Quantitative mass spectrometry for human melanocortin peptides in vitro and in vivo suggests prominent roles for β-MSH and desacetyl α-MSH in energy homeostasis

Abstract

Objective: The lack of pro-opiomelanocortin (POMC)-derived melanocortin peptides results in hypoadrenalism and severe obesity in both humans and rodents that is treatable with synthetic melanocortins. However, there are significant differences in POMC processing between humans and rodents, and little is known about the relative physiological importance of POMC products in the human brain. The aim of this study was to determine which POMC-derived peptides are present in the human brain, to establish their relative concentrations, and to test if their production is dynamically regulated.

Methods: We analysed both fresh post-mortem human hypothalamic tissue and hypothalamic neurons derived from human pluripotent stem cells (hPSCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the sequence and quantify the production of hypothalamic neuropeptides, including those derived from POMC.

Results: In both in vitro and in vivo hypothalamic cells, LC-MS/MS revealed the sequence of hundreds of neuropeptides as a resource for the field. Although the existence of β-melanocyte stimulating hormone (MSH) is controversial, we found that both this peptide and desacetyl α-MSH (d-α-MSH) were produced in considerable excess of acetylated α-MSH. In hPSC-derived hypothalamic neurons, these POMC derivatives were appropriately trafficked, secreted, and their production was significantly (P < 0.0001) increased in response to the hormone leptin.

Conclusions: Our findings challenge the assumed pre-eminence of α-MSH and suggest that in humans, d-α-MSH and β-MSH are likely to be the predominant physiological products acting on melanocortin receptors.

Keywords: Human pluripotent stem cell; Leptin; MSH; Neuropeptide; Obesity; POMC.

Figure 1

Regulation of POMC processing and secretion. Human POMC is translated as a 267-amino acid protein that, after removal of the signal peptide, undergoes successive rounds of cleavage and trimming at dibasic residues (blue) in a tissue-specific manner; the hypothalamic pattern is illustrated. Additional levels of post-translational modification include C-terminal amidation (orange) and N-terminal acetylation (magenta). The most extensively characterised POMC-derived peptides that regulate food intake (green) include d-α-MSH(1-13), α-MSH(1-13), β-MSH(1-18), β-EP (1-31) and β-EP (1-27). Illustrated mutations in β-MSH have been associated with obesity, suggesting a role for this peptide in human body weight regulation. The concentrations of secreted POMC-derived peptides may be regulated at the levels of transcription, translation, processing, and secretion. ACTH, adrenocorticotropic hormone; CLIP, corticotropin-like intermediary peptide; EP, endorphin; LPH, lipotropin; MSH, melanocyte stimulating hormone; NPP, POMC N-terminal region; POMC, pro-opiomelanocortin.

Figure 2

Subcellular localisation of POMC and its derivatives in hPSC-derived hypothalamic neurons. A) Schematic diagram of the differentiation of human pluripotent stem cells into hypothalamic POMC neurons that mature over time in culture. Experiments were carried out between 25 and 90 days post-differentiation. B) Human hypothalamic differentiation yields predominantly neuronal cultures as indicated by immunostaining for Tuj1 and MAP2, of which approximately 6% are immunopositive for POMC. C) Confocal micrographs of punctate POMC-immunoreactive structures localised to cell bodies and neurites (inset). D) Some Tuj1-expressing axon-like processes are strongly immunopositive for POMC (red staining). E-G) Transmission electron micrographs showing specific cytoplasmic and punctate labelling by Immunogold staining for β-EP (F) or α-MSH (G) localised to rounded, vesicle-like structures (G, yellow arrows) in regions with abundant vesicles resembling dense core vesicles (G, black arrows), and adjacent to neurotransmitter-like vesicles (F, black arrows). H) Quantification of the diameters of neurotransmitter-like (NT-like, black circles) clear vesicles, POMC Immunogold-positive (POMC-IG, red circles), and dense core-like (DC-like, blue circles) vesicles in hPSC-derived hypothalamic neurons. Neurotransmitter-like vesicles were significantly smaller than POMC Immunogold-positive structures (p < 0.0001). Error bars show SEM. ****, P < .0001.

Figure 3

Identification of processed POMC peptides by LC-MS/MS. A) Schematic workflow for the quantification of POMC-derived peptides from hPSC-derived hypothalamic neurons. B) Liquid chromatographs of samples from hPSC-derived hypothalamic neurons (top), compared with stable isotope-labelled synthetic d-α-MSH(1-13), β-MSH(1-18) and β-EP (1-31) (bottom). Note that heavy isotope-labelled reference peptides have identical retention times as endogenous peptides but have slightly higher mass-to-charge (m/z) ratios, and that m/z values measured from Orbitrap and triple quadrupole mass spectrometers may differ. C) Schematic of human POMC protein and relevant dibasic cleavage sites (blue), expected POMC-derived peptides, and those peptides detected in hPSC-derived hypothalamic cultures by LC-MS/MS. Detected peptides included γ₁-MSH (Lys- γ₁-MSH), d-α-MSH(1-13), β-MSH(1-18) and β-EP (1-31). Quantified peptides are indicated in green. D) Summary of POMC-derived peptides detected by LC-MS/MS, where the colour intensity represents the relative abundance of each peptide species. Repl., replicates. Other abbreviations are as in Figure 1.

Figure 4

Quantification of POMC-derived peptides in hPSC-derived neurons and primary human hypothalamus. A) Schematic diagram of standard curve generation for peptide quantification. B) LC-MS/MS-based quantification of d-α-MSH, α-MSH, β-MSH and β-EP peptides in hPSC-derived POMC neurons. N = 5 independent experiments with 3–8 replicates per experiment. Concentrations were converted to molarity and then normalised to the mean concentration of d-α-MSH for each technical replicate. Error bars show SEM. C) Quantification of POMC-derived peptides in primary human brain samples. Note that while α-MSH was clearly detected in PVH Brain 1 Right and Left and MBH Brain 1 Right, the concentration was below the assay's limit of accurate quantification. N = 3 independent samples from N = 2 brains per brain region. PVH, dissected region encompassing the paraventricular hypothalamus; MBH, dissected region encompassing the mediobasal hypothalamus. Error bars show SEM.

Figure 5

Characterisation of the peptidome of the human hypothalamus. A) Sagittal view of the right hemisphere of a post-mortem human forebrain used in this study. Regions of approximately 3 × 3 × 3 mm from the indicated brain regions were dissected for analysis. B) Colour-coded schematic diagram of several neuropeptidergic cell types of interest. C) Summary of peptides associated with the regulated secretory pathway, synaptic proteins, and neuropeptide processing enzymes detected in hPSC-derived hypothalamic neurons in vitro or in the MBH, PVH, or CTX of the human brain. Filled boxes represent peptides corresponding to the indicated gene that were detected and automatically identified. D) Summary of select neuropeptides detected in hPSC-derived hypothalamic neurons or in the human brain. Sequences too long to readily display are denoted by an ellipsis. CTX, cortex (orbitofrontal gyrus); LHA, lateral hypothalamic area; MBH, mediobasal hypothalamus; Pit., pituitary gland; PVH, paraventricular nucleus of the hypothalamus, Unchar. Pep., uncharacterised peptide. An adjoining ‘p’ on the amino acid sequences denotes a pyroglutamate residue, while an ‘a’ denotes an amide group. N = 4 independent samples from N = 3 unrelated fresh post-mortem brains.

Figure 6

Regulated secretion of POMC-derived peptides. A) Experimental schematic for quantifying the stimulated secretion of POMC-derived peptides in hPSC-derived hypothalamic neurons by LC-MS/MS. B) Secreted d-α-MSH, β-MSH and β-EP (1-31) peptides in pre-stimulation controls (black circles), 30 mM KCl stimulations (red circles) and ACSF controls (blue circles). Molar peptide concentrations were normalised to the mean pre-stimulation β-MSH concentration. N = 4 independent experiments with 4–8 technical replicates per experiment. C) KCl-induced changes in secreted peptide concentration were calculated from data shown in (B) for each technical replicate by subtracting the pre-stimulation peptide concentrations from the ACSF control or KCl stimulation condition, and normalising to mean concentrations seen in ACSF control-stimulated cultures. KCl stimulation significantly (P < 3 × 10⁻⁵) increased the concentrations of d-α-MSH, β-MSH and β-EP (1-31). KCl, potassium chloride. Error bars show SEM. ****, P < .0001.

Figure 7

Regulation of POMC processing in vitro. A) Experimental schematic for measuring the regulation POMC and its derivates by leptin or Furin inhibitor I, which blocks prohormone convertases. B) Treatment with 25 uM Furin Inhibitor 1 (blue) significantly reduced d-α−MSH and β-MSH (P < 0.0001), and β-EP (P < 0.05) concentrations. C) Treatment with recombinant human leptin significantly increased the measured concentrations of d-α−MSH, β-MSH, and β-EP at all concentrations tested. N = 3 independent experiments with 4–8 technical replicates per experiment. Error bars show SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.