The preparation of specifically iodine-125 (125I)-labeled peptides of high purity and specific activity represents a key tool for the detailed characterization of their binding properties in interaction with their binding partners. Early synthetic methods for the incorporation of iodine faced challenges such as harsh reaction conditions, the use of strong oxidants and low reproducibility. Herein, we review well-established radiolabeling strategies available to incorporate radionuclide into a protein of interest, and our long-term experience with a mild, simple and generally applicable technique of 125I late-stage-labeling of biomolecules using the Pierce iodination reagent for the direct solid-phase oxidation of radioactive iodide. General recommendations, tips, and details of optimized chromatographic conditions to isolate pure, specifically 125I-mono-labeled biomolecules are illustrated on a diverse series of (poly)peptides, ranging up to 7.6 kDa and 67 amino acids (aa). These series include peptides that contain at least one tyrosine or histidine residue, along with those featuring disulfide crosslinking or lipophilic derivatization. This mild and straightforward late-stage-labeling technique is easily applicable to longer and more sensitive proteins, as demonstrated in the cases of the insulin-like growth factor binding protein-3 (IGF-BP-3) (29 kDa and 264 aa) and the acid-labile subunit (ALS) (93 kDa and 578 aa).

• Keywords
• 125I-labeling of peptides, High specific activity, Intramolecular effect of 125I decay, Late-stage peptide labeling, Radiochemical stability, Radiohalogenated prosthetic groups, Site-specific labeling,
• Publication type
• Journal Article MeSH
• Review MeSH

Obesity and type 2 diabetes mellitus (T2DM) are preconditions for the development of metabolic syndrome, which is reaching pandemic levels worldwide, but there are still only a few anti-obesity drugs available. One of the promising tools for the treatment of obesity and related metabolic complications is anorexigenic peptides, such as prolactin-releasing peptide (PrRP). PrRP is a centrally acting neuropeptide involved in food intake and body weight (BW) regulation. In its natural form, it has limitations for peripheral administration; thus, we designed analogs of PrRP lipidized at the N-terminal region that showed high binding affinities, increased stability and central anorexigenic effects after peripheral administration. In this review, we summarize the preclinical results of our chronic studies on the pharmacological role of the two most potent palmitoylated PrRP31 analogs in various mouse and rat models of obesity, glucose intolerance, and insulin resistance. We used mice and rats with diet-induced obesity fed a high-fat diet, which is considered to simulate the most common form of human obesity, or rodent models with leptin deficiency or disrupted leptin signaling in which long-term food intake regulation by leptin is distorted. The rodent models described in this review are models of metabolic syndrome with different severities, such as obesity or morbid obesity, prediabetes or diabetes and hypertension. We found that the effects of palmitoylated PrRP31 on food intake and BW but not on glucose intolerance require intact leptin signaling. Thus, palmitoylated PrRP31 analogs have potential as therapeutics for obesity and related metabolic complications.

• Keywords
• leptin resistance, obesity, prolactin-releasing peptide, rodent models, type 2 diabetes,
• Publication type
• Journal Article MeSH
• Review MeSH

The anorexigenic neuropeptide prolactin-releasing peptide (PrRP) is involved in the regulation of food intake and energy expenditure. Lipidization of PrRP stabilizes the peptide, facilitates central effect after peripheral administration and increases its affinity for its receptor, GPR10, and for the neuropeptide FF (NPFF) receptor NPFF-R2. The two most potent palmitoylated analogs with anorectic effects in mice, palm11-PrRP31 and palm-PrRP31, were studied in vitro to determine their agonist/antagonist properties and mechanism of action on GPR10, NPFF-R2 and other potential off-target receptors related to energy homeostasis. Palmitoylation of both PrRP31 analogs increased the binding properties of PrRP31 to anorexigenic receptors GPR10 and NPFF-R2 and resulted in a high affinity for another NPFF receptor, NPFF-R1. Moreover, in CHO-K1 cells expressing GPR10, NPFF-R2 or NPFF-R1, palm11-PrRP and palm-PrRP significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (Akt) and cAMP-responsive element-binding protein (CREB). Palm11-PrRP31, unlike palm-PrRP31, did not activate either c-Jun N-terminal kinase (JNK), p38, c-Jun, c-Fos or CREB pathways in cells expressing NPFF-1R. Palm-PrRP31 also has higher binding affinities for off-target receptors, namely, the ghrelin, opioid (KOR, MOR, DOR and OPR-L1) and neuropeptide Y (Y1, Y2 and Y5) receptors. Palm11-PrRP31 exhibited fewer off-target activities; therefore, it has a higher potential to be used as an anti-obesity drug with anorectic effects.

• Keywords
• GPR10, NPFF-R1, NPFF-R2, binding properties, neuropeptide FF, prolactin-releasing peptide, signaling pathways,
• MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Prolactin-Releasing Hormone chemistry   genetics   metabolism   MeSH
• Cricetinae MeSH
• Humans MeSH
• Lipoylation * MeSH
• Receptors, Neuropeptide genetics   metabolism   MeSH
• Receptors, G-Protein-Coupled genetics   metabolism   MeSH
• In Vitro Techniques MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Prolactin-Releasing Hormone MeSH
• neuropeptide FF receptor MeSH Browser
• PRLH protein, human MeSH Browser
• PRLHR protein, human MeSH Browser
• Receptors, Neuropeptide MeSH
• Receptors, G-Protein-Coupled MeSH
• Calcium MeSH

Prolactin-releasing peptide (PrRP), a natural ligand for the GPR10 receptor, is a neuropeptide with anorexigenic and antidiabetic properties. Due to its role in the regulation of food intake, PrRP is a potential drug for obesity treatment and associated type 2 diabetes mellitus (T2DM). Recently, the neuroprotective effects of lipidized PrRP analogs have been proven. In this study, we focused on the molecular mechanisms of action of natural PrRP31 and its lipidized analog palm11-PrRP31 in the human neuroblastoma cell line SH-SY5Y to describe their cellular signaling and possible anti-apoptotic properties. PrRP31 significantly upregulated the phosphoinositide-3 kinase-protein kinase B/Akt (PI3K-PKB/Akt) and extracellular signal-regulated kinase/cAMP response element-binding protein (ERK-CREB) signaling pathways that promote metabolic cell survival and growth. In addition, we proved via protein kinase inhibitors that activation of signaling pathways is mediated specifically by PrRP31 and its palmitoylated analog. Furthermore, the potential neuroprotective properties were studied through activation of anti-apoptotic pathways of PrRP31 and palm11-PrRP31 using the SH-SY5Y cell line and rat primary neuronal culture stressed with toxic methylglyoxal (MG). The results indicate increased viability of the cells treated with PrRP and palm11-PrRP31 and a reduced degree of apoptosis induced by MG, suggesting their potential use in the treatment of neurological disorders.

• Keywords
• SH-SY5Y, cellular signaling, inhibitors, methylglyoxal, neuroprotection, primary neuronal culture, prolactin-releasing peptide,
• MeSH
• Apoptosis * MeSH
• Prolactin-Releasing Hormone chemistry   pharmacology   MeSH
• Humans MeSH
• Tumor Cells, Cultured MeSH
• Neuroblastoma drug therapy   metabolism   pathology   MeSH
• Neuropeptides chemistry   pharmacology   MeSH
• Neuroprotective Agents chemistry   pharmacology   MeSH
• Apoptosis Regulatory Proteins metabolism   MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Prolactin-Releasing Hormone MeSH
• Neuropeptides MeSH
• Neuroprotective Agents MeSH
• Apoptosis Regulatory Proteins MeSH

Prolactin-releasing peptide (PrRP) belongs to the large RF-amide neuropeptide family with a conserved Arg-Phe-amide motif at the C-terminus. PrRP plays a main role in the regulation of food intake and energy expenditure. This review focuses not only on the physiological functions of PrRP, but also on its pharmacological properties and the actions of its G-protein coupled receptor, GPR10. Special attention is paid to structure-activity relationship studies on PrRP and its analogs as well as to their effect on different physiological functions, mainly their anorexigenic and neuroprotective features and the regulation of the cardiovascular system, pain, and stress. Additionally, the therapeutic potential of this peptide and its analogs is explored.

• Keywords
• GPR10, RF-amide peptides, energy expenditure, food intake regulation, neuroprotection, prolactin-releasing peptide, signaling,
• MeSH
• Energy Metabolism drug effects   MeSH
• Prolactin-Releasing Hormone chemistry   metabolism   pharmacology   MeSH
• Humans MeSH
• Neurodegenerative Diseases drug therapy   pathology   MeSH
• Neuroprotective Agents chemistry   pharmacology   therapeutic use   MeSH
• Eating drug effects   MeSH
• Receptors, G-Protein-Coupled chemistry   genetics   metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Prolactin-Releasing Hormone MeSH
• Neuroprotective Agents MeSH
• PRLHR protein, human MeSH Browser
• Receptors, G-Protein-Coupled MeSH

BACKGROUND/OBJECTIVES: Prolactin-releasing peptide (PrRP) has a potential to decrease food intake and ameliorate obesity, but is ineffective after peripheral administration. We have previously shown that our novel lipidized analogs PrRP enhances its stability in the circulation and enables its central effect after peripheral application. The purpose of this study was to explore if sub-chronic administration of novel PrRP analog palmitoylated in position 11 (palm11-PrRP31) to Koletsky-spontaneously hypertensive obese rats (SHROB) could lower body weight and glucose intolerance as well as other metabolic parameters. SUBJECTS/METHODS: The SHROB rats (n = 16) were used for this study and age-matched hypertensive lean SHR littermates (n = 16) served as controls. Palm11-PrRP31 was administered intraperitoneally to SHR and SHROB (n = 8) at a dose of 5 mg/kg once-daily for 3 weeks. During the dosing period food intake and body weight were monitored. At the end of the experiment the oral glucose tolerance test was performed; plasma and tissue samples were collected. Thereafter, arterial blood pressure was measured. RESULTS: At the end of the experiment, vehicle-treated SHROB rats showed typical metabolic syndrome parameters, including obesity, glucose intolerance, dyslipidemia, and hypertension. Peripheral treatment with palm11-PrRP31 progressively decreased the body weight of SHR rats but not SHROB rats, though glucose tolerance was markedly improved in both strains. Moreover, in SHROB palm11-PrRP31 ameliorated the HOMA index, insulin/glucagon ratio, and increased insulin receptor substrate 1 and 2 expression in fat and insulin signaling in the hypothalamus, while it had no effect on blood pressure. CONCLUSIONS: We demonstrated that our new lipidized PrRP analog is capable of improving glucose tolerance in obese SHROB rats after peripheral application, suggesting that its effect on glucose metabolism is independent of leptin signaling and body weight lowering. These data suggest that this analog has the potential to be a compound with both anti-obesity and glucose-lowering properties.

• MeSH
• Glucagon blood   MeSH
• Glucose Tolerance Test MeSH
• Prolactin-Releasing Hormone administration & dosage   analogs & derivatives   pharmacology   therapeutic use   MeSH
• Hypertension blood   drug therapy   MeSH
• Insulin blood   metabolism   MeSH
• Insulin Resistance MeSH
• Blood Glucose metabolism   MeSH
• Blood Pressure drug effects   MeSH
• Lipids blood   MeSH
• Metabolic Syndrome * blood   drug therapy   metabolism   MeSH
• Brain drug effects   metabolism   MeSH
• Obesity * blood   drug therapy   MeSH
• Glucose Intolerance * blood   drug therapy   MeSH
• Rats, Inbred SHR MeSH
• Insulin Receptor Substrate Proteins metabolism   MeSH
• Body Weight drug effects   MeSH
• Adipose Tissue drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glucagon MeSH
• Prolactin-Releasing Hormone MeSH
• Insulin MeSH
• Blood Glucose MeSH
• Lipids MeSH
• Insulin Receptor Substrate Proteins MeSH

Analogs of anorexigenic neuropeptides, such as prolactin-releasing peptide (PrRP), have a potential as new anti-obesity drugs. In our previous study, palmitic acid attached to the N-terminus of PrRP enabled its central anorexigenic effects after peripheral administration. In this study, two linkers, γ-glutamic acid at Lys11 and a short, modified polyethylene glycol at the N-terminal Ser and/or Lys11, were applied for the palmitoylation of PrRP31 to improve its bioavailability. These analogs had a high affinity and activation ability to the PrRP receptor GPR10 and the neuropeptide FF2 receptor, as well as short-term anorexigenic effect similar to PrRP palmitoylated at the N-terminus. Two-week treatment with analogs that were palmitoylated through linkers to Lys11 (analogs 1 and 2), but not with analog modified both at the N-terminus and Lys11 (analog 3) decreased body and liver weights, insulin, leptin, triglyceride, cholesterol and free fatty acid plasma levels in a mouse model of diet-induced obesity. Moreover, the expression of uncoupling protein-1 was increased in brown fat suggesting an increase in energy expenditure. In addition, treatment with analogs 1 and 2 but not analog 3 significantly decreased urinary concentrations of 1-methylnicotinamide and its oxidation products N-methyl-2-pyridone-5-carboxamide and N-methyl-4-pyridone-3-carboxamide, as shown by NMR-based metabolomics. This observation confirmed the previously reported increase in nicotinamide derivatives in obesity and type 2 diabetes mellitus and the effectiveness of analogs 1 and 2 in the treatment of these disorders.

• MeSH
• beta-Lactamases metabolism   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Diet * MeSH
• Prolactin-Releasing Hormone chemistry   metabolism   MeSH
• Binding, Competitive MeSH
• Cricetinae MeSH
• Metabolomics MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Obesity etiology   metabolism   MeSH
• Peptides chemistry   pharmacology   MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• beta-Lactamases MeSH
• Prolactin-Releasing Hormone MeSH
• Peptides MeSH

OBJECTIVES: Obesity is a frequent metabolic disorder but an effective therapy is still scarce. Anorexigenic neuropeptides produced and acting in the brain have the potential to decrease food intake and ameliorate obesity but are ineffective after peripheral application. We have designed lipidized analogs of prolactin-releasing peptide (PrRP), which is involved in energy balance regulation as demonstrated by obesity phenotypes of both PrRP- and PrRP-receptor-knockout mice. RESULTS: Lipidized PrRP analogs showed binding affinity and signaling in PrRP receptor-expressing cells similar to natural PrRP. Moreover, these analogs showed high binding affinity also to anorexigenic neuropeptide FF-2 receptor. Peripheral administration of myristoylated and palmitoylated PrRP analogs to fasted mice induced strong and long-lasting anorexigenic effects and neuronal activation in the brain areas involved in food intake regulation. Two-week-long subcutaneous administration of palmitoylated PrRP31 and myristoylated PrRP20 lowered food intake, body weight and improved metabolic parameters, and attenuated lipogenesis in mice with diet-induced obesity. CONCLUSIONS: Our data suggest that the lipidization of PrRP enhances stability and mediates its effect in central nervous system. Strong anorexigenic and body-weight-reducing effects make lipidized PrRP an attractive candidate for anti-obesity treatment.

• MeSH
• Energy Metabolism MeSH
• Prolactin-Releasing Hormone analogs & derivatives   pharmacology   MeSH
• Anti-Obesity Agents pharmacology   MeSH
• Lipids chemistry   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Obesity prevention & control   MeSH
• Half-Life MeSH
• Eating MeSH
• Appetite Regulation MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Prolactin-Releasing Hormone MeSH
• Anti-Obesity Agents MeSH
• Lipids MeSH