Orexin antagonism and substance-P: Effects and interactions on polycystic ovary syndrome in the wistar rats.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder without definitive treatments. Orexin and Substance-P (SP) neuropeptides can affect the ovarian steroidogenesis. Moreover, there are limited studies about the role of these neuropeptides in PCOS. We aimed here to clarify the effects of orexins and SP in PCOS as well as any possible interactions between them. METHODS: For this purpose, the animals (n = five rats per group) received intraperitoneally a single dose of SB-334,867-A (orexin-1 receptor antagonist; OX1Ra), JNJ-10,397,049 (orexin-2 receptor antagonist; OX2Ra), and CP-96,345 (neurokinin-1 receptor antagonist; NK1Ra), alone or in combination with each other after two months of PCOS induction. The blocking of orexin and SP receptors was studied in terms of ovarian histology, hormonal changes, and gene expression of ovarian steroidogenic enzymes. RESULTS: The antagonists' treatment did not significantly affect the formation of ovarian cysts. In the PCOS groups, the co-administration of OX1Ra and OX2Ra as well as their simultaneous injections with NK1Ra significantly reversed testosterone levels and Cyp19a1 gene expression when compared to the PCOS control group. There were no significant interactions between the PCOS groups that received NK1Ra together with one or both OX1R- and OX2R-antagonists. CONCLUSION: The blocking of the orexin receptors modulates abnormal ovarian steroidogenesis in the PCOS model of rats. This suggests that the binding of orexin-A and -B to their receptors reduces Cyp19a1 gene expression while increasing testosterone levels.

Antimicrobial and anti-inflammatory activities of the neuropeptide antagonist SPA.

Antimicrobial peptides have received increased attention due to the increasing prevalence of antibiotic-resistant bacteria. However, the development of antimicrobial peptides for clinical applications remains a huge challenge. SPA ([D-rg1 , D-Trp5,7,9 , Leu11 ]SP), an analog of substance P, is a broad-spectrum neuropeptide antagonist. In this study, we found that SPA could efficiently kill Gram-positive and Gram-negative bacteria by membrane disruption, similar to antimicrobial peptides. In addition, SPA showed high killing activity toward bacteria rather than mammalian cells. Our results also demonstrated that SPA could significantly decrease the expression of proinflammatory cytokines and rescue mice from lethal septic shock induced by lipopolysaccharide (LPS). The impressive therapeutic potential of SPA, as indicated in this study, makes it a good template for developing effective antibiotics. Meanwhile, our study provides a new idea for developing multifunctional therapeutic agents to combat bacterial infections.

Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling.

Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10-30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn2+-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca2+ only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.

Connections between Immune-Derived Mediators and Sensory Nerves for Itch Sensation.

Although histamine is a well-known itch mediator, histamine H1-receptor blockers often lack efficacy in chronic itch. Recent molecular and cellular based studies have shown that non-histaminergic mediators, such as proteases, neuropeptides and cytokines, along with their cognate receptors, are involved in evocation and modulation of itch sensation. Many of these molecules are produced and secreted by immune cells, which act on sensory nerve fibers distributed in the skin to cause itching and sensitization. This understanding of the connections between immune cell-derived mediators and sensory nerve fibers has led to the development of new treatments for itch. This review summarizes current knowledge of immune cell-derived itch mediators and neuronal response mechanisms, and discusses therapeutic agents that target these systems.

A potential role for somatostatin signaling in regulating retinal neurogenesis.

Neuropeptides have been reported to regulate progenitor proliferation and neurogenesis in the central nervous system. However, these studies have typically been conducted using pharmacological agents in ex vivo preparations, and in vivo evidence for their developmental function is generally lacking. Recent scRNA-Seq studies have identified multiple neuropeptides and their receptors as being selectively expressed in neurogenic progenitors of the embryonic mouse and human retina. This includes Sstr2, whose ligand somatostatin is transiently expressed by immature retinal ganglion cells. By analyzing retinal explants treated with selective ligands that target these receptors, we found that Sstr2-dependent somatostatin signaling induces a modest, dose-dependent inhibition of photoreceptor generation, while correspondingly increasing the relative fraction of primary progenitor cells. These effects were confirmed by scRNA-Seq analysis of retinal explants but abolished in Sstr2-deficient retinas. Although no changes in the relative fraction of primary progenitors or photoreceptor precursors were observed in Sstr2-deficient retinas in vivo, scRNA-Seq analysis demonstrated accelerated differentiation of neurogenic progenitors. We conclude that, while Sstr2 signaling may act to negatively regulate retinal neurogenesis in combination with other retinal ganglion cell-derived secreted factors such as Shh, it is dispensable for normal retinal development.

RAC1 controls progressive movement and competitiveness of mammalian spermatozoa.

Mammalian spermatozoa employ calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) signaling in generating flagellar beat. However, how sperm direct their movement towards the egg cells has remained elusive. Here we show that the Rho small G protein RAC1 plays an important role in controlling progressive motility, in particular average path velocity and linearity. Upon RAC1 inhibition of wild type sperm with the drug NSC23766, progressive movement is impaired. Moreover, sperm from mice homozygous for the genetically variant t-haplotype region (tw5/tw32), which are sterile, show strongly enhanced RAC1 activity in comparison to wild type (+/+) controls, and quickly become immotile in vitro. Sperm from heterozygous (t/+) males, on the other hand, display intermediate RAC1 activity, impaired progressive motility and transmission ratio distortion (TRD) in favor of t-sperm. We show that t/+-derived sperm consist of two subpopulations, highly progressive and less progressive. The majority of highly progressive sperm carry the t-haplotype, while most less progressive sperm contain the wild type (+) chromosome. Dosage-controlled RAC1 inhibition in t/+ sperm by NSC23766 rescues progressive movement of (+)-sperm in vitro, directly demonstrating that impairment of progressive motility in the latter is caused by enhanced RAC1 activity. The combined data show that RAC1 plays a pivotal role in controlling progressive motility in sperm, and that inappropriate, enhanced or reduced RAC1 activity interferes with sperm progressive movement. Differential RAC1 activity within a sperm population impairs the competitiveness of sperm cells expressing suboptimal RAC1 activity and thus their fertilization success, as demonstrated by t/+-derived sperm. In conjunction with t-haplotype triggered TRD, we propose that Rho GTPase signaling is essential for directing sperm towards the egg cells.

Contrasting conformational dynamics of ß-sheet A and helix F with implications in neuroserpin inhibition and aggregation.

Neuroserpin (NS) is an inhibitory protein of serpin super family, its shutter region variants have high propensity to aggregate leading to pathological disorders like familial encephalopathy with NS inclusion bodies (FENIB). Helix F and ß-sheet A of NS participate in the tissue plasminogen activator (tPA) inhibition but the mechanism is not yet completely understood. A microsecond (µs) molecular dynamics simulation of the helix F and strand 3A variants showed predominant fluctuations in the loop connecting the strands of ß-sheet A. Therefore to understand the role of helix F and strand 3A of ß-sheet A, cysteine was incorporated at the position N182 in stand 3A (N182C) and position W154 (W154C) in the helix F using site-directed mutagenesis. Purified variants were further labeled with Alexa Fluor488 C5 maleimide dye. Temperature dependent study using non-denaturing PAGE showed the formation of large aggregates of helix F variant W154C but not the strand 3A N182C variant. Interestingly tPA inhibition was found to be decreased in the labeled N182C with decreased tPA-complex formation as compared to labeled W154C NS variant. The fluorescence emission intensity of the labeled helix F variant W154C decreased in the presence of an increasing concentration of tPA, whereas an increase in emission intensity was observed in labeled strand 3A variant N182C, indicating more exposure of strand 3A and shielding of helix F. Taken together the data shows that helix F has a predominant role in the aggregation but a minor role in the inhibition mechanism.

Exogenous pyruvate alleviates UV-induced hyperpigmentation via restraining dendrite outgrowth and Rac1 GTPase activity.

BACKGROUND: Melanin is synthesized in melanocytes and transferred to keratinocytes through dendrites. Endogenous pyruvate is a key metabolite for ATP production in glycolysis, and the tricarboxylic acid (TCA) cycle and exogenous pyruvate provide protection against oxidative stress and acidosis in the intercellular space. The function of pyruvate in the regulation of dendrite outgrowth remains to be elucidated. OBJECTIVE: We examined the effect of pyruvate on dendritic elongation and skin pigmentation METHODS: Murine B16F10 melanoma cells and human primary melanocytes were used for in vitro analysis. Melanin quantitation and histochemical staining were performed in a 3D pigmented human skin model. RESULTS: We demonstrated the participation of monocarboxylate transporters (MCTs) responsible for the membrane transport of pyruvate in B16F10 melanoma cells. The accumulation of pyruvate occurred in a pH-dependent manner, which was highly sensitive to a specific MCT inhibitor (α-cyano-4-hydroxycinnamic acid). α-MSH-induced morphological changes, including dendrite elongation and growth-cone-like structure, were diminished in B16F10 cells upon treatment with pyruvate. In addition, the number of dendrite branches was reduced in normal human epidermal melanocytes. As the Rho-subfamily of monomeric GTP-binding proteins modulates dendrite formation, we subsequently examined the suppression of Rac1 activation by pyruvate, but not RhoA and Cdc42. Furthermore, pyruvate showed anti-melanogenic effects against UV-induced pigmentation in reconstructed pigmented epidermis, established by co-seeding autologous melanocytes and keratinocytes, which act similar to in vivo skin tissue. CONCLUSION: These results suggest that pyruvate treatment may be an alternative or additive therapeutic strategy to prevent hyperpigmentation.

Prokineticin Receptor Inhibition With PC1 Protects Mouse Primary Sensory Neurons From Neurotoxic Effects of Chemotherapeutic Drugs in vitro.

Neurotoxicity is a common side effect of chemotherapeutics that often leads to the development of chemotherapy-induced peripheral neuropathy (CIPN). The peptide Prokineticin 2 (PK2) has a key role in experimental models of CIPN and can be considered an insult-inducible endangering mediator. Since primary afferent sensory neurons are highly sensitive to anticancer drugs, giving rise to dysesthesias, the aim of our study was to evaluate the alterations induced by vincristine (VCR) and bortezomib (BTZ) exposure in sensory neuron cultures and the possible preventive effect of blocking PK2 signaling. Both VCR and BTZ induced a concentration-dependent reduction of total neurite length that was prevented by the PK receptor antagonist PC1. Antagonizing the PK system also reduced the upregulation of PK2, PK-R1, TLR4, IL-6, and IL-10 expression induced by chemotherapeutic drugs. In conclusion, inhibition of PK signaling with PC1 prevented the neurotoxic effects of chemotherapeutics, suggesting a promising strategy for neuroprotective therapies against the sensory neuron damage induced by exposure to these drugs.

Silencing PROK2 Inhibits Invasion of Human Cervical Cancer Cells by Targeting MMP15 Expression.

Cervical cancer is the second most frequent type of gynecologic cancer worldwide. Prokineticin 2 (PROK2) is reported to be involved in tumor progression in some malignant tumors. However, the role of PROK2 in the development of cervical cancer remains unknown. Our results indicate that PROK2 is overexpressed in the human cervical cancer. Cervical cancer patients with high PROK2 expression have a shorter overall survival rate (OS) and disease-free survival rate (DFS). PROK2 acts as a potential biomarker for predicting OS and DFS of cervical cancer patients. We further show that PROK2 is important factor for oncogenic migration and invasion in human cervical cancer cells. Knockdown PROK2 significantly inhibited cell migration, invasion, and MMP15 protein expression in HeLa cells. High expression of MMP15 is confirmed in the human cervical cancer, is significantly associated with the shorter overall survival rate (OS) and is correlated with PROK2 expression. Overexpression of PROK2 using PROK2 plasmid significantly reverses the function of knockdown PROK2, and further upregulates MMP15 expression, migration and invasion of human cervical cancer cells. In conclusion, our findings are the first to demonstrate the role of PROK2 as a novel and potential biomarker for clinical use, and reveal the oncogenic functions of PROK2 as therapeutic target for cervical cancer.

RNA silencing of hormonal biosynthetic genes impairs larval growth and development in cotton bollworm, Helicoverpa armigera.

The cotton bollworm, Helicoverpa armigera, is a highly polyphagous pest, causing enormous losses to various economically important crops. The identification and in vitro functional validation of target genes of a pest is a prerequisite to combat pest via host-mediated RNA interference (RNAi). In the present study, six hormonal biosynthesis genes of H. armigera were chosen and evaluated by feeding insect larvae with dsRNAs corresponding to each target gene, viz., juvenile hormone acid methyltransferase (HaJHAMT), prothoracicotropic hormone (HaPTTH), pheromone biosynthesis-activating peptide (HaPBAP), molt regulating transcription factor (HaHR3), activated protein 4 (HaAP-4) and eclosion hormone precursor (HaEHP). The loss of function phenotypes for these hormonal genes were observed by releasing second instar larvae on to artificial diet containing target gene-specific dsRNAs. Ingestion of dsRNAs resulted in mortality ranging from 60% to 90%, reduced larval weight, phenotypic deformities and delayed pupation. The quantitative real-time PCR (qRT-PCR) analysis showed that the target gene transcript levels were decreased drastically (31% to 77%) as compared to control or unrelated control (GFP-dsRNA), and correlated well with the mortality and developmental defects of larvae. Also, a comparison of the silencing efficacy of un-diced long HaPTTH -dsRNAwith RNase III diced HaPTTH-dsRNA (siRNAs) revealed that long dsRNAs were more efficient in silencing the target gene. These results indicated that the hormonal biosynthesis genes have varied sensitivity towards RNAi and could be the vital targets for insect resistance in crop plants like cotton which are infested by H. armigera.

MicroRNA-203-mediated inhibition of doublecortin underpins cardioprotection conferred by sevoflurane in rats after myocardial ischaemia-reperfusion injury.

Myocardial ischaemia-reperfusion (I/R) injury is a serious illness with high morbidity and mortality. Mounting evidence indicates the utility of sevoflurane (SEV) in the treatment of myocardial I/R injury. This study aimed to explore the molecular mechanisms underlying the protective action of SEV against myocardial I/R injury. A rat model of myocardial I/R injury was established, and I/R rats were treated with different concentrations of SEV. MicroRNA-203 (miR-203) and doublecortin (DCX) expression levels were determined using reverse transcription-quantitative polymerase chain reaction. Putative target relationship between miR-203 and DCX was explored using dual-luciferase reporter gene assay and RNA-binding protein immunoprecipitation assay. Ischaemia-reperfusion rats were treated with SEV, miR-203 antagomir or sh-DCX, followed by determination of oxidative stress- and inflammation-related factor levels using nitrite and enzyme-linked immunosorbent assays, and that of apoptosis-related factors using Western blot analysis. The apoptotic rate of myocardial tissues was determined using TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, and the infract area was evaluated using triphenyltetrazolium chloride staining. The results showed miR-203 was poorly expressed and DCX was highly expressed in myocardial tissues of I/R rats. Sevoflurane was found to elevate miR-203, and miR-203, in turn, could target and reduce DCX expression. Sevoflurane, miR-203 overexpression or DCX silencing resulted in declined oxidative stress, inflammation, apoptosis and infarct area, ultimately alleviating myocardial I/R injury. Collectively, these findings showed that SEV-activated miR-203 exhibited suppressive effects on myocardial I/R injury in rats and highlighted the SEV/miR-203/DCX axis as a promising therapeutic target for myocardial I/R injury management.

Centchroman prevents metastatic colonization of breast cancer cells and disrupts angiogenesis via inhibition of RAC1/PAK1/ß-catenin signaling axis.

AIMS: We have previously reported that Centchroman (CC), an oral contraceptive drug, inhibits breast cancer progression and metastasis. In this study, we investigated whether CC inhibits local invasion of tumor cells and/or their metastatic colonization with detailed underlying mechanisms. MAIN METHODS: The effect of CC on the experimental metastasis and spontaneous metastasis was demonstrated by using tail-vein and orthotopic 4T1-syngeneic mouse tumor models, respectively. The anti-angiogenic potential of CC was evaluated using well established in vitro and in vivo models. The role of RAC1/PAK1/ß-catenin signaling axis in the metastasis was investigated and validated using siRNA-mediated knockdown of PAK1 as well as by pharmacological PAK1-inhibitor. KEY FINDINGS: The oral administration of CC significantly suppressed the formation of metastatic lung nodules in the 4T1-syngeneic orthotopic as well as experimental metastatic models. More importantly, CC treatment suppressed the tube formation and migration capacities of human umbilical vein endothelial cells (HUVEC) and inhibited pre-existing vasculature as well as the formation of neovasculature. The suppression of migration and invasion capacities of metastatic breast cancer cells upon CC treatment was associated with the inhibition of small GTPases (Rac1 and Cdc42) concomitant with the downregulation of PAK1 and downstream ß-catenin signaling. In addition, CC upregulated the expression of miR-145, which is known to target PAK1. SIGNIFICANCE: This study warrants the repurposing of CC as a potential therapeutic agent against metastatic breast cancer.

Stress Pathway Modulation Is Detrimental or Ineffective for Functional Recovery after Spinal Cord Injury in Mice.

A mounting body of evidence suggests that stress plays a major role in the injury progression after spinal cord injury (SCI). Injury activates the stress systems; this in turn may augment the generation of pro-inflammatory cytokines, stimulate pro-inflammatory immune cells, and alter the balance between the pro- and anti-inflammatory immune response. As a result, it is suggested that stress pathways may augment neuronal damage and loss after SCI. Considering these potential detrimental effects of stress after SCI, we hypothesized that inhibition of stress pathways immediately after SCI may offer protection from damage and improve recovery. To investigate the relevance of stress responses in SCI recovery, we investigated the effects of blocking three well-studied stress response axes in a mouse model of SCI. Propranolol, RU-486, and CP-99994 were administered to inhibit the sympathetic axis, the hypothalamus-pituitary-adrenal axis, and the neuropeptide axis, respectively. Surprisingly, assessing functional recovery by the Basso Mouse Scale revealed that RU-486 and CP-99994 did not affect functional outcome, indicating that these pathways are dispensable for neuroprotection or repair after SCI. Moreover, the beta-blocker propranolol worsened functional outcome in the mouse SCI model. In conclusion, immediate inhibition of three major stress axes has no beneficial effects on functional recovery after SCI in mice. These results suggest that injury-induced stress responses do not interfere with the healing process and hence, pharmacological targeting of stress responses is not a recommended treatment option for SCI. These findings are of great importance for other researchers to avoid unnecessary and potentially futile animal experiments.

A bidirectional effect of Rac1 inhibition-Protects radiation-induced intestinal injury while inhibits tumor.

AIMS: To investigate whether Rac1 inhibition can alleviate radiation-induced intestinal injury (RIII), meanwhile exist no protection on tumors. MATERIALS AND METHODS: Rac1 inhibition was achieved by its specific inhibitor, NSC23766. Mice were pretreated with different intraperitoneal injections, which were normal saline for NS group (N = 9), and 2.5 mg/kg and 5 mg/kg of NSC23766 for Low-Dose group (N = 9) and High-Dose group (N = 9), respectively. After total body irritation (10Gy), small intestinal tissues were collected for Hematoxylin-Eosin (H&E) staining and Terminal-deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling (TUNEL). Intestinal epithelial and tumor cell lines, namely MODE-k and CT-26, were used to further study the role of Rac1 inhibition on radiation damage. Flow cytometry was used to detect changes in reactive oxygen species production, cell cycles and mitochondrial membrane potential, the latter was also checked by fluorescence microscope. Changes of protein-expression associated with apoptosis and cell cycles were detected by Western blotting to explain the possible molecular mechanism. KEY FINDINGS: Height of intestine villi and depth of crypt were higher (P < 0.01) and apoptosis ratio lower (P < 0.01) in High-Dose group compared with those in NS group. After radiation, Rac1 inhibition pre-treatment improved the vitality (P < 0.01) and reduced the apoptosis (P < 0.01) in MODE-k while yielded opposite results in CT-26, and reduced ROS production of MODE-k (P < 0.01) while had little effect on that of CT-26. Rac1 inhibition differently affected the cell cycles of normal cells and that of tumor cells. SIGNIFICANCE: Inhibition of Rac1 could alleviate RIII, meanwhile assist the killing effect of radiation on tumor cells.

Neuroserpin regulates human T cell-T cell interactions and proliferation through inhibition of tissue plasminogen activator.

T cells play a key role in mounting an adaptive immune response. T cells are activated upon recognition of cognate Ag presented by an APC. Subsequently, T cells adhere to other activated T cells to form activation clusters, which lead to directed secretion of cytokines between communicating cells. T cell activation clusters have been implicated in regulating activation, proliferation, and memory formation in T cells. We previously reported the expression of the protease inhibitor neuroserpin by human T cells and showed that expression and intracellular localization is regulated following T cell activation. To gain a better understanding of neuroserpin in the proteolytic environment postactivation we assessed its role in human T cell clustering and proliferation. Neuroserpin knockdown increased T cell proliferation and cluster formation following T cell activation. This increased cluster formation was dependent on the proteases tissue plasminogen activator (tPA) and plasmin. Furthermore, neuroserpin knockdown or plasmin treatment of T cells increased the cleavage of annexin A2, a known plasmin target that regulates the actin cytoskeleton. Live cell imaging of activated T cells further indicated a role of the actin cytoskeleton in T cell clustering. The inhibition of actin regulators myosin ATPase and Rho-associated protein kinase signaling completely reversed the neuroserpin knockdown-induced effects. The results presented in this study reveal a novel role for neuroserpin and the proteolytic environment in the regulation of T cell activation biology.

Interplay between α2-chimaerin and Rac1 activity determines dynamic maintenance of long-term memory.

Memory consolidation theory suggests that once memory formation has been completed, memory is maintained at a stable strength and is incapable of further enhancement. However, the current study reveals that even long after formation, contextual fear memory could be further enhanced. Such unexpected enhancement is possible because memory is dynamically maintained at an intermediate level that allows for bidirectional regulation. Here we find that both Rac1 activation and expression of α2-chimaerin are stimulated by single-trial contextual fear conditioning. Such sustained Rac1 activity mediates reversible forgetting, and α2-chimaerin acts as a memory molecule that reverses forgetting to sustain memory through inhibition of Rac1 activity during the maintenance stage. Therefore, the balance between activated Rac1 and expressed α2-chimaerin defines dynamic long-term memory maintenance. Our findings demonstrate that consolidated memory maintains capacity for bidirectional regulation.

New roles for prokineticin 2 in feeding behavior, insulin resistance and type 2 diabetes: Studies in mice and humans.

OBJECTIVE: Prokineticin 2 (PROK2) is a hypothalamic neuropeptide that plays a critical role in the rhythmicity of physiological functions and inhibits food intake. PROK2 is also expressed in the main olfactory bulb (MOB) as an essential factor for neuro-and morphogenesis. Since the MOB was shown to be strongly involved in eating behavior, we hypothesized that PROK2 could be a new target in the regulation of food intake and energy homeostasis, through its effects in the MOB. We also asked whether PROK2 could be associated with the pathophysiology of obesity, the metabolic syndrome (MetS), and type 2 diabetes (T2D) in humans. METHODS: We assessed in wild type mice whether the expression of Prok2 in the MOB is dependent on the nutritional status. We measured the effect of human recombinant PROK2 (rPROK2) acute injection in the MOB on food intake and olfactory behavior. Then, using a lentivirus expressing Prok2-shRNA, we studied the effects of Prok2 underexpression in the MOB on feeding behavior and glucose metabolism. Metabolic parameters and meal pattern were determined using calorimetric cages. In vivo 2-deoxyglucose uptake measurements were performed in mice after intraperitoneally insulin injection. Plasmatic PROK2 dosages and genetic associations studies were carried out respectively on 148 and more than 4000 participants from the D.E.S.I.R. (Data from an Epidemiologic Study on the Insulin Resistance Syndrome) cohort. RESULTS: Our findings showed that fasting in mice reduced Prok2 expression in the MOB. Acute injection of rPROK2 in the MOB significantly decreased food intake whereas Prok2-shRNA injection resulted in a higher dietary consumption characterized by increased feeding frequency and decreased meal size. Additionally, Prok2 underexpression in the MOB induced insulin resistance compared to scrambled shRNA-injected mice. In the human D.E.S.I.R. cohort, we found a significantly lower mean concentration of plasma PROK2 in people with T2D than in those with normoglycemia. Interestingly, this decrease was no longer significant when adjusted for Body Mass Index (BMI) or calorie intake, suggesting that the association between plasma PROK2 and diabetes is mediated, at least partly, by BMI and feeding behavior in humans. Moreover, common Single Nucleotide Polymorphisms (SNPs) in PROK2 gene were genotyped and associated with incident T2D or impaired fasting glycemia (IFG), MetS, and obesity. CONCLUSIONS: Our data highlight PROK2 as a new target in the MOB that links olfaction with eating behavior and energy homeostasis. In humans, plasma PROK2 is negatively correlated with T2D, BMI, and energy intake, and PROK2 genetic variants are associated with incident hyperglycemia (T2D/IFG), the MetS and obesity.