Evidence for the ectopic synthesis of melanin in human adipose tissue

Abstract

Melanin is a common pigment in animals. In humans, melanin is produced in melanocytes, in retinal pigment epithelium (RPE) cells, in the inner ear, and in the central nervous system. Previously, we noted that human adipose tissue expresses several melanogenesis-related genes. In the current study, we confirmed the expression of melanogenesis-related mRNAs and proteins in human adipose tissue using real-time polymerase chain reaction and immunohistochemical staining. TYR mRNA signals were also detected by in situ hybridization in visceral adipocytes. The presence of melanin in human adipose tissue was revealed both by Fontana-Masson staining and by permanganate degradation of melanin coupled with liquid chromatography/ultraviolet/mass spectrometry determination of the pyrrole-2,3,5-tricarboxylic acid (PTCA) derivative of melanin. We also compared melanogenic activities in adipose tissues and in other human tissues using the L-[U-(14)C] tyrosine assay. A marked heterogeneity in the melanogenic activities of individual adipose tissue extracts was noted. We hypothesize that the ectopic synthesis of melanin in obese adipose may serve as a compensatory mechanism that uses its anti-inflammatory and its oxidative damage-absorbing properties. In conclusion, our study demonstrates for the first time that the melanin biosynthesis pathway is functional in adipose tissue.

Figure 1.

Fontana-Masson stain of human adipose tissue demonstrates melanin pigment (black staining) mainly in the periphery of the adipocytes. A, B) Multiple conglomerates of melanin granules are present at the periphery of the adipocytes in adipose tissue from morbidly obese subjects (×20). C, D) Melanin granules are scarce in the adipocytes of adipose tissue from nonobese subjects (×20). E) No melanin granules were observed in the microvessels located in the adipose tissue (×20). F) Melanin staining in skin tissue used as a positive control (×10).

Figure 2.

Presence of melanin in extracts of adipose tissue as revealed by LC-UV-MS. A) Homogenized samples of adipose tissue separated into three phases: supernatant (fat), aqueous, and sediment. Cell debris sediments from adipose tissue samples from morbidly obese subjects contain visible amounts of black pigment. A, B) Adipose sediments of nonobese subjects. C, D) Adipose sediments from obese subjects. B) CAD of the PTCA precursor ion at mass-to-charge ratio (m/z) 198 produces abundant product ions at m/z 154 and 110 peaks. C) LC-MS multi-ion SIM chromatogram of PTCA peak at a retention time of 6 min. D) Negative ESI mass spectra of PTCA peak at 6 min. E) HPLC-UV/VIS chromatograms at 270 nm.

Figure 3.

Results of the L-[U-¹⁴C] tyrosine assay. OA1-7, adipose samples from morbidly obese individuals; NOA, adipose sample from nonobese subject; Ga, gastric sample; Liv, liver sample; MNT1, highly pigmented human melanoma cells. Recorded values of melanogenic pathway activity for blanks (no protein extract added) and for gastric and liver side controls were similar (Blanks, 234±37 cpm; Ga, 237±15 cpm; Liv, 238±23 cpm). Recorded values for obese adipose samples were different from those of nonadipose samples (OA1-7, 845±99; Ga&Liv, 237±1). Recorded value for nonobese sample was 428 ± 25. For representation purposes, the recorded count per minute values were adjusted by subtraction of blank values and were transformed to picomole product per microgram per hour values (left-hand scale). Activity of melanogenic pathway per microgram of protein extract of MNT1 cells was ∼20 times higher that that of the adipose samples (right-hand scale).

Figure 4.

Relative abundance of MC1R, TYR, TYRP1, and TYRP2 transcripts in adipose samples collected from morbidly obese (OA) and nonobese (NOA) subjects after normalization against 18S RNA. Height of each square corresponds to the level of gene expression in the given sample. MCR1, dark blue; TYR, purple; TYRP1, yellow; TYRP2, light blue. Mann-Whitney P values: P < 0.05 for TYR and MC1R; nonsignificant for TYRP1 and TYRP2.

Figure 5.

In situ hybridization of a human TYR RNA probe on sections of visceral adipose tissues from morbidly obese and nonobese subjects demonstrated cytoplasmic and membrane-associated staining pattern (×20). A, B) T7 (cTYR) probe, visceral adipose tissue from a morbidly obese subject. C) T7 (cTYR) probe, visceral adipose tissue from a lean subject. D) SP6 (control) probe, visceral adipose tissue from a morbidly obese subject.

Figure 6.

Immunohistochemical staining of visceral adipose tissue sections from morbidly obese and from nonobese subjects for human TYR, TYRP1, and TYRP2 proteins (×20). Red: TYR, TYRP1 or TYRP2 staining. Blue: DAPI (nuclei). A, C, E, G) Visceral adipose tissue from a morbidly obese subject. B, D, F, H) Visceral adipose tissue from a nonobese subject. A, B) Tyrosinase. C, D) Tyrosinase-related protein Tyrp2 (dopachrome tautomerase). E, F) Tyrosinase-related protein Tyrp1. G, H) Negative control (secondary antibodies) and DAPI.

Figure 7.

Immunohistochemical staining of visceral adipose tissue sections from morbidly obese subjects for human TYR, adipocyte-specific markers LPL, and endothelial marker CD31. Red denotes TYR staining. Green denotes LPL or CD31 staining. A, D) Tyrosinase. B) LPL. C) Tyrosinase and LPL counterstaining. E) CD31. F) Tyrosinase and CD31 counterstaining.