The effect of melanocortin peptides on Interleukin 1-beta induced chondrocyte cell death and inflammation

ntroduction: Osteoarthritis (OA) is a degenerative joint disease affecting more than 8.5 million people in the UK. Disruption in the catabolic and anabolic balance, with the catabolic cytokine Interleukin 1 beta (IL-1β) being involved in the initiation and progression of OA (1). Melanocortin peptides (α-MSH and D[Trp8]-γ-MSH) exert their anti-inflammatory effects via activation of melanocortin receptors (MC), with both MC1 and MC3 being identified as promising candidates as novel targets for OA (2). This study aims to assess the chondroprotective and anti-inflammatory effects of the pan melanocortin receptor agonist α-MSH and MC3 agonist D[Trp8]-γ-MSH following IL-1β chondrocyte stimulation. Methods: RT-PCR/ Western Blot: Human C-20/A4 chondrocytic cell-line were cultured in 6 well plates (1x106 cells/well) and harvested to determine MC and IL-1β expression by RT-PCR, and Western Blot. Cell-Culture: Cells were cultured in 96 well plates (1x106 cells/well) and stimulated with H2O2 (0.3%), TNF-α (60 pg/ml) or IL-1β (0-5000pg/ml) for 0-72h and cell viability determined. Drug Treatment: In separate experiments cells were pre-treated with 3 μg/ml α-MSH (Sigma-Aldrich Inc. Poole, UK), or D[Trp8]-γ-MSH (Phoenix Pharmaceuticals, Karlsrhue, Germany) (all dissolved in PBS) for 30 minutes prior to IL-1β (5000pg/ml) stimulation for 6-24h. Analysis: Cell viability was determined by using the three cell viability assays; Alamar Blue, MTT and the Neutral Red (NR) assay. Cell-free supernatants were collected and analysed for Interleukin -6 (IL-6) and IL-8 release by ELISA. Data expressed as Mean ± SD of n=4-8 determination in quadruplicate. *p≤ 0.05 vs. control. Results: Both RT-PCR, and Western Blot showed MC1 and MC3 expression on C-20/A4 cells. Cell viability analysis: IL-1β stimulation led to a maximal cell death of 35% at 6h (Alamar Blue), and 40% and 75% with MTT and Neutral Red respectively at 24h compared to control. The three cell viability assays have different cellular uptake pathways, which accounts for the variations observed in cell viability in response to the concentration of IL-1β, and time. Cytokine analysis by ELISA: IL-1β (5000pg/ml) stimulation for 6 and 24h showed maximal IL-6 production 292.3 ±3.8 and 275.5 ±5.0 respectively, and IL-8 production 353.3 ±2.6 and 598.3 ±8.6 respectively. Pre-treatment of cells with α-MSH and D[Trp8]-γ-MSH caused significant reductions in both IL-6 and IL-8 respectively following IL-1β stimulation at 6h. Conclusion: MC1/3 are expressed on C-20/A4 cells, activation by melanocortin peptides led to an inhibition of IL-1β induced cell death and pro-inflammatory cytokine release.

Laurdan Spectrum Decomposition as a Tool for the Analysis of Surface Bilayer Structure and Polarity: a Study with DMPG, Peptides and Cholesterol

The highly hydrophobic fluorophore Laurdan (6-dodecanoyl-2-(dimethylaminonaphthalene)) has been widely used as a fluorescent probe to monitor lipid membranes. Actually, it monitors the structure and polarity of the bilayer surface, where its fluorescent moiety is supposed to reside. The present paper discusses the high sensitivity of Laurdan fluorescence through the decomposition of its emission spectrum into two Gaussian bands, which correspond to emissions from two different excited states, one more solvent relaxed than the other. It will be shown that the analysis of the area fraction of each band is more sensitive to bilayer structural changes than the largely used parameter called Generalized Polarization, possibly because the latter does not completely separate the fluorescence emission from the two different excited states of Laurdan. Moreover, it will be shown that this decomposition should be done with the spectrum as a function of energy, and not wavelength. Due to the presence of the two emission bands in Laurdan spectrum, fluorescence anisotropy should be measured around 480 nm, to be able to monitor the fluorescence emission from one excited state only, the solvent relaxed state. Laurdan will be used to monitor the complex structure of the anionic phospholipid DMPG (dimyristoyl phosphatidylglycerol) at different ionic strengths, and the alterations caused on gel and fluid membranes due to the interaction of cationic peptides and cholesterol. Analyzing both the emission spectrum decomposition and anisotropy it was possible to distinguish between effects on the packing and on the hydration of the lipid membrane surface. It could be clearly detected that a more potent analog of the melanotropic hormone alpha-MSH (Ac-Ser(1)-Tyr(2)-Ser(3)-Met(4)-Glu(5)-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2)) was more effective in rigidifying the bilayer surface of fluid membranes than the hormone, though the hormone significantly decreases the bilayer surface hydration.

Laurdan Spectrum Decomposition as a Tool for the Analysis of Surface Bilayer Structure and Polarity: a Study with DMPG, Peptides and Cholesterol

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

α-Melanocyte stimulating hormone promotes muscle glucose uptake via melanocortin 5 receptors

OBJECTIVE: Central melanocortin pathways are well-established regulators of energy balance. However, scant data exist about the role of systemic melanocortin peptides. We set out to determine if peripheral α-melanocyte stimulating hormone (α-MSH) plays a role in glucose homeostasis and tested the hypothesis that the pituitary is able to sense a physiological increase in circulating glucose and responds by secreting α-MSH.

METHODS: We established glucose-stimulated α-MSH secretion using humans, non-human primates, and mouse models. Continuous α-MSH infusions were performed during glucose tolerance tests and hyperinsulinemic-euglycemic clamps to evaluate the systemic effect of α-MSH in glucose regulation. Complementary ex vivo and in vitro techniques were employed to delineate the direct action of α-MSH via the melanocortin 5 receptor (MC5R)-PKA axis in skeletal muscles. Combined treatment of non-selective/selective phosphodiesterase inhibitor and α-MSH was adopted to restore glucose tolerance in obese mice.

RESULTS: Here we demonstrate that pituitary secretion of α-MSH is increased by glucose. Peripheral α-MSH increases temperature in skeletal muscles, acts directly on soleus and gastrocnemius muscles to significantly increase glucose uptake, and enhances whole-body glucose clearance via the activation of muscle MC5R and protein kinase A. These actions are absent in obese mice, accompanied by a blunting of α-MSH-induced cAMP levels in skeletal muscles of obese mice. Both selective and non-selective phosphodiesterase inhibition restores α-MSH induced skeletal muscle glucose uptake and improves glucose disposal in obese mice.

CONCLUSION: These data describe a novel endocrine circuit that modulates glucose homeostasis by pituitary α-MSH, which increases muscle glucose uptake and thermogenesis through the activation of a MC5R-PKA-pathway, which is disrupted in obesity.

Evidence of a possible role for Lys-gamma 3-MSH in the regulation of adipocyte function

Lys-gamma 3-MSH is a melanocortin peptide derived from the C-terminal of the 16 kDa fragment of POMC. The physiological role of Lys-gamma 3-MSH is unclear, although it has previously been shown that, although not directly steroidogenic, it can act to potentiate the steroidogenic response of adrenal cortical cells to ACTH. This synergistic effect appears to be correlated with an ability to increase the activity of hormone sensitive lipase (HSL) and therefore the rate of cholesterol ester hydrolysis. Ligand binding studies have suggested that high-affinity binding sites for Lys-gamma 3-MSH exist in the adrenal gland and a number of other rat tissues that express HSL, including adipose, skeletal muscle and testes. To investigate the hypothesis that Lys-gamma 3-MSH may play a wider role in cholesterol and lipid metabolism, we tested the effect of Lys-gamma 3-MSH on lipolysis, an HSL-mediated process, in 3T3-L1 adipocytes. In comparison with other melanocortin peptides, Lys-gamma 3-MSH was found to be a potent stimulator of lipolysis. It was also able to phosphorylate HSL at key serine residues and stimulate the hyper-phosphorylation of perilipin A. The receptor through which the lipolytic actions of Lys-gamma 3-MSH are being mediated is not clear. Attempts to characterise this receptor suggest that either the pharmacology of the melanocortin receptor 5 in 3T3-L1 adipocytes is different from that described when expressed in heterologous systems or the possibility that a further, as yet uncharacterised, receptor exists.

Neuroendocrine control of a dynamic communication signal

Multiple physiological systems regulate the electric communication signal of the weakly electric gymnotiform fish, Brachyhypopomus pinnicaudatus. Fish were injected with neuroendocrine probes which identified pharmacologically relevant serotonin (5-HT) receptors similar to the mammalian 5-HT1AR and 5-HT2AR. Peptide hormones of the hypothalamic-pituitary-adrenal/interrenal axis also augment the electric waveform. These results indicate that the central serotonergic system interacts with the hypothalamic-pituitary-interrenal system to regulate communication signals in this species. The same neuroendocrine probes were tested in females before and after introducing androgens to examine the relationship between sex steroid hormones, the serotonergic system, melanocortin peptides, and EOD modulations. Androgens caused an increase in female B. pinnicaudatus responsiveness to other pharmacological challenges, particularly to the melanocortin peptide adrenocorticotropic hormone (ACTH). A forced social challenge paradigm was administered to determine if androgens are responsible for controlling the signal modulations these fish exhibit when they encounter conspecifics. Males and females responded similarly to this social challenge construct, however introducing androgens caused implanted females to produce more exaggerated responses. These results confirm that androgens enhance an individual's capacity to produce an exaggerated response to challenge, however another unidentified factor appears to regulate sex-specific behaviors in this species. These results suggest that the rapid electric waveform modulations B. pinnicaudatus produces in response to conspecifics are situation-specific and controlled by activation of different serotonin receptor types and the subsequent effect on release of pituitary hormones.

Neuroendocrine control of a dynamic communication signal

Multiple physiological systems regulate the electric communication signal of the weakly electric gymnotiform fish, Brachyhypopomuspinnicaudatus. Fish were injected with neuroendocrine probes which identified pharmacologically relevant serotonin (5-HT) receptors similar to the mammalian 5-HT1AR and 5-HT2AR. Peptide hormones of the hypothalamic-pituitary-adrenal/interrenal axis also augment the electric waveform. These results indicate that the central serotonergic system interacts with the hypothalamic-pituitaryinterrenal system to regulate communication signals in this species. The same neuroendocrine probes were tested in females before and after introducing androgens to examine the relationship between sex steroid hormones, the serotonergic system, melanocortin peptides, and EOD modulations. Androgens caused an increase in female B. pinnicaudatus responsiveness to other pharmacological challenges, particularly to the melanocortin peptide adrenocorticotropic hormone (ACTH). A forced social challenge paradigm was administered to determine if androgens are responsible for controlling the signal modulations these fish exhibit when they encounter conspecifics. Males and females responded similarly to this social challenge construct, however introducing androgens caused implanted females to produce more exaggerated responses. These results confirm that androgens enhance an individual's capacity to produce an exaggerated response to challenge, however another unidentified factor appears to regulate sex-specific behaviors in this species. These results suggest that the rapid electric waveform modulations B. pinnicaudatus produces in response to conspecifics are situation-specific and controlled by activation of different serotonin receptor types and the subsequent effect on release of pituitary hormones.

The role of the melanocortin 3 receptor in mediating the effects of gamma-MSH peptides on the adrenal

The pro-opiomelanocortin (POMC)-derived peptides, pro-gamma-MSH (16K fragment), and Lys-gamma(3)-MSH, have been shown to potentiate the steroidogenic action of corticotrophin (ACTH) on the adrenal cortex. Using a continuously perfused adrenal cell column system, we have tested the hypothesis that gamma-MSH peptides exert their effect through the Melanocortin 3 Receptor (MC3-R), since this is the only known receptor to have high affinity for gamma-MSH peptides and has been suggested to be expressed in the rat adrenal. To investigate this hypothesis we tested whether the MC3-R agonist MTII and antagonist SHU9119 could mimic or block the actions of pro-gamma-MSH. We found that MTII could not mimic, and SHU9119 could not block pro-gamma-MSH mediated potentiation of ACTH-induced steroidogenesis. These results suggest that the MC3-R is not involved in mediating the potentiation effect, adding further evidence to the argument that another melanocortin receptor exists.

Ghrelin gene-related peptides : multifunctional endocrine/autocrine modulators in health and disease

Ghrelin is a multi-functional peptide hormone which affects various processes including growth hormone and insulin release, appetite regulation, gut motility, metabolism and cancer cell proliferation. Ghrelin is produced in the stomach and in other normal and pathological cell types. It may act as an endocrine or autocrine/paracrine factor. The ghrelin gene encodes a precursor protein, preproghrelin, from which ghrelin and other potentially active peptides are derived by alternative mRNA splicing and/or proteolytic processing. The metabolic role of the peptide obestatin, derived from the preproghrelin C-terminal region, is controversial. However, it has direct effects on cancer cell proliferation. The regulation of ghrelin expression and the mechanisms through which the peptide products arise are unclear. We have recently re-examined the organisation of the ghrelin gene and identified several novel exons and transcripts. One transcript, which lacks the ghrelin-coding region of preproghrelin, contains the coding sequence of obestatin. Furthermore, we have identified an overlapping gene on the antisense strand of ghrelin, GHRLOS, which generates transcripts that may function as non-coding regulatory RNAs or code for novel, short bioactive peptides. The identification of these novel ghrelin-gene related transcripts and peptides raises critical questions regarding their physiological function and their role in obesity, diabetes and cancer.

Establishment of a mouse model of colitis and its use to evaluate the anti-inflammatory effects of two ghrelin peptides

Ghrelin is a gut-brain peptide hormone that induces appetite, stimulates the release of growth hormone, and has recently been shown to ameliorate inflammation. Recent studies have suggested that ghrelin may play a potential role in inflammation-related diseases such as inflammatory bowel diseases (IBD). A previous study with ghrelin in the TNBS mouse model of colitis demonstrated that ghrelin treatment decreased the clinical severity of colitis and inflammation and prevented the recurrence of disease. Ghrelin may be acting at the immunological and epithelial level as the ghrelin receptor (GHSR) is expressed by immune cells and intestinal epithelial cells. The current project investigated the effect of ghrelin in a different mouse model of colitis using dextran sodium sulphate (DSS) – a luminal toxin. Two molecular weight forms of DSS were used as they give differing effects (5kDa and 40kDa). Ghrelin treatment significantly improved clinical colitis scores (p=0.012) in the C57BL/6 mouse strain with colitis induced by 2% DSS (5kDa). Treatment with ghrelin suppressed colitis in the proximal colon as indicated by reduced accumulative histopathology scores (p=0.03). Whilst there was a trend toward reduced scores in the mid and distal colon in these mice this did not reach significance. Ghrelin did not affect histopathology scores in the 40kDa model. There was no significant effect on the number of regulatory T cells or TNF-α secretion from cultured lymph node cells from these mice. The discovery of C-terminal ghrelin peptides, for example, obestatin and the peptide derived from exon 4 deleted proghrelin (Δ4 preproghrelin peptide) have raised questions regarding their potential role in biological functions. The current project investigated the effect of Δ4 peptide in the DSS model of colitis however no significant suppression of colitis was observed. In vitro epithelial wound healing assays were also undertaken to determine the effect of ghrelin on intestinal epithelial cell migration. Ghrelin did not significantly improve wound healing in these assays. In conclusion, ghrelin treatment displays a mild anti-inflammatory effect in the 5kDa DSS model. The potential mechanisms behind this effect and the disparity between these results and those published previously will be discussed.

The effects of exercise-induced weight loss on appetite-related peptides and motivation to eat

Context: The magnitude of exercise-induced weight loss depends on the extent of compensatory responses. An increase in energy intake is likely to result from changes in the appetite control system toward an orexigenic environment; however, few studies have measured how exercise impacts on both orexigenic and anorexigenic peptides. ---------- Objective: The aim of the study was to investigate the effects of medium-term exercise on fasting/postprandial levels of appetite-related hormones and subjective appetite sensations in overweight/obese individuals. ---------- Design and Setting: We conducted a longitudinal study in a university research center. ---------- Participants and Intervention: Twenty-two sedentary overweight/obese individuals (age, 36.9 ± 8.3 yr; body mass index, 31.3 ± 3.3 kg/m2) took part in a 12-wk supervised exercise programme (five times per week, 75% maximal heart rate) and were requested not to change their food intake during the study. ---------- Main Outcome Measures: We measured changes in body weight and fasting/postprandial plasma levels of glucose, insulin, total ghrelin, acylated ghrelin (AG), peptide YY, and glucagon-like peptide-1 and feelings of appetite. ---------- Results: Exercise resulted in a significant reduction in body weight and fasting insulin and an increase in AG plasma levels and fasting hunger sensations. A significant reduction in postprandial insulin plasma levels and a tendency toward an increase in the delayed release of glucagon-like peptide-1 (90–180 min) were also observed after exercise, as well as a significant increase (127%) in the suppression of AG postprandially. ---------- Conclusions: Exercise-induced weight loss is associated with physiological and biopsychological changes toward an increased drive to eat in the fasting state. However, this seems to be balanced by an improved satiety response to a meal and improved sensitivity of the appetite control system.

Ghrelin axis genes, peptides and receptors : recent findings and future challenges

The ghrelin axis consists of the gene products of the ghrelin gene (GHRL), and their receptors, including the classical ghrelin receptor GHSR. While it is well-known that the ghrelin gene encodes the 28 amino acid ghrelin peptide hormone, it is now also clear that the locus encodes a range of other bioactive molecules, including novel peptides and non-coding RNAs. For many of these molecules, the physiological functions and cognate receptor(s) remain to be determined. Emerging research techniques, including proteogenomics, are likely to reveal further ghrelin axis-derived molecules. Studies of the role of ghrelin axis genes, peptides and receptors, therefore, promises to be a fruitful area of basic and clinical research in years to come.

Challenges in mass spectrometry-based quantification of bioactive peptides: A case study exploring the neuropeptide Y family

The study of biologically active peptides is critical to the understanding of physiological pathways, especially those involved in the development of disease. Historically, the measurement of biologically active endogenous peptides has been undertaken by radioimmunoassay, a highly sensitive and robust technique that permits the detection of physiological concentrations in different biofluid and tissue extracts. Over recent years, a range of mass spectrometric approaches have been applied to peptide quantification with limited degrees of success. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to the NPY family exhibiting regulatory effects on appetite and feeding behavior. The physiological significance of these peptides depends on their molecular forms and in vivo concentrations systemically and at local sites within tissues. In this report, we describe an approach for quantification of individual peptides within mixtures using high-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the NPY family peptides. Aspects of quantification including sample preparation, the use of matrix-matched calibration curves, and internal standards will be discussed. This method for the simultaneous determination of NPY, PYY, and PP was accurate and reproducible but lacks the sensitivity required for measurement of their endogenous concentration in plasma. The advantages of mass spectrometric quantification will be discussed alongside the current obstacles and challenges. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 357–366, 2012.