Jinkui Shenqi pills ameliorate diabetes by regulating hypothalamic insulin resistance and POMC/AgRP expression and activity.

BACKGROUND: Research on the imbalance of proopiomelanocortin (POMC)/agouti-related protein (AgRP) neurons in the hypothalamus holds potential insights into the pathophysiology of diabetes. Jinkui Shenqi pills (JSP), a prevalent traditional Chinese medicine, regulate hypothalamic function and treat diabetes. PURPOSE: To investigate the hypoglycemic effect of JSP and explore the probable mechanism in treating diabetes. METHODS: A type 2 diabetes mouse model was used to investigate the pharmacodynamics of JSP. The glucose-lowering efficacy of JSP was assessed through various metrics including body weight, food consumption, fasting blood glucose (FBG), serum insulin levels, and an oral glucose tolerance test (OGTT). To elucidate the modulatory effects of JSP on hypothalamic mechanisms, we quantified the expression and activity of POMC and AgRP and assessed the insulin-mediated phosphoinositide 3-kinase (PI3K)/protein kinase A (AKT)/forkhead box O1 (FOXO1) pathway in diabetic mice via western blotting and immunohistochemistry. Additionally, primary hypothalamic neurons were exposed to high glucose and palmitic acid levels to induce insulin resistance, and the influence of JSP on POMC/AgRP protein expression and activation was evaluated by PI3K protein inhibition using western blotting and immunofluorescence. RESULTS: Medium- and high-dose JSP treatment effectively inhibited appetite, resulting in a steady declining trend in body weight, FBG, and OGTT results in diabetic mice (p < 0.05). These JSP groups also had significantly increased insulin levels (p < 0.05). Importantly, the medium-dose group exhibited notable protection of hypothalamic neuronal and synaptic structures, leading to augmentation of dendritic length and branching (p < 0.05). Furthermore, low-, medium-, and high-dose JSP groups exhibited increased phosphorylated (p) INSR, PI3K, pPI3K, AKT, and pAKT expression, as well as decreased FOXO1 and increased pFOXO1 expression, indicating improved hypothalamic insulin resistance in diabetic mice (p < 0.05). Treatment with 10% JSP-enriched serum produced a marked elevation of both expression and activation of POMC (p < 0.05), with a concurrent reduction in AgRP expression and activation within primary hypothalamic neurons (p < 0.05). Intriguingly, these effects could be attributed to the regulatory dynamics of PI3K activity. CONCLUSION: Our findings suggest that JSP can ameliorate diabetes by regulating POMC/AgRP expression and activity. The insulin-mediated PI3K/AKT/FOXO1 pathway plays an important regulatory role in this intricate process.

Nuclear receptor 5A2 regulation of Agrp underlies olanzapine-induced hyperphagia.

Antipsychotic (AP) drugs are efficacious treatments for various psychiatric disorders, but excessive weight gain and subsequent development of metabolic disease remain serious side effects of their use. Increased food intake leads to AP-induced weight gain, but the underlying molecular mechanisms remain unknown. In previous studies, we identified the neuropeptide Agrp and the transcription factor nuclear receptor subfamily 5 group A member 2 (Nr5a2) as significantly upregulated genes in the hypothalamus following AP-induced hyperphagia. While Agrp is expressed specifically in the arcuate nucleus of the hypothalamus and plays a critical role in appetite stimulation, Nr5a2 is expressed in both the CNS and periphery, but its role in food intake behaviors remains unknown. In this study, we investigated the role of hypothalamic Nr5a2 in AP-induced hyperphagia and weight gain. In hypothalamic cell lines, olanzapine treatment resulted in a dose-dependent increase in gene expression of Nr5a2 and Agrp. In mice, the pharmacological inhibition of NR5A2 decreased olanzapine-induced hyperphagia and weight gain, while the knockdown of Nr5a2 in the arcuate nucleus partially reversed olanzapine-induced hyperphagia. Chromatin-immunoprecipitation studies showed for the first time that NR5A2 directly binds to the Agrp promoter region. Lastly, the analysis of single-cell RNA seq data confirms that Nr5a2 and Agrp are co-expressed in a subset of neurons in the arcuate nucleus. In summary, we identify Nr5a2 as a key mechanistic driver of AP-induced food intake. These findings can inform future clinical development of APs that do not activate hyperphagia and weight gain.

Combination of chronic stress with fructose diet increases AMP-activated protein kinase phosphorylation and affects agouti-related protein and proopiomelanocortin expression in the hypothalamus of male Wistar rats.

Appetite regulation in the hypothalamus is dependent on hormonal signals from the periphery, such as insulin and leptin, and can be modulated by both sugar-rich diet and stress. Our aim was to explore the effects of 9-week feeding with 20% fructose solution combined with 4-week chronic unpredictable stress, on appetite-regulating neuropeptides and modulatory role of leptin and insulin signalling in the hypothalamus of male Wistar rats. Energy intake, body mass and adiposity, as well as circulatory leptin and insulin concentrations were assessed. Hypothalamic insulin signalling was analysed at the level of glucose transporters, as well as at the protein level and phosphorylation of insulin receptor, insulin receptor supstrate-1, Akt and ERK. Phosphorylation of AMP-activated protein kinase (AMPK), level of protein tyrosine phosphatase 1B (PTP1B) and expression of leptin receptor (ObRb) and suppressor of cytokine signalling 3 (SOCS3) were also analysed, together with the expression of orexigenic agouti-related protein (AgRP) and anorexigenic proopiomelanocortin (POMC) neuropeptides. The results revealed that stress decreased body mass and adiposity, blood leptin level and expression of ObRb, SOCS3 and POMC, while combination with fructose diet led to marked increase of AgRP, associated with AMPK phosphorylation despite increased plasma insulin. Reduced Akt, enhanced ERK activity and elevated PTP1B were also observed in the hypothalamus of these animals. In conclusion, our results showed that joint effects of fructose diet and stress are more deleterious than the separate ones, since inappropriate appetite control in the hypothalamus may provide a setting for the disturbed energy homeostasis in the long run.

Functional Mixture-Based Positional Scan Identifies a Library of Antagonist Tetrapeptide Sequences (LAtTeS) with Nanomolar Potency for the Melanocortin-4 Receptor and Equipotent with the Endogenous AGRP(86-132) Antagonist.

The melanocortin-4 receptor (MC4R) plays an important role in appetite. Agonist ligands that stimulate the MC4R decrease appetite, while antagonist compounds increase food consumption. Herein, a functional mixture-based positional scan identified novel MC4R antagonist sequences. Mixtures comprising a library of 12,960,000 tetrapeptides were screened in the presence and absence of the NDP-MSH agonist. These results led to the synthesis of 48 individual tetrapeptides, of which 40 were screened for functional activity at the melanocortin receptors. Thirteen compounds were found to possess nanomolar antagonist potency at the MC4R, with the general tetrapeptide sequence Ac-Aromatic-Basic-Aromatic-Basic-NH2. The most notable results include the identification of tetrapeptide 48 [COR1-25, Ac-DPhe(pI)-Arg-Nal(2')-Arg-NH2], an equipotent MC4R antagonist to agouti-related protein [AGRP(86-132)], more potent than miniAGRP(87-120), and possessing 15-fold selectivity for the MC4R versus the MC3R. These tetrapeptides may serve as leads for novel appetite-inducing therapies to treat states of negative energy balance, such as cachexia and anorexia.

Leukemia inhibitory factor as a mediator of lipopolysaccharide effects on appetite and selected hormones and metabolites.

Leukemia inhibitory factor (LIF) has been suggested to function as a potent inhibitor of feed intake in rodents. In sheep, intravenous injection of lipopolysaccharide (LPS) resulted in an increase in gene expression for LIF in the arcuate nucleus ( < 0.01). In the same experiment, agouti related protein (AgRP) expression was elevated ( < 0.05) but there were no effects on proopiomelanocortin expression. Another group of sheep were provided intracerebroventricular (ICV) injections of LIF at 250, 500, 1,000, and 2,500 ng per sheep. Cumulative feed intake was inhibited by the 1,000- and 2,500-ng doses at 8 and 10 h after ICV injection ( < 0.03). All doses of LIF elevated temperature above 40°C, indicating a fever. When AgRP was intracerebroventricularly injected before LIF, there was no effect of LIF to reduce feed intake, suggesting the LIF inhibition of feed intake is consistent with the concept that the effect is mediated by the melanocortin-4 receptor. In an experiment to determine whether endocrine and metabolic effects of LIF were similar to reported effects of LPS, sheep were intracerebroventricularly injected with 2,500 ng LIF, and blood samples were collected at 10-min intervals for 6 h for assay of LH, samples from the first 3 h were assayed for GH, and samples at 30-min intervals were assayed for glucose and free fatty acids. The effect of treatment and treatment × time interaction was significant, indicating elevated plasma free fatty acids ( < 0.03 and < 0.001, respectively) and glucose ( < 0.01 and < 0.0001, respectively). There was also a treatment × time interaction on circulating concentrations of LH such that LIF caused LH to decrease ( < 0.0001). Additionally, there was a tendency for LIF treatment to increase circulating concentrations of GH (P = 0.0874). The effects of LIF on feed intake and other parameters was similar to the effects of LPS and leads to a hypothesis that LIF expression in response to LPS may be a component of the mechanism for feed intake inhibition and perhaps for changes in selected hormone and metabolites in disease models.

Importance of Adult Dmbx1 in Long-Lasting Orexigenic Effect of Agouti-Related Peptide.

Dmbx1 is a brain-specific homeodomain transcription factor expressed primarily during embryogenesis, and its systemic disruption (Dmbx1(-/-)) in the ICR mouse strain resulted in leanness associated with impaired long-lasting orexigenic effect of agouti-related peptide (AgRP). Because spatial and temporal expression patterns of Dmbx1 change dramatically during embryogenesis, it remains unknown when and where Dmbx1 plays a critical role in energy homeostasis. In the present study, the physiological roles of Dmbx1 were examined by its conditional disruption (Dmbx1(loxP/loxP)) in the C57BL/6 mouse strain. Although Dmbx1 disruption in fetal brain resulted in neonatal lethality, its disruption by synapsin promoter-driven Cre recombinase, which eliminated Dmbx1 expression postnatally, exempted the mice (Syn-Cre;Dmbx1(loxP/loxP) mice) from lethality. Syn-Cre;Dmbx1(loxP/loxP) mice show mild leanness and impaired long-lasting orexigenic action of AgRP, demonstrating the physiological relevance of Dmbx1 in the adult. Visualization of Dmbx1-expressing neurons in adult brain using the mice harboring tamoxifen-inducible Cre recombinase in the Dmbx1 locus (Dmbx1(CreERT2/+) mice) revealed Dmbx1 expression in small numbers of neurons in restricted regions, including the lateral parabrachial nucleus (LPB). Notably, c-Fos expression in LPB was increased at 48 hours after AgRP administration in Dmbx1(loxP/loxP) mice but not in Syn-Cre;Dmbx1(loxP/loxP) mice. These c-Fos-positive neurons in LPB did not coincide with neurons expressing Dmbx1 or melanocortin 4 receptor but did coincide with those expressing calcitonin gene-related peptide. Accordingly, Dmbx1 in the adult LPB is required for the long-lasting orexigenic effect of AgRP via the neural circuitry involving calcitonin gene-related peptide neurons.

Temporal cAMP Signaling Selectivity by Natural and Synthetic MC4R Agonists.

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain, where it controls energy balance through pathways including α-melanocyte-stimulating hormone (α-MSH)-dependent signaling. We have reported that the MC4R can exist in an active conformation that signals constitutively by increasing cAMP levels in the absence of receptor desensitization. We asked whether synthetic MC4R agonists differ in their ability to increase intracellular cAMP over time in Neuro2A cells expressing endogenous MC4R and exogenous, epitope-tagged hemagglutinin-MC4R-green fluorescent protein. By analyzing intracellular cAMP in a temporally resolved Förster resonance energy transfer assay, we show that withdrawal of α-MSH leads to a quick reversal of cAMP induction. By contrast, the synthetic agonist melanotan II (MTII) induces a cAMP signal that persists for at least 1 hour after removal of MTII from the medium and cannot be antagonized by agouti related protein. Similarly, in mHypoE-42 immortalized hypothalamic neurons, MTII, but not α-MSH, induced persistent AMP kinase signal, which occurs downstream of increased cAMP. By using a fluorescence recovery after photobleaching assay, it appears that the receptor exposed to MTII continues to signal after being internalized. Similar to MTII, the synthetic MC4R agonists, THIQ and BIM-22511, but not LY2112688, induced prolonged cAMP signaling after agonist withdrawal. However, agonist-exposed MC4R desensitized to the same extent, regardless of the ligand used and regardless of differences in receptor intracellular retention kinetics. In conclusion, α-MSH and LY2112688, when compared with MTII, THIQ, and BIM-22511, vary in the duration of the acute cAMP response, showing distinct temporal signaling selectivity, possibly linked to specific cell compartments from which cAMP signals may originate.

Exposure of MC4R to agonist in the endoplasmic reticulum stabilizes an active conformation of the receptor that does not desensitize.

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in neurons of the hypothalamus where it regulates food intake. MC4R responds to an agonist, α-melanocyte-stimulating hormone (α-MSH) and to an antagonist/inverse agonist, agouti-related peptide (AgRP), which are released by upstream neurons. Binding to α-MSH leads to stimulation of receptor activity and suppression of food intake, whereas AgRP has opposite effects. MC4R cycles constantly between the plasma membrane and endosomes and undergoes agonist-mediated desensitization by being routed to lysosomes. MC4R desensitization and increased AgRP expression are thought to decrease the effectiveness of MC4R agonists as an antiobesity treatment. In this study, α-MSH, instead of being delivered extracellularly, is targeted to the endoplasmic reticulum (ER) of neuronal cells and cultured hypothalamic neurons. We find that the ER-targeted agonist associates with MC4R at this location, is transported to the cell surface, induces constant cAMP and AMP kinase signaling at maximal amplitude, abolishes desensitization of the receptor, and promotes both cell-surface expression and constant signaling by an obesity-linked MC4R variant, I316S, that otherwise is retained in the ER. Formation of the MC4R/agonist complex in the ER stabilizes the receptor in an active conformation that at the cell surface is insensitive to antagonism by AgRP and at the endosomes is refractory to routing to the lysosomes. The data indicate that targeting agonists to the ER can stabilize an active conformation of a G protein-coupled receptor that does not become desensitized, suggesting a target for therapy.

Activation of MAPK by inverse agonists in six naturally occurring constitutively active mutant human melanocortin-4 receptors.

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor that plays an essential role in regulating energy homeostasis. Defects in MC4R are the most common monogenic form of obesity, with about 170 distinct mutations identified in human. In addition to the conventional Gs-stimulated adenylyl cyclase pathway, it has been recently demonstrated that MC4R also activates mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2). Herein, we investigated the potential of four MC4R ligands that are inverse agonists at the Gs-cAMP signaling pathway, including agouti-related peptide (AgRP), MCL0020, Ipsen 5i and ML00253764, to regulate ERK1/2 activation (pERK1/2) in wild type and six naturally occurring constitutively active mutant (CAM) MC4Rs. We showed that these four inverse agonists acted as agonists for the ERK1/2 signaling cascade in wild type and CAM MC4Rs. Three mutants (P230L, L250Q and F280L) had significantly increased pERK1/2 level upon stimulation with all four inverse agonists, with maximal induction ranging from 1.6 to 4.2-fold. D146N had significantly increased pERK1/2 level upon stimulation with AgRP, MCL0020 or ML00253764, but not Ipsen 5i. The pERK1/2 levels of H76R and S127L were significantly increased only upon stimulation with AgRP or MCL0020. In summary, our studies demonstrated for the first time that MC4R inverse agonists at the Gs-cAMP pathway could serve as agonists in the MAPK pathway. These results suggested that there were multiple activation states of MC4R with ligand-specific and/or mutant-specific conformations capable of differentially coupling the MC4R to distinct signaling pathways.

Direct regulation of GnRH neuron excitability by arcuate nucleus POMC and NPY neuron neuropeptides in female mice.

Hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons act to sense and coordinate the brain's responses to metabolic cues. One neuronal network that is very sensitive to metabolic status is that controlling fertility. In this study, we investigated the impact of neuropeptides released by NPY and POMC neurons on the cellular excitability of GnRH neurons, the final output cells of the brain controlling fertility. The majority (∼70%) of GnRH neurons were activated by α-melanocyte-stimulating hormone, and this resulted from the direct postsynaptic activation of melanocortin receptor 3 and melanocortin receptor 4. A small population of GnRH neurons (∼15%) was excited by cocaine and amphetamine-regulated transcript or inhibited by ß-endorphin. Agouti-related peptide, released by NPY neurons, was found to have variable inhibitory (∼10%) and stimulatory (∼25%) effects upon subpopulations of GnRH neurons. A variety of NPY and pancreatic polypeptide analogs was used to examine potential NPY interactions with GnRH neurons. Although porcine NPY (Y1/Y2/Y5 agonist) directly inhibited the firing of approximately 45% of GnRH neurons, [Leu(31),Pro(34)]-NPY (Y1/Y4/Y5 agonist) could excite (56%) or inhibit (19%). Experiments with further agonists indicated that Y1 receptors were responsible for suppressing GnRH neuron activity, whereas postsynaptic Y4 receptors were stimulatory. These results show that the activity of GnRH neurons is regulated in a complex manner by neuropeptides released by POMC and NPY neurons. This provides a direct route through which different metabolic cues can regulate fertility.

Melanocortin antagonism ameliorates muscle wasting and inflammation in chronic kidney disease.

Aberrant melanocortin signaling has been implicated in the pathogenesis of wasting in chronic kidney disease (CKD). Previously, we demonstrated that agouti-related peptide (AgRP), a melenocortin-4 receptor antagonist, reduced CKD-associated cachexia in CKD mice. Our previous studies with AgRP utilized dual energy X-ray (DXA) densitometry to assess the body composition in mice (Cheung W, Kuo HJ, Markison S, Chen C, Foster AC, Marks DL, Mak RH. J Am Soc Nephrol 18: 2517-2524, 2007; Cheung W, Yu PX, Little BM, Cone RD, Marks DL, Mak RH. J Clin Invest 115: 1659-1665, 2005). DXA is unable to differentiate water content in mice, and fluid retention in CKD may lead to an overestimate of lean mass. In this study, we employed quantitative magnetic resonance technique to evaluate body composition change following central administration of AgRP in a CKD mouse model. AgRP treatment improved energy expenditure, total body mass, fat mass, and lean body mass in CKD mouse. We also investigated the effect of CKD-associated cachexia on the signaling pathways leading to wasting in skeletal muscle, as well as whether these changes can be ameliorated by central administration of AgRP. AgRP treatment caused an overall decrease in proinflammatory cytokines, which may be one important mechanism of its effects. Muscle wasting in CKD may be due to the activation of proteolytic pathways as well as inhibition of myogenesis and muscle regeneration processes. Our results suggest that these aberrant pathological pathways leading to muscle wasting in CKD mice were ameliorated by central administration of AgRP.

Third ventricular coinjection of subthreshold doses of NPY and AgRP stimulate food hoarding and intake and neural activation.

We previously demonstrated that 3rd ventricular (3V) neuropeptide Y (NPY) or agouti-related protein (AgRP) injection potently stimulates food foraging/hoarding/intake in Siberian hamsters. Because NPY and AgRP are highly colocalized in arcuate nucleus neurons in this and other species, we tested whether subthreshold doses of NPY and AgRP coinjected into the 3V stimulates food foraging, hoarding, and intake, and/or neural activation [c-Fos immunoreactivity (c-Fos-ir)] in hamsters housed in a foraging/hoarding apparatus. In the behavioral experiment, each hamster received four 3V treatments by using subthreshold doses of NPY and AgRP for all behaviors: 1) NPY, 2) AgRP, 3) NPY+AgRP, and 4) saline with a 7-day washout period between treatments. Food foraging, intake, and hoarding were measured 1, 2, 4, and 24 h and 2 and 3 days postinjection. Only when NPY and AgRP were coinjected was food intake and hoarding increased. After identical treatment in separate animals, c-Fos-ir was assessed at 90 min and 14 h postinjection, times when food intake (0-1 h) and hoarding (4-24 h) were uniquely stimulated. c-Fos-ir was increased in several hypothalamic nuclei previously shown to be involved in ingestive behaviors and the central nucleus of the amygdala (CeA), but only in NPY+AgRP-treated animals (90 min and 14 h: magno- and parvocellular regions of the hypothalamic paraventricular nucleus and perifornical area; 14 h only: CeA and sub-zona incerta). These results suggest that NPY and AgRP interact to stimulate food hoarding and intake at distinct times, perhaps released as a cocktail naturally with food deprivation to stimulate these behaviors.

Agouti-related protein segments outside of the receptor binding core are required for enhanced short- and long-term feeding stimulation.

The agouti-related protein (AgRP) plays a central role in energy balance by reducing signaling through the hypothalamic melanocortin receptors (McRs) 3 and 4, in turn stimulating feeding and decreasing energy expenditure. Mature AgRP(83-132), produced by endoproteolytic processing, contains a central region that folds as an inhibitor cystine knot (ICK) stabilized by a network of disulfide bonds; this domain alone carries the molecular features for high affinity McR binding and inverse agonism. Outside of the ICK domain are two polypeptide segments, an N-terminal extension and a C-terminal loop, both completely conserved but of unknown function. Here we examine the physiological roles of these non-ICK segments by developing a panel of modified AgRPs that were administered to rats through intracerebroventricular (ICV) injection. Analysis of food consumption demonstrates that basic (positively charged) residues are essential for potent short- and long-term AgRP stimulated feeding. Moreover, we demonstrate an approximate linear relationship between protein charge density and 24 h food intake. Next, we developed artificial AgRP(83-132) analogues with increased positive charge and found that these species were substantially more potent than wild type. A single dose of one protein, designated AgRP-4K, results in enhanced feeding for well over a week and weight gain that is nearly double that of AgRP(83-132). These studies suggest new strategies for the development of potent orexigenic species and may serve as leads for the development of therapeutics for treating wasting conditions such as cachexia.

Cystine-knot peptides engineered with specificities for α(IIb)ß(3) or α(IIb)ß(3) and α(v)ß(3) integrins are potent inhibitors of platelet aggregation.

A truncated form of the Agouti-related protein (AgRP), a member of the cystine-knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg-Gly-Asp integrin recognition motif, and randomized the neighboring residues to create a library of approximately 20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high-throughput fluorescence-activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α(IIb)ß(3), a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K(D)) constants for α(IIb)ß(3) integrin ranging from 60 to 90 nM, and did not bind to α(v)ß(3), α(v)ß(5), or α(5)ß(1) integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α(IIb)ß(3) and α(v)ß(3) integrins with K(D) values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α(v)ß(5) or α(5)ß(1) integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA-approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine-knot peptides can be generated with high affinity and specificity to closely-related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors.

Central nervous system melanocortin-3 receptors are required for synchronizing metabolism during entrainment to restricted feeding during the light cycle.

Melanocortin-3 receptors (Mc3rs) in the central nervous system are involved in expression of anticipatory rhythms and synchronizing clocks maintaining circadian rhythms during restricted feeding (RF) [mice housed under a 12-h light-dark cycle with lights on between zeitgeber time (ZT) 0 to ZT12 fed 60% of normal calories between ZT7 and ZT11]. Because the systems governing circadian rhythms are important for adaptation to RF, we investigated whether Mc3rs are required for metabolic adaption to RF. Mc3r(-/-) mice subjected to RF exhibited normal weight loss; however, they developed hyperinsulinemia, glucose intolerance, increased expression of lipogenic genes, and increased ketogenesis relative to controls. Rhythmic expression of transcription factors regulating liver clock activity and energy metabolism (Bmal1, Rev-erbalpha, Pgc1, Foxo1, Hnf4alpha, and Pck1) was severely compromised in Mc3r(-/-) mice during RF. Inhibition of neural melanocortin receptors by agouti-related peptide also attenuated rhythmicity in the hepatic expression of these genes during RF. Collectively, these data suggest that neural Mc3rs are important for adapting metabolism and maintaining rhythms of liver metabolism during periods when feeding is restricted to the light cycle.-Sutton, G. M., Begriche, K., Kumar, K. G., Gimble, J. M., Perez-Tilve, D., Nogueiras, R., McMillan, R. P., Hulver, M. W., Tschöp, M. H., Butler, A. A. Central nervous system melanocortin-3 receptors are required for synchronizing metabolism during entrainment to restricted feeding during the light cycle.

Hormonal regulation of the mouse adrenal melanocortinergic system.

Adrenocortical cells of several species have been reported to express significant levels of Agouti-related protein (Agrp) as well as melanocortin 4-receptor (MC4-R). In this study, we used the mouse tumoral adrenal cell line ATC7- L that secretes corticosterone in basal conditions with a 2- fold increase in response to ACTH treatment. We reported that these cells expressed functional MC4-R. They also expressed Agrp mRNA and secreted immunoreactive Agrp in the culture medium. Long-term treatment of ATC7-L with (Nle4,D-Phe7)-alpha MSH (NDP-alpha MSH) or forskolin as well as Agrp strongly reduced MC4-R level by more than 30%. On the contrary, leptin treatment did not modify this level although it significantly reduced MC2-R level. These results could be correlated to some data obtained in vivo on adrenal glands removed from diet-induced obese mice exhibiting a hyperleptinemia, where the level of both MC2-R and MC4-R appeared to be reduced as Agrp mRNA expression level was increased compared to Control mice. All these data would suggest the existence of a link between the metabolic status and the activation of the adrenal melanocortinergic system.

Administration of IL-1beta to the 4th ventricle causes anorexia that is blocked by agouti-related peptide and that coincides with activation of tyrosine-hydroxylase neurons in the nucleus of the solitary tract.

Inflammation-associated cachexia is associated with multiple chronic diseases and involves activation of appetite regulating centers in the arcuate nucleus of the hypothalamus (ARH). The nucleus of the solitary tract (NTS) in the brainstem has also been implicated as an important nucleus involved in appetite regulation. We set out to determine whether the NTS may be involved in inflammation-associated anorexia by injecting IL-1 beta into the 4th ventricle and assessing food intake and NTS neuronal activation. Injection of IL-1 beta produced a decrease in food intake at 3 and 12h after injection which was ameliorated at the 12h time point by a sub-threshold dose of agouti-related peptide (AgRP). Investigation into neuron types in the NTS revealed that IL-1 beta injection was associated with an increase in c-Fos activity in NTS neurons expressing tyrosine hydroxylase (TH). Additionally, injection of IL-1 beta into the 4th ventricle did not produce c-Fos activation of neurons expressing pro-opiomelanocortin (POMC) in the ARH, cells known to be involved in producing anorexia in response to systemic inflammation. Double-label in situ hybridization revealed that TH neurons did not express IL-1 receptor I (IL1-RI) transcript, demonstrating that c-Fos activation of TH neurons in this setting was not via direct stimulation of IL-1 beta on TH neurons themselves. We conclude that IL-1 beta injection into the 4th ventricle produces anorexia and is accompanied by an increase in activation in TH neurons in the NTS. This provides evidence that the brainstem may be an important mediator of anorexia in the setting of inflammation.

Estradiol decreases the orexigenic effect of neuropeptide Y, but not agouti-related protein, in ovariectomized rats.

Available data suggest that estradiol exerts an inhibitory effect on food intake by modulating the actions of multiple gut- and brain-derived peptides implicated in the control of food intake. For example, recent studies have shown that estradiol decreases the orexigenic effects of ghrelin and melanin-concentrating hormone. In the present study, we examined estradiol's ability to decrease the actions of two additional orexigenic peptides, neuropeptide Y (NPY) and agouti-related protein (AgRP). Food intake was monitored following lateral ventricular infusions of 5 microg NPY, 10 microg AgRP, or saline vehicle in ovariectomized rats treated with either 1 microg estradiol or sesame oil vehicle. NPY increased food intake for 2h in both oil- and estradiol-treated ovariectomized rats. During this interval, the orexigenic effect of NPY was significantly greater in oil-treated rats, relative to estradiol-treated rats. In contrast to the short-term action of NPY, a single injection of AgRP increased food intake for 3 days in oil- and estradiol-treated rats. Meal pattern analysis revealed that the orexigenic effect of AgRP is mediated by an increase in meal size, not meal number. Unlike that observed following NPY treatment, estradiol failed to modulate the magnitude by which AgRP increased food intake and meal size. We conclude that a physiological regimen of estradiol treatment decreases the orexigenic effect of NPY, but not AgRP, in ovariectomized rats.

In vivo evidence for inverse agonism of Agouti-related peptide in the central nervous system of proopiomelanocortin-deficient mice.

OBJECTIVE: Melanocyte-stimulating hormone (MSH) peptides processed from proopiomelanocortin (POMC) regulate energy homeostasis by activating neuronal melanocortin receptor (MC-R) signaling. Agouti-related peptide (AgRP) is a naturally occurring MC-R antagonist but also displays inverse agonism at constitutively active melanocortin-4 receptor (MC4-R) expressed on transfected cells. We investigated whether AgRP functions similarly in vivo using mouse models that lack all neuronal MSH, thereby precluding competitive antagonism of MC-R by AgRP. RESEARCH DESIGN AND METHODS: Feeding and metabolic effects of the MC-R agonist melanotan II (MTII), AgRP, and ghrelin were investigated after intracerebroventricular injection in neural-specific POMC-deficient (Pomc(-/-)Tg/+) and global POMC-deficient (Pomc(-/-)) mice. Gene expression was quantified by RT-PCR. RESULTS: Hyperphagic POMC-deficient mice were more sensitive than wild-type mice to the anorectic effects of MTII. Hypothalamic melanocortin-3 (MC3)/4-R mRNAs in POMC-deficient mice were unchanged, suggesting increased receptor sensitivity as a possible mechanism for the heightened anorexia. AgRP reversed MTII-induced anorexia in both mutant strains, demonstrating its ability to antagonize MSH agonists at central MC3/4-R, but did not produce an acute orexigenic response by itself. The action of ghrelin was attenuated in Pomc(-/-)Tg/+ mice, suggesting decreased sensitivity to additional orexigenic signals. However, AgRP induced delayed and long-lasting modifications of energy balance in Pomc(-/-)Tg/+, but not glucocorticoid-deficient Pomc(-/-) mice, by decreasing oxygen consumption, increasing the respiratory exchange ratio, and increasing food intake. CONCLUSIONS: These data demonstrate that AgRP can modulate energy balance via a mechanism independent of MSH and MC3/4-R competitive antagonism, consistent with either inverse agonist activity at MC-R or interaction with a distinct receptor.