An autonomous activation of interleukin-17 receptor signaling sustains inflammation and promotes disease progression.

Anti-interleukin-17 (IL-17) therapy has been used in various autoimmune diseases. However, the efficacy is unexpectedly limited in several IL-17-associated diseases, and the mechanism of limited efficacy remains unclear. Here, we show that a molecular complex containing the adaptor molecule Act1 and tyrosine phosphatase SHP2 mediated autonomous IL-17R signaling that accelerated and sustained inflammation. SHP2, aberrantly augmented in various autoimmune diseases, was induced by IL-17A itself in astrocytes and keratinocytes, sustaining chemokine production even upon anti-IL-17 therapies. Mechanistically, SHP2 directly interacted with and dephosphorylated Act1, which replaced Act1-TRAF5 complexes and induced IL-17-independent activation of IL-17R signaling. Genetic or pharmacologic inactivation of SHP2, or blocking Act1-SHP2 interaction, paralyzed both IL-17-induced and IL-17-independent signaling and attenuated primary or relapsing experimental autoimmune encephalomyelitis. Therefore, Act1-SHP2 complexes mediate an alternative pathway for autonomous activation of IL-17R signaling, targeting which could be a therapeutic option for IL-17-related diseases in addition to current antibody therapies.

Molecular dissection of an intronic enhancer governing cold-induced expression of the vacuolar invertase gene in potato.

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to minimize sprouting and losses due to disease. However, cold temperatures strongly induce the expression of the potato vacuolar invertase gene (VInv) and cause reducing sugar accumulation. This process, referred to as "cold-induced sweetening," is a major postharvest problem for the potato industry. We discovered that the cold-induced expression of VInv is controlled by a 200 bp enhancer, VInvIn2En, located in its second intron. We identified several DNA motifs in VInvIn2En that bind transcription factors involved in the plant cold stress response. Mutation of these DNA motifs abolished VInvIn2En function as a transcriptional enhancer. We developed VInvIn2En deletion lines in both diploid and tetraploid potato using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing. VInv transcription in cold-stored tubers was significantly reduced in the deletion lines. Interestingly, the VInvIn2En sequence is highly conserved among distantly related Solanum species, including tomato (Solanum lycopersicum) and other non-tuber-bearing species. We conclude that the VInv gene and the VInvIn2En enhancer have adopted distinct roles in the cold stress response in tubers of tuber-bearing Solanum species.

Systematic Analysis of Galactinol Synthase and Raffinose Synthase Gene Families in Potato and Their Expression Patterns in Development and Abiotic Stress Responses.

Raffinose family oligosaccharides (RFOs) are very important for plant growth, development, and abiotic stress tolerance. Galactinol synthase (GolS) and raffinose synthase (RFS) are critical enzymes involved in RFO biosynthesis. However, the whole-genome identification and stress responses of their coding genes in potato remain unexplored. In this study, four StGolS and nine StRFS genes were identified and classified into three and five subgroups, respectively. Remarkably, a total of two StGolS and four StRFS genes in potato were identified to form collinear pairs with those in both Arabidopsis and tomato, respectively. Subsequent analysis revealed that StGolS4 exhibited significantly high expression levels in transport-related tissues, PEG-6000, and ABA treatments, with remarkable upregulation under salt stress. Additionally, StRFS5 showed similar responses to StGolS4, but StRFS4 and StRFS8 gene expression increased significantly under salt treatment and decreased in PEG-6000 and ABA treatments. Overall, these results lay a foundation for further research on the functional characteristics and molecular mechanisms of these two gene families in response to ABA, salt, and drought stresses, and provide a theoretical foundation and new gene resources for the abiotic-stress-tolerant breeding of potato.

Transcriptome analysis reveals the proline metabolic pathway and its potential regulation TF-hub genes in salt-stressed potato.

Potato (Solanum tuberosum) is currently the third most important food crop in the world. However, the production of potato is seriously threatened by salt stress, which often occurs in the facility cultivation environment, and the mining of salt tolerance genes in potato remains to be further studied. In this study, test-tube plantlets of DM potato were treated with 200-mM NaCl to simulate salt stress, and 15 cDNA libraries were constructed for RNA-seq analysis. A total of 8383 DEGs were identified, of which 3961 DEGs were shared among all the salt treatments, and 264 (7.15%) TF-coding genes were identified from these shared DEGs. KEGG enrichment analysis showed that most DEGs identified from the "arginine and proline metabolism" (ko00330) were enriched in the proline metabolic pathway, and their functions almost covered the whole proline metabolic process. Further analysis showed that expression levels of all the 13 structural DEGs in the pathway were significantly up-regulated and proline accumulation was also significantly increased under salt stress, and 13 TF-hub genes were discovered by WGCNA in the lightcyan and tan modules which were highly positively correlated with the proline contents. Correlation analysis revealed that the four TF-hub genes of the lightcyan module and seven structural DEGs of the proline metabolic pathway might be the potential candidate genes, especially the potential and novel regulatory gene StGLK014720. Furthermore, the dual-luciferase reporter assay confirmed that the key protein StGLK014720 could activate the promoters of both structural genes StAST021010 and StAST017480. In conclusion, these results lay the foundation for further study on the salt tolerance mechanism of potato, and provide a theoretical basis and new genetic resources for salt tolerance breeding of potato.

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist.

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.

Optimization of privileged structures for selective and potent melanocortin subtype-4 receptor ligands.

Design, syntheses and structure-activity relationships of N-acetylated piperazine privileged structures containing MC4R agonist compounds were described. The most potent derivatives were low nM MC4R selective full agonists. Several compounds from the series had modest pharmacokinetic properties.

Discovery of potent, selective, and orally bioavailable 3H-spiro[isobenzofuran-1,4'-piperidine] based melanocortin subtype-4 receptor agonists.

Design, synthesis, and SAR of a series of 3H-spiro[isobenzofuran-1,4'-piperidine] based compounds as potent, selective and orally bioavailable melanocortin subtype-4 receptor (MC4R) agonists are disclosed.

Discovery of highly potent and efficacious MC4R agonists with spiroindane N-Me-1,2,4-triazole privileged structures for the treatment of obesity.

We report an SAR study of MC4R analogs containing spiroindane heterocyclic privileged structures. Compound 26 with N-Me-1,2,4-triazole moiety possesses exceptional potency at MC4R and potent anti-obesity efficacy in a mouse model. However, the efficacy is not completely mediated through MC4R. Additional SAR studies led to the discovery of compound 32, which is more potent at MC4R. Compound 32 demonstrates MC4R mediated anti-obesity efficacy in rodent models.

Discovery of a spiroindane based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor agonist.

We report the design, synthesis and properties of spiroindane based compound 1, a potent, selective, orally bioavailable, non-peptide melanocortin subtype-4 receptor agonist. Compound 1 shows excellent erectogenic activity in the rodent models.

Spiroindane based amides as potent and selective MC4R agonists for the treatment of obesity.

We report a series of potent and selective MC4R agonists based on spiroindane amide privileged structures for potential treatments of obesity. Among the synthetic methods used, Method C allows rapid synthesis of the analogs. The series of compounds can afford high potency on MC4R as well as good rodent pharmacokinetic profiles. Compound 1r (MK-0489) demonstrates MC4R mediated reduction of food intake and body weight in mouse models. Compound 1r is efficacious in 14-day diet-induced obese (DIO) rat models.

Melanocortin 3 receptors control crystal-induced inflammation.

In this study we have characterized the anti-inflammatory profile of a selective melanocortin type 3 receptor (MC3-R) ligand [D-Trp8]-gamma-MSH, validating in vitro results with analyses in mice deficient for this receptor subtype. In wild-type (WT) macrophages, [D-Trp8]-gamma-MSH activated MC3-R (as tested by accumulation of cyclic AMP) and inhibited (approximately 50%) the release of interleukin (IL)-1 and the chemokine KC (CXCL1), but was ineffective in cells taken from MC3-R null mice. In vivo, administration of 3-30 microg [D-Trp8]-gamma-MSH significantly inhibited leukocyte influx and cytokine production in a model of crystal-induced peritonitis, and these effects were absent in MC3-R null mice or blocked by coadministration of an MC3-R antagonist. Finally, in a model of gouty arthritis, direct injection of urate crystals into the rat joint provoked a marked inflammatory reaction that was significantly inhibited (approximately 70%) by systemic or local administration of [D-Trp8]-gamma-MSH. In conclusion, using an integrated transgenic and pharmacological approach, we provide strong proof of concept for the development of selective MC3-R agonists as novel anti-inflammatory therapeutics.

Neither agouti-related protein nor neuropeptide Y is critically required for the regulation of energy homeostasis in mice.

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp(-/-)) mice to examine the physiological role of AgRP. Agrp(-/-) mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp(-/-) mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp(-/-) mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp(-/-);Npy(-/-)) mice to determine whether NPY or AgRP plays a compensatory role in Agrp(-/-) or NPY-deficient (Npy(-/-)) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp(-/-);Npy(-/-) mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.

The role of melanocortins in body weight regulation: opportunities for the treatment of obesity.

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

The role of melanocortins in body weight regulation: opportunities for the treatment of obesity.

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

Design, synthesis, and evaluation of conformationally restricted acetanilides as potent and selective ß3 adrenergic receptor agonists for the treatment of overactive bladder.

A series of conformationally restricted acetanilides were synthesized and evaluated as ß3-adrenergic receptor agonists (ß3-AR) for the treatment of overactive bladder (OAB). Optimization studies identified a five-membered ring as the preferred conformational lock of the acetanilide. Further optimization of both the aromatic and thiazole regions led to compounds such as 19 and 29, which have a good balance of potency and selectivity. These compounds have significantly reduced intrinsic clearance compared to our initial series of pyridylethanolamine ß3-AR agonists and thus have improved unbound drug exposures. Both analogues demonstrated dose dependent ß3-AR mediated responses in a rat bladder hyperactivity model.

Therapeutic potential of histamine H3 receptor agonist for the treatment of obesity and diabetes mellitus.

Histamine H3 receptors (H3Rs) are located on the presynaptic membranes and cell soma of histamine neurons, where they negatively regulate the synthesis and release of histamine. In addition, H3Rs are also located on nonhistaminergic neurons, acting as heteroreceptors to regulate the releases of other amines such as dopamine, serotonin, and norepinephrine. The present study investigated the effects of H3R ligands on appetite and body-weight regulation by using WT and H3R-deficient mice (H3RKO), because brain histamine plays a pivotal role in energy homeostasis. The results showed that thioperamide, an H3R inverse agonist, increases, whereas imetit, an H3R agonist, decreases appetite and body weight in diet-induced obese (DiO) WT mice. Moreover, in DiO WT mice, but not in DiO H3RKO mice, imetit reduced fat mass, plasma concentrations of leptin and insulin, and hepatic triglyceride content. The anorexigenic effects of imetit were associated with a reduction in histamine release, but a comparable reduction in histamine release with alpha-fluoromethylhistidine, an inhibitor of histamine synthesis, increased appetite. Moreover, the anorexigenic effects of imetit were independent of the melanocortin system, because imetit comparably reduced appetite in melanocortin 3 and 4 receptor-deficient mice. The results provide roles of H3Rs in energy homeostasis and suggest a therapeutic potential for H3R agonists in the treatment of obesity and diabetes mellitus.

Mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to peroxisome proliferator-activated receptor gamma agonists.

The adipose tissue-derived hormone adiponectin improves insulin sensitivity and its circulating levels are decreased in obesity-induced insulin resistance. Here, we report the generation of a mouse line with a genomic disruption of the adiponectin locus. We aimed to identify whether these mice develop insulin resistance and which are the primary target tissues affected in this model. Using euglycemic/insulin clamp studies, we demonstrate that these mice display severe hepatic but not peripheral insulin resistance. Furthermore, we wanted to test whether the lack of adiponectin magnifies the impairments of glucose homeostasis in the context of a dietary challenge. When exposed to high fat diet, adiponectin null mice rapidly develop glucose intolerance. Specific PPARgamma agonists such as thiazolidinediones (TZDs) improve insulin sensitivity by mechanisms largely unknown. Circulating adiponectin levels are significantly up-regulated in vivo upon activation of PPARgamma. Both TZDs and adiponectin have been shown to activate AMP-activated protein kinase (AMPK) in the same target tissues. We wanted to address whether the ability of TZDs to improve glucose tolerance is dependent on adiponectin and whether this improvement involved AMPK activation. We demonstrate that the ability of PPARgamma agonists to improve glucose tolerance in ob/ob mice lacking adiponectin is diminished. Adiponectin is required for the activation of AMPK upon TZD administration in both liver and muscle. In summary, adiponectin is an important contributor to PPARgamma-mediated improvements in glucose tolerance through mechanisms that involve the activation of the AMPK pathway.

Diet induction of monocyte chemoattractant protein-1 and its impact on obesity.

OBJECTIVE: To examine the effect of a high-fat diet on gene expression in adipose tissues and to determine induction kinetics of adipose monocyte chemoattractant protein-1 and -3 (MCP-1 and MCP-3) in diet-induced obesity (DIO) and the effect of a lack of MCP-1 signaling on DIO susceptibility and macrophage recruitment into adipose tissue. RESEARCH METHODS AND PROCEDURES: Obese and lean adipose tissues were profiled for expression changes. The time-course of MCP-1 and MCP-3 expression was examined by reverse transcriptase-polymerase chain reaction. Plasma MCP-1 levels were determined by enzyme-linked immunosorbent assay (ELISA). Chemokine receptor-2 (CCR2) knockout mice were placed on the high-fat diet to determine DIO susceptibility. Macrophage infiltration in adipose tissue was examined by immunohistochemistry with F4/80 antibody. RESULTS: DIO elevated adipose expression of many inflammatory genes, including MCP-1 and MCP-3. Adipose MCP-1 and MCP-3 mRNA levels increased within 7 days of starting a high-fat diet, with elevation of plasma MCP-1 detected after 4 weeks on the diet. The induction of MCP-1 and MCP-3 expression preceded that of tumor necrosis factor-alpha. The elevated plasma MCP-1 concentration in obese mice was partially reversed by treatment with AM251. No change in DIO susceptibility and macrophage accumulation in adipose tissue were observed in CCR2 knockout mice, which lack the MCP-1 receptor CCR2. DISCUSSION: A high-fat diet elevated adipose expression of inflammatory genes, including early induction of MCP-1 and MCP-3, supporting the view that obese adipose tissues contribute to systemic inflammation. However, despite increased MCP-1 in obesity, disruption of MCP-1 signaling did not confer resistance to DIO in mice or reduce adipose tissue macrophage infiltration.

Effects of melanocortin receptor activation and blockade on ethanol intake: a possible role for the melanocortin-4 receptor.

BACKGROUND: The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor pro-opiomelanocortin. A growing body of literature suggests that the MC system modulates neurobiological responses to drugs of abuse. Because ethanol has direct effects on central pro-opiomelanocortin activity, it is possible that MC neuropeptides participate in the control of voluntary ethanol consumption. Here we assessed the possibility that MC receptor (MCR) agonists modulate ethanol intake via the MC3 receptor (MC3R) and/or the MC4 receptor (MC4R) and whether the MCR antagonist AgRP-(83-132) controls ethanol consumption. METHODS: Mc3r-deficient (Mc3r) and wild-type (Mc3r) littermate mice were given intraperitoneal (10 mg/kg) and intracerebroventricular (1.0 microg ICV) doses of melanotan II (MTII), a nonselective MCR agonist. To assess the role of MC4R, C57BL/6J mice were given an ICV infusion of the highly selective MC4R agonist cyclo(NH-CH2-CH2-CO-His-d-Phe-Arg-Trp-Glu)-NH2 (1.0 or 3.0 microg). Finally, naïve C57BL/6J mice were given an ICV infusion of AgRP-(83-132) (0.05 and 1.0 microg). RESULTS: MTII was similarly effective at reducing ethanol drinking in Mc3r-deficient (Mc3r) and wild-type (Mc3r) littermate mice. Furthermore, ICV infusion of the MC4R agonist significantly reduced ethanol drinking, whereas ICV infusion of AgRP-(83-132) significantly increased ethanol drinking in C57BL/6J mice. Neither MTII nor AgRP-(83-132) altered blood ethanol levels at doses that modulated ethanol drinking. CONCLUSIONS: The present results suggest that MC4R, and not MC3R, modulates MCR agonist-induced reduction of ethanol consumption and that ethanol intake is increased by the antagonistic actions of AgRP-(83-132). These findings strengthen the argument that MCR signaling controls ethanol consumption and that compounds directed at MCR may represent promising targets for treating alcohol abuse disorders in addition to obesity.

Discovery and activity of (1R,4S,6R)-N-[(1R)-2-[4-cyclohexyl-4-[[(1,1-dimethylethyl)amino]carbonyl]-1-piperidinyl]-1-[(4-fluorophenyl)methyl]-2-oxoethyl]-2-methyl-2-azabicyclo[2.2.2]octane-6-carboxamide (3, RY764), a potent and selective melanocortin subtype-4 receptor agonist.

A novel isoquinuclidine containing selective melanocortin subtype-4 receptor small molecule agonist, 3 (RY764), is reported. Its in vivo characterization revealed mechanism-based food intake reduction and erectile activity augmentation in rodents.