. 5.8 +/- 0.5 g) MCHR1-/- mice than that of the wild-type control (P < 0.01), but the lean mass remains constant. When normalized to body weight, female mice are hyperphagic, and male mice are hyperphagic and hypermetabolic, compared with wild-type mice. Consistent with the lower fat mass, both leptin and insulin levels are significantly lower in male MCHR1-/- mice than in the wild-type controls. Our data firmly establish MCHR1 as a mediator of MCH effects on energy homeostasis and suggest that inactivation of MCHR1 alone is capable to counterbalance obesity induced by a high-fat diet.

The prohormone convertase PC2 requires the aid of a helper protein, known as 7B2, for production of active enzyme. Deletion of 7B2 results in a lethal phenotype resembling Cushing's disease. In this study, we have investigated the effect of a single low dose of recombinant adenovirus vector encoding 7B2 and delivered directly to the pituitary of 7B2 nulls on pituitary ACTH, plasma ACTH, corticosterone, alpha MSH and glucose, and survival time. We show that after injection of recombinant adenovirus encoding 27-kDa 7B2 into 7B2 nulls, transgene expression, as measured by RIA for 7B2, exhibits a transient elevation in the pituitary and blood, with a slight but significant elevation of PC2 activity in pituitaries of 7B2 nulls and a drop in the level of circulating ACTH concomitant with a small increase in circulating alpha MSH. The level of circulating blood glucose was increased, and that of corticosterone was decreased. Lastly, slight but significantly prolonged survival times were observed. These data showing partial rescue of 7B2 nulls support the idea that adenoviral administration of 7B2 will represent an effective means to study the role of this interesting neuroendocrine protein on endocrine function in vivo.

The neuroendocrine-specific protein 7B2, which serves as a molecular escort for proPC2 in the secretory pathway, promotes the production of enzymatically active PC2 and may have non-PC2 related endocrine roles. Mice null for 7B2 exhibit a lethal phenotype with a complex Cushing's-like pathology, which develops from intermediate lobe ACTH hypersecretion as a consequences of interruption of PC2-mediated peptide processing as well as undefined consequences of the loss of 7B2. In this study we investigated the endocrine and metabolic alterations of 7B2 null mice from pathological and biochemical points of view. Our results show that 7B2 nulls exhibit a multisystem disorder that includes severe pathoanatomical and histopathologic alterations of vital organs, including the heart and spleen but most notably the liver, in which massive steatosis and necrosis are observed. Metabolic derangements in glucose metabolism result in glycogen and fat deposition in liver under conditions of chronic hypoglycemia. Liver failure is also likely to contribute to abnormalities in blood coagulation and blood chemistry, such as lactic acidosis. A hypoglycemic crisis coupled with respiratory distress and intensive internal thrombosis most likely results in rapid deterioration and death of the 7B2 null.

The serine protease prohormone convertase 2 (PC2), principally involved in the processing of polypeptide hormone precursors in neuroendocrine tissues, requires interaction with the neuroendocrine protein 7B2 to generate an enzymatically active form. 7B2 null mice express no PC2 activity and release large quantities of uncleaved ACTH, resulting in a lethal endocrine condition that resembles pituitary Cushing's (Westphal, C. H., Muller, L., Zhou, A., Bonner-Weir, S., Schambelan, M., Steiner, D. F., Lindberg, I. & Leder, P. (1999) Cell 96, 689). Here, we have compared the 7B2 and PC2 null mouse models to determine why the 7B2 null, but not the PC2 null, exhibits a lethal disease state. Both 7B2 and PC2 nulls contained highly elevated pituitary adrenocorticotropic hormone (ACTH); the neurointermediate lobe content of ACTH in 7B2 nulls was 13-fold higher than in WT mice; that of the PC2 null was 65-fold higher. However, circulating ACTH levels were much higher in the 7B2 null than in the PC2 null. Because hypothalamic inhibitory dopaminergic control represents the major influence on intermediate lobe proopiomelanocortin-derived peptide secretion, dopamine levels were measured, and they revealed that 7B2 null pituitaries contained only one-fourth of WT pituitary dopamine. Adrenalectomized 7B2 null animals survived past the usual time of death at 5 weeks; a month after adrenalectomy, they exhibited normal levels of pituitary dopamine, circulating ACTH, and corticosterone. Elevated corticosterone, therefore, seems to play a central role in the lethal phenotype of the 7B2 null, whereas a 7B2-mediated dopaminergic deficiency state may be involved in the actual ACTH hypersecretion phenomenon. Interestingly, adrenalectomized 7B2 nulls also developed unexpectedly severe obesity.