Melanin-concentrating Hormone mRNA Research Articles

Various Dietary Fats Differentially Change the Gene Expression of Neuropeptides Involved in Body Weight Regulation in Rats

Various high-fat diets are obesogenic but not to the same extent. The aim of the present study was to investigate the effects of saturated fat n-6 and n-3 polyunsaturated fatty acids (PUFAs) on the central neuropeptidergic system in adult rats. Using reverse transcriptase-polymerase chain reaction and in situ hybridisation, we evaluated the net effect of feeding in these fats, comparing the effects of a high- to low-fat diet, and the diversity of the effects of these fats in the same amount within the diet. We also determined plasma lipids, glucose, insulin and leptin concentrations. Six-week feeding with high-saturated fat evoked hyperpahagia and the largest weight gain compared to both high-PUFA diets. Rats fed high-saturated fat were found to have decreased neuropeptide Y (NPY) mRNA expression in the arcuate nucleus (ARC) and the compact zone of the dorsomedial nucleus (DMHc), unchanged pro-opiomelanocortin (POMC), galanin-like peptide (GALP) mRNA expression in the ARC, as well as melanin-concentrating hormone (MCH) and prepro-orexin (preORX) mRNA expression in the lateral hypothalamus, compared to low-saturated fed rats. By contrast, feeding with both high-PUFA diets increased POMC and GALP mRNA expression in the ARC compared to the corresponding low-fat diet and the high-saturated fat diet. Furthermore, feeding with both low-PUFA diets reduced NPY mRNA expression compared to the low-saturated fat diet exclusively in the DMHc. Uniquely, the high n-3 PUFA feeding halved MCH and preORX mRNA expression in the lateral hypothalamus compared to the other high-fat and low n-3 PUFA diets. In rats fed three high-fat diets, plasma insulin and leptin concentrations were significantly increased and the type of fat had no effect on these hormone levels. Rats fed high-saturated fat had both hyperglycaemia and hypertriacylglycerolemia and rats fed high n-3 PUFA only had hyperglycaemia. The present study demonstrates that various forms of dietary fat differentially change the expression of neuropeptide genes involved in energy homeostasis.

Intracerebroventricular melanin-concentrating hormone stimulates food intake in sheep

Melanin-concentrating hormone (MCH) stimulates feeding when injected intracerebroventricularly (ICV) in rats. At present it is not clear whether the function of MCH is similar in ruminants, which are species with a continuous delivery of nutrients. Therefore the current investigation sought to determine the role of MCH in sheep. In the first experiment, six, castrate male sheep were satiated and received one of four treatments [saline, 0.1, or 1.0 nmol/kg MCH, and NPY (0.1 nmol/kg)] injected ICV over 30 s, then infused ICV for 6 h (∼500 μl/h). Food intake was measured for 2 h before and at 2, 4, 6, 8, 12 and 24 h. In this experiment, feed intake was increased ( P ≤ 0.05) in NPY treated sheep only. In the second experiment, the same sheep were fed to satiety and then randomized to receive one of six treatments [saline and either 0.1, 1.0 or 5.0 nmol/kg MCH, 0.1 nmol/kg NPY, or MCH + NPY (0.1 nmol/kg)] injected ICV over 30 s. Food intake was measured for 2 h before and at 2, 4, 6, 8, 12, and 24 h after ICV injection. All doses of MCH as well as NPY resulted in greater ( P ≤ 0.05) food intake than saline. In order to determine whether MCH expression was regulated by fasting, brains from fed and 3-day fasted sheep were fixed in situ, sectioned in the coronal plane, and subjected to dual-label immunohistochemistry using Fos as a marker for neuronal activity. Nutritional state (fed or fasted) did not alter Fos expression in MCH neurons. Finally, using real time PCR, MCH mRNA was unchanged by fasting. In this study we found bolus ICV MCH to be a potent stimulus to food intake in sheep, but MCH was not regulated by fasting.

Increases in melanin-concentrating hormone and MCH receptor levels in the hypothalamus of dietary-obese rats

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that stimulates feeding and increases body weight in rodents. We studied the role of the system in energy homeostasis and its regulation by the satiety signals, leptin and insulin. We used real-time PCR to measure the hypothalamic expression of MCH and its receptor (MCHR1) in two contrasting models of altered nutritional status, namely, obesity induced by 8 weeks' voluntary overeating and food restriction for 10 days. Diet-fed rats were stratified according to final total fat-pad mass into a ‘high fat gain’ group (HG) and ‘low fat gain' group (LG). MCH mRNA levels were increased by 31% ( p>0.05) and 49% ( p<0.05) in the LG and HG, respectively, compared with controls. MCHR1 mRNA levels rose by 118% in the LG ( p<0.01) and 85% in the HG ( p<0.01). There were significant positive correlations ( p<0.05) between plasma leptin concentration and both MCH and MCHR1 mRNA levels, and between plasma insulin and MCHR1 expression. A positive correlation was also observed between MCH and MCHR1 mRNA levels ( p<0.05). Food-restricted rats showed no significant alterations in the levels of either MCH mRNA or MCHR1 mRNA. In a second experiment, we measured MCH peptide levels in five discrete hypothalamic areas of dietary-obese rats. MCH concentrations were significantly increased in the arcuate nuclei of the HG ( p<0.05) and the paraventricular nuclei of both the LG ( p<0.05) and HG ( p<0.05), compared with their lean counterparts. These results suggest that the MCH system becomes more active in dietary obesity and could be involved in enhancing appetite for palatable food. The possibility that MCH and MCHR1 expression are positively regulated by leptin and insulin, which normally inhibit feeding, is a putative explanation for how appetite for palatable food is able to override mechanisms that prevent the development of obesity.

Comparison of hypocretin/orexin and melanin-concentrating hormone neurons and axonal projections in the embryonic and postnatal rat brain

Hypocretin/orexin (H/O) and melanin-concentrating hormone (MCH) are peptide neuromodulators found in separate populations of neurons located within the lateral and perifornical hypothalamic regions. H/O has been linked to sleep-wakefulness regulation and to the sleep disorder narcolepsy, and both systems have been implicated in energy homeostasis, including the regulation of food intake. In the present study we compared the development of H/O and MCH-expressing neuronal populations with in situ hybridization and immunohistochemistry on adjacent sections in the embryonic and postnatal rat brain. We found that MCH mRNA and protein were present in developing neurons of the hypothalamus by embryonic day 16 (E16), whereas H/O mRNA and protein were not detected until E18. We also identified previously undescribed populations of MCH-immunoreactive cells in the lateral septum, paraventricular hypothalamic nucleus, lateral zona incerta, and ventral lateral geniculate nucleus that may play a specific role in the development of these regions. MCH immunoreactive axonal processes were also evident earlier than H/O stained fibers and at the time H/O immunoreactive processes were first identified in the hypothalamus at E20, extensive MCH axonal fiber systems were already present in many brain regions. Interestingly, however, the density of axonal fibers immunoreactive for H/O in the locus coeruleus reached peak levels at the same developmental age (P21) as MCH immunoreactive axons in the diagonal band of Broca (DBB). The peak of axon density coincided with the developmental stage at which adult patterns of feeding and sleep–waking activity become established. The present results demonstrate developmental differences and similarities between the MCH and H/O systems that may relate to their respective roles in feeding and sleep regulation.

Single-cell RT-PCR gene expression profiling of acutely dissociated and immunocytochemically identified central neurons

Identification of neurons for single-cell mRNA profiling is difficult when cells of interest are located in heterogeneous brain regions. We developed a protocol in which acutely dissociated neurons are immunocytochemically labeled prior to single-cell reverse transcription-polymerase chain reaction (RT-PCR). We tested the protocol on hypothalamic melanin-concentrating hormone (MCH) and prepro-orexin (PPO) neurons, which are similarly distributed but functionally different. Cells dissociated from the perifornical region of the posterior hypothalamus of juvenile or adult rats were incubated with anti-MCH or anti-PPO primary antibodies, followed by washout and incubation with fluorescein-tagged secondary antibodies. Individual labeled cells were subjected to RT-PCR with primers for PPO and MCH. MCH mRNA was detected in 26 out of the 38 successfully reverse-transcribed cells identified as MCH-containing, and 28 cells out of the 42 identified as PPO-containing expressed PPO mRNA. No cell expressed both mRNAs. Most MCH neurons tested (five out of six) expressed the adrenergic α 2A receptor mRNA, whereas it was absent from all seven PPO neurons tested. Neither PPO ( n=11) nor MCH ( n=6) cells expressed the type 2 orexin receptor mRNA. Thus, the method allows, with at least 66% confidence, immunocytochemical cell identification prior to mRNA studies of single neurons located in heterogeneous brain regions.

Mice lacking pro-opiomelanocortin are sensitive to high-fat feeding but respond normally to the acute anorectic effects of peptide-YY(3-36).

Inactivating mutations of the pro-opiomelanocortin (POMC) gene in both mice and humans leads to hyperphagia and obesity. To further examine the mechanisms whereby POMC-deficiency leads to disordered energy homeostasis, we have generated mice lacking all POMC-derived peptides. Consistent with a previously reported model, Pomc(-/-) mice were obese and hyperphagic. They also showed reduced resting oxygen consumption associated with lowered serum levels of thyroxine. Hypothalami from Pomc(-/-) mice showed markedly increased expression of melanin-concentrating hormone mRNA in the lateral hypothalamus, but expression of neuropeptide Y mRNA in the arcuate nucleus was not altered. Provision of a 45% fat diet increased energy intake and body weight in both Pomc(-/-) and Pomc(+/-) mice. The effects of leptin on food intake and body weight were blunted in obese Pomc(-/-) mice whereas nonobese Pomc(-/-) mice were sensitive to leptin. Surprisingly, we found that Pomc(-/-) mice maintained their acute anorectic response to peptide-YY(3-36) (PYY(3-36)). However, 7 days of PYY(3-36) administration had no effect on cumulative food intake or body weight in wild-type or Pomc(-/-) mice. Thus, POMC peptides seem to be necessary for the normal response of energy balance to high-fat feeding, but not for the acute anorectic effect of PYY(3-36) or full effects of leptin on feeding. The finding that the loss of only one copy of the Pomc gene is sufficient to render mice susceptible to the effects of high fat feeding emphasizes the potential importance of this locus as a site for gene-environment interactions predisposing to obesity.

Hypothalamic Neuropeptide Y/Y1 Receptor Pathway Activated by a Reduction in Circulating Leptin, but Not by an Increase in Circulating Ghrelin, Contributes to Hyperphagia Associated with Triiodothyronine-Induced Thyrotoxicosis

Food intake is regulated by hypothalamic neuropeptides which respond to peripheral signals. Plasma ghrelin and leptin levels reflect peripheral energy balance and regulate hypothalamic neuropeptides such as neuropeptide Y (NPY), pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and orexins. Thyroid hormone stimulates food intake in humans and rodents. However, the mechanisms responsible for this stimulation have not been fully elucidated. To investigate the hyperphagic response to triiodothyronine (T₃)-induced thyrotoxicosis, adult male rats were studied 7 days after daily intraperitoneal injections of T₃ or vehicle. T₃-treated rats were markedly hyperphagic. During this hyperphagia, plasma leptin levels were markedly decreased. However, the expression of the ghrelin gene in the stomach and the plasma ghrelin concentrations did not differ between the 2 groups. Hypothalamic NPY mRNA levels were significantly increased and associated with a marked decreased in both hypothalamic POMC and CART mRNA levels in the T₃-treated rats. Hypothalamic MCH and orexin mRNA levels did not differ between the 2 groups. In addition, hyperphagia was partially reversed by intracerebroventricular administration of the NPY Y1 receptor antagonist BIBO3304. Therefore, the decreased plasma leptin levels could contribute to hyperphagia in T₃-induced thyrotoxicosis. However, plasma ghrelin levels did not contribute to this hyperphagia.

Diencephalic neurons producing melanin-concentrating hormone are influenced by local and multiple extra-hypothalamic tachykininergic projections through the neurokinin 3 receptor

As melanin-concentrating hormone (MCH) neurons express the neurokinin 3 receptor (NK3) in the rat diencephalon, their innervation by tachykininergic fibers, the origin of this innervation and the effect of a NK3 agonist on MCH mRNA expression were researched. The obtained results show that the tachykininergic system develops complex relationships with MCH neurons. Overall, MCH cell bodies appeared targeted by both NKB- and SP-inputs. These afferents have multiple hypothalamic and extra-hypothalamic origins, but a local (intra-lateral hypothalamic area) origin from small interneurons was suspected as well. MCH cell bodies do not express NK1, but around 2.7% of the MCH neurons contained SP after colchicine injection. Senktide, a NK3 agonist, produced an increase of the MCH mRNA expression in cultured hypothalamic slices. This effect was reversed by two NK3 antagonists. Tachykinins enhance MCH mRNA expression, and, thus, may modulate the effect of MCH in functions such as feeding and reproductive behaviors in which this peptide has been experimentally involved.

Orexin/hypocretin neurons: chemical phenotype and possible interactions with melanin-concentrating hormone neurons

We showed earlier that a specific neuron population of the rat lateral hypothalamus, differing from the codistributed melanin-concentrating hormone (MCH) neurons, express both dynorphin (DYN) and secretogranin II (SgII) genes. We demonstrated later that this population corresponds in fact to the newly identified orexin/hypocretin (OX/Hcrt) neurons. In the present study, by revisiting the chemical phenotype of these neurons, we confirm that all of them contain DYN B- and SgII-immunoreactive materials. The roles played by these peptide/protein in OX/Hcrt neurons are still unclear. Double immunocytochemical stainings highlight putative somasomatic, axosomatic and axodendritic contacts between OX/Hcrt and MCH neurons. Adding OX/Hcrt to the culture medium of hypothalamic slices from 8-day-old rats results either in a significant increase of MCH mRNA after 24 h survival or a strong fall after 10 days culture. These results taken together suggest that OX/Hcrt can directly and/or indirectly affect MCH expression, and that both OX/Hcrt and MCH neuron populations interact to respond in a coordinated manner to central and peripheral signals.

Central and peripheral dysregulation of melanin-concentrating hormone in obese Zucker rats

Melanin concentrating hormone (MCH) is a peptide synthesized in the lateral hypothalamus which stimulates food ingestion and leptin secretion in rodents. In this experiment, we measured the expressions of MCH as well as of its receptor (SLC-1) in the hypothalamus of obese hyperphagic and lean Zucker rats by quantitative real time RT-PCR. MCH mRNA expression in the obese rats was significantly increased by a factor of five ( P<0.01) whereas expression of SLC-1 was decreased by more than 50% ( P<0.05). Circulating levels of leptin and MCH were increased in the plasma of obese Zucker rats when compared to lean rats (38-fold and 1.7-fold, respectively, P<0.001 and P<0.01). However, individual MCH levels were not directly correlated to leptin levels in the lean (functional leptin receptor) or in the obese (non-functional leptin receptor) Zucker rats. These results indicate that the absence of leptin signaling in rats is associated with an increased hypothalamic expression and circulating release of MCH, contributing to their obesity syndrome.

Characterization of melanin concentrating hormone and preproorexin expression in the murine hypothalamus

Melanin concentrating hormone (MCH) and the orexins (A and B) have been identified as neuropeptides localized to the lateral hypothalamic area (LHA) and are potential regulators of energy homeostasis. Potential factors regulating expression of both MCH and the orexins include fasting and leptin. Previous studies have generated conflicting data and, as there is little leptin receptor expressed in the lateral hypothalamus, it is likely that any observed leptin effects on these peptides are indirect. In this study, we examined MCH and preproorexin expression in mice in physiological states of starvation, with or without leptin administration, in addition to characterizing MCH and preproorexin expression in well-known obesity models, including ob/ ob and UCP-DTA mice. Neuropeptide Y (NPY) expression in the arcuate nucleus was used as a positive control. After a 60-h fast, expression of both NPY and MCH mRNA was increased (by 148 and 33%, respectively) while preproorexin expression in the murine LHA did not change. Leptin administration to fasted mice blunted the rise in MCH and NPY expression towards control levels. In contrast, there was a 78% increase in preproorexin expression in fasted mice in response to peripheral leptin administration. MCH expression was increased (by 116%) in ob/ ob mice at baseline, as we have previously reported. In addition, leptin treatment of ob/ ob mice blunted the increase in MCH expression. In contrast, preproorexin expression did not differ in the leptin-deficient ob/ ob mice or in the obese hyperleptinemic brown adipose tissue deficient (UCP-DTA) mice in comparison with controls. In summary, MCH expression is increased in two states of decreased leptin, fasting and ob/ ob mice, and leptin replacement blunts MCH expression in both paradigms. Thus, MCH expression appears to be regulated by leptin. In contrast, preproorexin expression does not respond acutely to fasting, although it is acutely increased by leptin treatment during fasting. These preproorexin responses are in contrast to those seen with well-characterized orexigenic neuropeptides, such as NPY and AgRP, suggesting that appetite regulation may not be a significant physiological role of orexins. This conclusion is further supported by the observation that orexin ablated mice have arousal and not feeding deficits.

Differential neuronal expression and projections of melanin-concentrating hormone (MCH) and MCH-gene-overprinted-polypeptide (MGOP) in the rat brain.

The rat melanin-concentrating hormone (MCH) gene may produce, through alternative splicing, either the precursor of MCH and neuropeptide EI, two neuropeptides coexpressed in the zona incerta (ZI) and lateral hypothalamus (LHA), or a putative protein we named previously MCH-gene-overprinted-polypeptide (MGOP). First, we investigated the distribution and relative expression of MCH and MGOP mRNA in the rat brain by Northern blotting, RT-PCR and in situ hybridization. MGOP gene transcripts were detected mainly in the hypothalamus only by RT-PCR. Second, different antisera were raised toward the C-terminus of MGOP and used to identify the translational products. In the rat brain, no MGOP-processed peptide could be detected based on RP-HPLC coupled to specific RIA. A polypeptide of 14 kDa was found in the secretory pathway of transfected monkey COS7 cells expressing recombinant MGOP. In the rat hypothalamus, a specific protein of 12 kDa was identified by Western blot analysis. Finally, distribution of MGOP-immunoreactivity (IR) was investigated in the rat brain. Colocalization studies demonstrated that 98% of the MGOP-expressing perikarya in ZI/LHA also synthesized MCH. In addition, numerous, strongly stained MGOP-containing neurons were encountered in the hypothalamic periventricular nucleus. Perikarya labelled with MGOP antiserum were also found scattered in the cortex, caudate putamen, amygdala and lateral septal nucleus. MCH was not detected in these MGOP-containing neurons. Strikingly, dense staining of terminals was observed with MGOP antiserum but not with MCH antibodies in the suprachiasmatic, ventromedial and arcuate nuclei, and also in the external layer of the median eminence. These results demonstrated that MGOP and MCH-IR overlapped in LHA/ZI but displayed a differential distribution in other areas. Based on this cerebral distribution, MGOP may act as a new secreted protein in regulating many neuroendocrine functions, such as nursing, feeding and growth control in associated behavioural components.

Hypothalamic Melanin-Concentrating Hormone and Estrogen-Induced Weight Loss

Melanin-concentrating hormone (MCH) is an orexigenic neuropeptide produced by neurons of the lateral hypothalamic area (LHA). Because genetic MCH deficiency induces hypophagia and loss of body fat, we hypothesized that MCH neurons may represent a specific LHA pathway that, when inhibited, contributes to the pathogenesis of certain anorexia syndromes. To test this hypothesis, we measured behavioral, hormonal, and hypothalamic neuropeptide responses in two models of hyperestrogenemia in male rats, a highly reproducible anorexia paradigm. Whereas estrogen-induced weight loss engaged multiple systems that normally favor recovery of lost weight, the expected increase of MCH mRNA expression induced by energy restriction was selectively and completely abolished. These findings identify MCH neurons as specific targets of estrogen action and suggest that inhibition of these neurons may contribute to the hypophagic effect of estrogen.

Differential Regulation of Melanin-Concentrating Hormone and Orexin Genes in the Agouti-Related Protein/Melanocortin-4 Receptor System

Agouti protein and agouti-related protein (AGRP) antagonize α-melanocyte-stimulating hormone that binds to and activates the melanocortin-4 receptor (MC4-R) in the hypothalamus, thereby stimulating food intake. Melanin-concentrating hormone (MCH) and orexin are orexigenic peptides that specifically are synthesized in the lateral hypothalamus. MCH gene expression was augmented in Ay/a (agouti) mice which overexpress agouti protein, but orexin mRNA was not. AGRP administered intracerebroventricularly into wild-type rats augmented MCH but not orexin gene expression. Also, SHU9119, a peptidergic antagonist of MC4-R, increased only MCH mRNA. These findings indicate that interruption of signaling at MC4-R activates the MCH but not the orexin gene. The biosyntheses of MCH and orexin are regulated through different pathways.

Effect of 2-mercaptoacetate and 2-deoxy-D-glucose administration on the expression of NPY, AGRP, POMC, MCH and hypocretin/orexin in the rat hypothalamus.

Using in situ hybridization, the mRNA levels encoding neuropeptide Y (NPY), agouti gene-related protein (AGRP), proopiomelanocortin (POMC), melanin-concentrating hormone (MCH) and hypocretin/orexin (HC/ORX) were investigated in the rat arcuate nucleus (Arc) and lateral hypothalamic area (LHA) 2 h after a single dose of the glucose antimetabolite 2-deoxy-D-glucose (2-DG; 600 mg/kg) or of the fatty acid oxidation inhibitor mercaptoacetate (MA; 600 mumol/kg). Two hours after 2-DG or MA injection food intake was significantly increased. NPY and AGRP mRNA levels in the Arc were increased by 2-DG but not affected by MA, and MCH mRNA levels in the LHA were increased by both antimetabolites. These results suggest that Arc neurons expressing NPY and AGRP are regulated by changes in glucose, but not fatty acid availability, whereas both factors affect MCH neurons in the LHA.

Survival of rat MCH (melanin-concentrating hormone) neurons in hypothalamus slice culture: histological, pharmacological and molecular studies.

Hypothalamic slices containing the lateral hypothalamic area (LHA) were prepared from 6- to 8-day-old rats and maintained in stationary culture for up to 35 days in order to analyse how well the melanin-concentrating hormone (MCH) neurons survived. As previously reported for other brain areas, this method yielded a long-term well-preserved organotypic organization. Light- and electron-microscopic investigations showed that differentiation continued and that synaptic contacts developed in vitro. After a period of elimination of damaged cells and fibres, most of the remaining neurons and glial cells retained a normal morphology throughout the culture period. MCH neurons, in particular, survived well as attested by the strong immunocytochemical and in situ hybridization signals still observed after several weeks. In a comparison with the day of explantation, competitive reverse transcription/polymerase chain reaction demonstrated the remarkable stability of the level of MCH mRNA at least until the 20th day in culture; after 30 days, the clear decrease in this level seemed to be correlated with a loss of MCH neurons, rather than with a decrease in MCH expression. After 10 days of culture, the incubation of slices in the presence of the hormone leptin (50 ng/ml) resulted in a strong decrease of MCH gene expression, suggesting that MCH neurons retained their physiological properties. Thus, the LHA slice stationary culture, especially between one and three weeks (i.e. after tissue stabilization and before extensive cell loss), appears to be a suitable method for physiological and pharmacological studies of these neurons.

17β-Estradiol regulation of melanin-concentrating hormone and neuropeptide-E-I contents in cynomolgus monkeys: a preliminary study

Melanin-concentrating hormone (MCH) and neuropeptide-E-I (NEI) regulate several behaviors and neuroendocrine functions in rats. Possible influence of these peptides on sexual behavior and reproduction in mammals other than rodents prompted us to investigate: 1) The sites of synthesis of MCH and NEI in the brain of a non-human primate ( M. fascicularis); 2) The effect of 17β-estradiol (E 2) benzoate (E 2B) on pro-MCH-derived peptide concentrations in the hypothalamus of the ovariectomized (OVX) cynomolgus monkeys ( M. fascicularis). Expression of MCH mRNA and peptides was examined by Northern blotting, RT-PCR and RP-HPLC/RIA. Our results demonstrate that the MCH gene is predominantly expressed in hypothalamus of macaque. E 2B exposure of OVX monkeys provoked parallel phasic variations in the MCH-immunoreactivity (IR) and NEI-IR. NEI-IR and to a lesser extent MCH-IR, showed a transient increase (associated with the estradiol peak) at 30 h with a final rise of both MCH-IR and NEI-IR observed at the time (72 h post E 2B) of the luteinizing hormone (LH) surge. RP-HPLC analysis of peptide extracts revealed the presence, in addition to mature MCH and NEI, of different MCH-IR and NEI-IR forms in the hypothalami of control and E 2B-treated monkeys. Taken together, our results indicated that hypothalamic MCH and NEI contents are regulated after E 2B treatment and they suggest the possible involvement of these peptides in the regulation of the pre-ovulatory midcycle LH surge in primates.

Melanin-concentrating hormone expression in slice cultures of rat hypothalamus is not affected by 2-deoxyglucose

In rats, melanin-concentrating hormone (MCH) neurons are mainly located within the lateral hypothalamic area (LHA). This area is known to be involved in the control of feeding and to contain glucose-sensitive cells. As a role for MCH in the regulation of food intake has been reported, we investigated the effects of 2-deoxyglucose (2DG) on MCH expression in cultured LHA slices, to verify if MCH neurons are sensitive to local glucoprivation through a modulation of MCH synthesis. After a 2–10 h 2DG incubation, competitive reverse transcription-polymerase chain reaction (RT-PCR) did not show any variation of MCH mRNA; no change was also observed in MCH immunocytochemical labeling. A slight decrease of MCH mRNA (5–15%) after a 17 h 2DG treatment might be due to a general degradation of neurons induced by long-term glucoprivation. In conclusion, we suggest that MCH neurons are not the glucose-sensitive cells previously described in the LHA and that the signals inducing their previously reported response to glycemia variations do not arise from the LHA itself.

Cellular Localization and Role of Prohormone Convertases in the Processing of Pro-melanin Concentrating Hormone in Mammals

Melanin concentrating hormone (MCH) and neuropeptide EI (NEI) are two peptides produced from the same precursor in mammals, by cleavage at the Arg145-Arg146 site and the Lys129-Arg130 site, respectively. We performed co-localization studies to reveal simultaneously the expression of MCH mRNA and proconvertases (PCs) such as PC1/3 or PC2. In the rat hypothalamus, PC2 was present in all MCH neurons, and PC1/3 was present in about 15-20% of these cells. PC1/3 or PC2 was not found in MCH-positive cells in the spleen. In GH4C1 cells co-infected with vaccinia virus (VV):pro-MCH along with VV:furin, PACE4, PC1/3, PC2, PC5/6A, PC5/6B, or PC7, we observed only efficient cleavage at the Arg145-Arg146 site to generate mature MCH. Co-expression of pro-MCH together with PC2 and 7B2 resulted in very weak processing to NEI. Comparison of pro-MCH processing patterns in PC1/3- or PC2-transfected PC12 cells showed that PC2 but not PC1/3 generated NEI. Finally, we analyzed the pattern of pro-MCH processing in PC2 null mice. In the brain of homozygotic mutants, the production of mature NEI was dramatically reduced. In contrast, MCH content was increased in the hypothalamus of PC2 null mice. In the spleen, a single large MCH-containing peptide was identified in both wild type and PC2 null mice. Together, our data suggest that pro-MCH is processed differently in the brain and in peripheral organs of mammals. PC2 is the key enzyme that produces NEI, whereas several PCs may cleave at the Arg145-Arg146 site to generate MCH in neuronal cell types.

Characterization of expression of hypothalamic appetite-regulating peptides in obese hyperleptinemic brown adipose tissue-deficient (uncoupling protein-promoter-driven diphtheria toxin A) mice.

Brown adipose tissue-deficient [uncoupling protein (UCP)-promoter-driven diphtheria toxin A (DTA)] mice develop obesity as a result of both decreased energy expenditure and hyperphagia. The hyperphagia occurs despite high serum leptin levels. Hence, this is a model of leptin-resistant obesity in which the mechanism driving hyperphagia is unknown. Leptin is a regulator of a number of hypothalamic neuropeptides involved in energy homeostasis. In ob/ob mice, leptin deficiency results in increased expression of neuropeptide Y (NPY), agouti-related protein (AGRP), and melanin-concentrating hormone (MCH), and decreased expression of POMC. We have previously shown that NPY is reduced in the UCP-DTA mouse, suggesting a normal NPY response to leptin. To define other potential sites of leptin resistance, we used in situ hybridization to evaluate the expression of messenger RNAs (mRNAs) encoding a number of peptides, including NPY, AGRP, MCH, and POMC. We confirmed that the decrease in NPY expression previously detected by Northern blots reflects a decrease in NPY expression in the arcuate nucleus. AGRP mRNA was also decreased, whereas POMC mRNA levels in the arcuate nucleus were the same as control. MCH mRNA levels in the lateral hypothalamic area were also decreased. In contrast, there was induction of NPY expression in the dorsomedial hypothalamic nucleus in the UCP-DTA animals but not in the controls. The results indicate that these neuropeptides generally respond to leptin and that the hyperphagia seen in the UCP-DTA mice is likely the result of dysregulated expression of other, as yet unexamined, hypothalamic peptides, or lies at sites distal to the hypothalamus.