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Lipopolysaccharides (LPS) are major components of Gram-negative bacteria. LPS not only induce endotoxemia and inflammation, but also contribute to various diseases. In experimental settings, LPS administration serves as a model for acute inflammatory responses. This study aims to evaluate the anti-inflammatory potential and mechanism of action of palmitoylated prolactin-releasing peptide (palm11-PrRP31) in a rat model of LPS-induced inflammation. Palm11-PrRP31 has demonstrated its efficacy in mitigating LPS-induced weight loss and anorexia, emphasizing its potential protective effects. Cytokine profiles revealed a consistent reduction in tumor necrosis factor α, highlighting the potent anti-inflammatory effects of palm11-PrRP31. The peptide also modulated key cytokines and chemokines in plasma, the liver, and the hypothalamus, reflecting its broad-spectrum anti-inflammatory properties. Palm11-PrRP31 also effectively attenuated the expression levels of TLR4 signaling components in the liver, suggesting its suppressive effects on the activation of these pathways during LPS-induced inflammation. These anti-inflammatory effects were specific to palm11-PrRP31, whereas natural PrRP31 had minimal impact. In conclusion, this study reveals the efficacy of palm11-PrRP31 in modulating LPS-induced inflammation, offering insights into its immunomodulatory properties. The abilities of the peptide to suppress proinflammatory responses and attenuate relevant signaling pathways indicate its potential use as a therapeutic agent for inflammatory disorders.

In our project, ghrelin analogs possessing enhanced stability and potential to significantly increase food intake were used. Three newly synthesized ghrelin analogs with fatty acid residues consisting of 8, 10, and 14 carbon atoms were studied. The main goal of this work was to develop a suitable analytical method for the determination of the stability of the novel ghrelin analogs in plasma. An appropriate liquid chromatography-mass spectrometry method was developed and optimized. The results obtained were compared with the data measured by using a commercial enzyme-linked immunosorbent assay kit, and a good correlation was found. A preparation strategy for plasma samples was optimized and consisted of simple dilution of the plasma samples followed by direct injection onto a very short monolithic column in combination with mass spectrometric detection. The developed analytical method was utilized for the determination of the stability of the prepared lipopeptides in plasma and for the quantification of the lipopeptides in a preliminary pharmacokinetic study. The feasibility of the developed separation method was clearly demonstrated. Accuracy and precision were within 80-120% and ±20% limits, respectively. Calibration curves were constructed in the range of 1-250 μg/mL., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Prolactin-releasing peptide (PrRP) is an anorexigenic neuropeptide expressed in the brain where it regulates food intake and energy expenditure. The C-terminal Arg-Phe-NH2 of PrRP is crucial for its biological activity. In our previous study, we showed that PrRP analogs myristoylated or palmitoylated at the N- terminus seem to cross the blood-brain barrier and lower food intake following peripheral administration. In this study, myristoylated and palmitoylated PrRP31 analogs with a modified C-terminal Phe were designed and tested. Lipidized analogs containing Phe(31) replaced by aromatic non-coded amino acids or tyrosine revealed high binding affinity to rat pituitary RC-4B/C cells with endogenous PrRP and neuropeptide FF 2 receptors and to CHO-K1 cells overexpressing either PrRP or neuropeptide FF 2 receptors. The analogs also showed strong agonistic properties at the GPR10 receptor using the beta-lactamase reporter gene assay. Moreover, lipidized PrRP analogs, especially those that were palmitoylated, demonstrated strong and long-lasting anorexigenic effects in fasted mice after subcutaneous administration. The most efficient PrRP31 analogs with PheCl2(31), either palmitoylated or myristoylated at the N-terminus, are promising candidates for the study of food disorders, possibly for anti-obesity treatment. Despite the therapeutic potential in targeting central GPR10, the endogenous ligand PrRP cannot cross the blood-brain barrier. Understanding biological activity and transport of novel structural analogs of PrRP with a potential central anorexigenic effect is of key therapeutic significance.

Catecholamines participate in the food intake regulation, however, there are no literature data available, dealing with the activity of tyrosine hydroxylase (TH) neurons in response to stimulation or inhibition of GHS-R (growth hormone secretagogue receptor) in the hypothalamic arcuate nucleus (ARC). The present study was focused to reveal whether [Dpr(N-octanoyl) 3ghrelin], a stable GHS-R agonist, itself in doses of 5 or 10 mg/kg (s.c.) or in combination with GHS-R receptor antagonist ([DLys3]GHRP-6) in dose of 10 mg/kg (s.c.), may affect the activity of ARC TH-containing neurons in C57BL/6 male mice fed either with standard (SD) or high fat diet (HFD) that developed a diet-induced obesity (DIO). The data of the present study clearly indicate that both doses of GHS-R agonist stimulated food intake in SD mice and GHS-R antagonist significantly reduced GHS-R agonist orexinergic effect in SD mice and suppressed the voluntary food intake in HFD mice. Both doses of the GHS-R agonist stimulated Fos expression in ARC neurons in both diet groups of mice which was not abolished by GHS-R antagonist pretreatment. Moreover, both doses of the GHS-R agonist significantly influenced the activation of TH neurons in the ARC of SD mice. The GHS-R antagonist also significantly increased TH neurons activation after GHS-R agonist although this effect was less powerful in HFD mice. This is the first study demonstrating response of local ARC TH neurons to peripherally applied GHS-R agonist and antagonist. The present data point out that the response of TH neurons to GHS-R agonist and antagonist is different in normal and DIO mice and extend our knowledge about the further ARC neuronal phenotype responding to peripheral ghrelin. To bring insight into the understanding of the functional significance of the activated TH neurons in ARC, in the context of the ghrelin peripheral increase, further studies are required.

Objective: It is generally known that hypocretin (Hcrt) neurons in lateral hypothalamus (LH) are involved in feeding behaviour. The aim of this study was to reveal the activity response of Hcrt neurons, as measured by Fos protein incidence, to prolonged high fat (HF) diet in the LH of both genders of C57B1/6 mice., Methods: Standard (St) and high fat (HF) diets were available to mice for 16 weeks and thereafter the animals were perfused transcardially with fixative. Then the brains were removed, soaked with 15 % sucrose in 0.1 M phosphate buffer (PB), and cryo-sectioned throughout the hypothalamus into 35 microm thick coronal sections. Fos/Hcrt co-localizations were processed by employing avidin-biotin-peroxidase (ABC) complex and diaminobenzidine chromogen for Hcrt labeling. Fos immunoproduct was intensified by nickel chloride as a black color inducer. Evaluation of the incidence of Fos/Hcrt co-labeled perikarya was performed using a computerized Leica light microscopy., Results: The effect of of mice gender, applied diet, and gender plus applied diet on the activation of Hcrt neurons was found. Turkey s test revealed significant (p<0.05) rise in Fos labeled Hcrt neurons in male vs. female mice after consumption of both types of diets: St (44.64 +/- 2.28 % vs. 1.47 +/- 0.195 %, resp.) and HF (44.15 +/- 3.77 % vs. 24.32 +/- 0.7 %, resp.). This showed that HF diet significantly elevated the number of activated Hcrt neurons only in female mice (24.32 +/- 0.7 % in HF fed vs. 1.47 +/- 0.195 % in St fed, p<0.05). The body weight and accumulation of body fat in animals (body fat weight expressed as % of body weight) were influenced by gender and applied diet, although the body fat weight was influenced by HF diet (more noticeably in females)., Conclusion: The data indicated a positive correlation between body weight, fat gain, and Hcrt activities in females but not in males, thus accentuating the importance of the gender impact.