Metformin inhibits melanin synthesis and melanosome transfer through the cAMP pathway

Abstract

Several studies have demonstrated the inhibitory effect of metformin on pigmentation. However, the effect of metformin on melanosome transfer remains unknown. The goals of this study were to elucidate the effects of metformin on melanogenesis and melanosome transfer and explore the related mechanisms. We determined that, compared with those in the control zebrafish, the area occupied by pigment granules, melanin content, tyrosinase activity, and the expression levels of melanogenesis genes and melanosome transfer-related genes were reduced in metformin-treated zebrafish. In human primary melanocytes, MNT1 cells/B16F10 cells, metformin also plays a negative role in melanin synthesis regardless of health status and α-MSH-induced pigmentation. Unlike arbutin, metformin inhibited the formation of dendrites and filopodia-like structures and suppressed melanosome transfer. After treatment with metformin, the cAMP content was reduced, the expression of MITF and downstream molecules was downregulated, and the expression of Rho GTPases was changed. Metformin partially abrogated the changes in genes regulating melanin synthesis, melanosome transfer and the cytoskeleton induced by a cAMP activator. Furthermore, the Nrf2 expression was decreased upon metformin intervention, and metformin partially abrogated the changes in genes regulating melanogenesis caused by a Nrf2 activator. Our study revealed that metformin can serve as a candidate depigmentation agent.

Full text

Metformin inhibits melanin
synthesis and melanosome transfer
through the cAMP pathway
Xing Liu1,4, Xiaojie Sun1,4, Yunyao Liu2, Wenzhu Wang1, Hedan Yang1, Yiping Ge1,
Yin Yang1, Xu Chen2,3 & Tong Lin2
Several studies have demonstrated the inhibitory eect of metformin on pigmentation. However,
the eect of metformin on melanosome transfer remains unknown. The goals of this study were to
elucidate the eects of metformin on melanogenesis and melanosome transfer and explore the related
mechanisms. We determined that, compared with those in the control zebrash, the area occupied by
pigment granules, melanin content, tyrosinase activity, and the expression levels of melanogenesis
genes and melanosome transfer-related genes were reduced in metformin-treated zebrash. In human
primary melanocytes, MNT1 cells/B16F10 cells, metformin also plays a negative role in melanin
synthesis regardless of health status and α-MSH-induced pigmentation. Unlike arbutin, metformin
inhibited the formation of dendrites and lopodia-like structures and suppressed melanosome
transfer. After treatment with metformin, the cAMP content was reduced, the expression of MITF
and downstream molecules was downregulated, and the expression of Rho GTPases was changed.
Metformin partially abrogated the changes in genes regulating melanin synthesis, melanosome
transfer and the cytoskeleton induced by a cAMP activator. Furthermore, the Nrf2 expression was
decreased upon metformin intervention, and metformin partially abrogated the changes in genes
regulating melanogenesis caused by a Nrf2 activator. Our study revealed that metformin can serve as a
candidate depigmentation agent.
Keywords Metformin, Melanogenesis, Melanosome transfer, cAMP
Melanin pigments, which are produced by melanocytes, mainly determine skin colour and protect the skin
from external damage1. Melanin metabolism includes melanin synthesis, melanin transfer to neighbouring
keratinocytes and melanin degradation. Abnormal metabolism of melanin pigment leads to skin pigmentary
disorders, including hyperpigmentary diseases such as melasma, postinammatory hyperpigmentation (PIH),
and hypopigmentary conditions such as vitiligo. ese conditions are not life-threatening, but they cause
cosmetic troubles and psychological burdens, negatively aecting quality of life, especially in exposed areas,
including the face and arms2,3. Melanin is produced via a series of complex and delicate enzymatic biochemical
reactions, beginning with the amino acid tyrosine and its metabolite DOPA, which occur mainly in lysosome-
related organelles called melanosomes1. Microphthalmia transcription factor (MITF) is pivotal not only for
melanocyte survival4 but also for the expression of several pigmentation enzymes involved in melanogenesis and
dierentiation factors, such as tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and tyrosinase-related
protein-2 (TYRP2)/dopachrome tautomerase (DCT)5–7.
Upon ultraviolet radiation, α-MSH secreted by keratinocytes binds to the melanocortin-1 receptor (MC1R)
on the surface of melanocytes, stimulating the upregulation of cAMP in melanocytes6–8. cAMP mostly functions
through the activation of cAMP-dependent protein kinase A (PKA) and its phosphorylation of cAMP response
element (CREB)7,9. Phosphorylated CREB promotes the transcription of genes whose promoters have a cAMP-
response element sequence, including MITF7,8. e cAMP-CREB-MITF pathway is the classic melanogenesis
1Department of Cosmetic Laser Surgery, Hospital for Skin Diseases, Institute of Dermatology, Chinese Academy
of Medical Sciences & Peking Union Medical College, Jiangwangmiao Street 12, Xuanwu District, Nanjing 210042,
China. 2Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Institute of Dermatology, Chinese
Academy of Medical Sciences and Peking Union Medical College, Jiangwangmiao Street 12, Xuanwu District,
Nanjing 210042, China. 3Key Laboratory of Basic and Translational Research on Immune-Mediated Skin Diseases,
Chinese Academy of Medical Sciences, Jiangwangmiao Street 12, Xuanwu District, Nanjing 210042, China. 4Xing
Liu and Xiaojie Sun contributed equally to this study. email: yushiyy@163.com; chenx@pumcderm.cams.cn;
ddlin@hotmail.com
OPEN
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pathway6–8. Moreover, the dendrites of melanocytes contribute not only to specialized cellular morphology but
also, more importantly, to the eect of melanosome transfer to adjacent keratinocytes. Studies have revealed that
the cAMP pathway aects the formation of dendrites and the redistribution of the actin cytoskeleton by actin
stress bers10. e formation and regulation of stress bres are regulated by small GTP-binding proteins of the
Rho family, which include Rho, Rac and Cdc4211,12.
Melasma is a common hyperpigmentary disorder aecting millions of people worldwide. Melasma occurs
primarily in the facial area of darker-skinned individuals with skin types IV-VI, with at least 90% of the aected
individuals being female13. Melasma is associated with hormonal changes, mainly estrogen changes during
pregnancy or contraception13. Multiple treatments, including oral agents, topical agents, chemical peels, and
laser- and light-based therapies, have been used in the clinic but are partially ineective or unsatisfactory,
resulting in high rates of treatment failure and recurrence14. erefore, developing more eective and safe drugs
is important for the treatment of melasma.
Metformin, which is the mainstay of diabetes mellitus treatment, has been found to have therapeutic eects
on several cutaneous diseases in recent years, including hyperinsulinaemia, hormonal acne, hidradenitis
suppurativa, acanthosis nigricans, and polycystic ovarian syndrome, as well as cancer and aging15,16. Metformin
suppresses hepatic glucagon signalling to achieve antidiabetic eects, leading to the accumulation of AMP and
relevant nucleotides, which inhibit adenylate cyclase, decrease the production of cAMP and reduce the activity of
PKA17. A previous study revealed that metformin inhibited melanogenesis by decreasing cAMP accumulation18,
and two studies have shown that topical treatment with 30% metformin is eective in alleviating melasma19,20.
Additionally, ere was a study suggesting that estrogen altered cAMP levels that may be responsible for
hyperpigmentation21. Accordingly, it is reasonable to hypothesize that metformin may have a valuable function
in preventing and treating melasma. A recent study demonstrated that metformin exerted a substantial
neuroprotective eect and decelerated aging partially mediated by the nuclear factor erythroid-derived 2-like 2
(Nrf2)22. However, the eect of metformin on melanosome transfer is still not understood. is research aims to
elucidate the role of melanin in melanin synthesis and transport and explore its mechanism.
Results
Metformin inhibits melanin synthesis and downregulates genes expression involved in
melanosome transfer in zebrash
e development and morphology of zebrash embryos treated with 10 mM metformin were normal and
did not dier from those of the control group (Fig.1a). At 48 hpf, 72 hpf, and 96 hpf, an obvious reduction
in melanin content was observed in metformin-treated zebrash (Fig.1a), and the mean percentage area of
melanin granules in the head region signicantly decreased, according to the ImageJ analysis (Fig.1b). Melanin
content and tyrosinase activity were both markedly lower in the metformin group than in the control group
(Fig.1c,d). To determine whether treatment with metformin aects key molecules of the melanogenesis pathway
and melanosome transfer, we examined the mRNA expression of MITF, TYR, TYRP1, DCT, MLPH, Rab27a and
Myo5a via q-PCR. All genes were signicantly downregulated in the metformin-treated zebrash (Fig.1e,f).
Metformin inhibits melanin synthesis in melanocytes, MNT1 cells and B16F10 cells
To determine whether metformin can aect cellular viability, primary human melanocytes, MNT1 cells and
B16F10 cells were treated with four concentrations of metformin (5 mM, 10 mM, 20 mM, 40 mM) for 24h,
48–72h, and the results of the cell counting kit-8 (CCK8) assay are shown in Figure S1. e relative cell viability
aer treatment with 5 mM or 10 mM metformin was approximately 90% or greater, so these two concentrations
were used in subsequent research. Arbutin (Arb) is known for its inhibitory eect on tyrosinase activity and
is commonly used as a skin-whitening agent23. Previous studies demonstrated that 1 mM arbutin inhibited
melanogenesis at a noncytotoxic concentration. Accordingly, the concentration of 1 mM of arbutin was used in
the research24,25.
To study the inhibitory eect of metformin on melanogenesis, arbutin was also used for comparison. In
melanocytes and B16F10 cells, both 5 mM and 10 mM metformin signicantly reduced melanin content and
tyrosinase activity (Fig. S2a–f), whereas the inhibitory eect of 1 mM arbutin was more obvious in MNT1
cells (Fig. S2b,e). To assess the inhibitory eect on melanogenesis under hyperpigmented conditions, melanin
content and tyrosinase activity were detected in melanocytes and B16F10 cells aer α-MSH stimulation in the
presence or absence of metformin. As shown in Fig. S2g,h, 5 mM metformin, 10 mM metformin and 1 mM
arbutin signicantly reduced the melanin content compared with that in aer α-MSH treatment. Tyrosinase
activity was also markedly decreased in hyperpigmented conditions (Fig. S2i,j).
Metformin inhibits melanogenesis in part via the cAMP-Nrf2 pathway
e cAMP-MITF pathway is the main pathway involved in the regulation of pigment production. To elucidate
the mechanism by which metformin aects melanogenesis and verify whether metformin plays a role in this
pathway, we detected the cAMP content of cell supernatants and cell lysates treated with 10 mM metformin
for 72h. e results showed that the extracellular and intracellular cAMP levels were lower in the metformin-
treated group than in the control group (Fig.2a). MITF plays a crucial role in melanogenesis, regulating the
expression of key molecules downstream and promoting melanin synthesis, including TYR and TYRP1. e
mRNA levels of these molecules were also determined aer intervention for 12h; only the expression of MITF
and TYR signicantly decreased, whereas TYRP1 expression did not signicantly dier (Fig.2b). Treatment
of melanocytes with metformin or arbutin signicantly reduced the protein levels of MITF, TYR and TYRP1
(Fig.2c). In B16F10 cells, the melanin content and tyrosinase activity increased aer treatment with 20 µM
Forskolin, a cAMP activator, whereas 10 mM metformin partially abrogated the upregulation induced by
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Fig. 1. Metformin inhibits melanin synthesis and downregulates genes expression involved in melanosome
transfer in zebrash. (a) Melanin granules in the head regions of zebrash at 48hpf, 72hpf, and 96hpf (scale
bar = 500μm). (b) e percentage area of melanin granules in the head regions was signicantly decreased
aer the eect of 10 mM metformin at 48hpf, 72hpf, and 96hpf by Image J soware. (c) Metformin inhibited
melanin content at 96hpf. (d) Metformin inhibited tyrosinase activity at 96hpf. (e) e mRNA expression levels
of MITF, TYR, TYRP1a, and DCT in melanogenesis were down-regulated aer the treatment of metformin at
96hpf by RT-qPCR. (f) e mRNA expression levels of MLPHa, Rab27a, and Myo5a in melanosome transfer
were down-regulated aer the treatment of metformin at 96hpf by RT-qPCR. Results were presented as mean ±
SD. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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Forskolin (Fig.2d,e). Similarly, in melanocytes, metformin abrogated the upregulation of TYR and TYRP1
expression induced by Forskolin (Fig.2f).
In melanocytes, the active form of Nrf2 (phosphorylated Nrf2, p-Nrf2) was decreased aer metformin
treatment (Fig.3a), the expression of p-Nrf2, TYRP1 TYR and the cAMP content were increased aer treatment
with 20 µM Oltipraz, a Nrf2 activator, whereas 10 mM metformin partially abrogated the upregulation induced
by Oltipraz (Fig.3b,c). Additionally, the p-Nrf2 expression was increased upon 20 µM Forskolin treatment, while
the up-regulation caused by Forskolin was partially decreased aer incubated with metformin (Fig.3d).
Metformin alters cellular morphology and inhibits melanosome transfer
e morphology and formation of dendrites are essential for melanocyte function and especially melanosome
transfer. Aer culture with medium containing 5 mM or 10 mM metformin for 24h, the dendrites of B16F10
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Fig. 2. Metformin inhibits melanogenesis partly dependent on the cAMP-MITF pathway. (a) Treated
with metformin for 72h in melanocytes, the extracellular and intracellular cAMP levels were signicantly
decreased. (b) e mRNA expression levels of MITF, TYR, TYRP1 in melanogenesis were detected by
RT-qPCR (12h) in melanocytes. (c) e protein expression of MITF, TYR, TYRP1 was reduced aer the
incubation with metformin for 72h in melanocytes by Western blot (the gels/blots was from the same protein
sample which extracted from the same batch of cells). (d,e) In B16F10 cells, treated with 20 µM cAMP
activator (Forskolin) for 48h, the melanin content (d), tyrosinase activity (e) were elevated, while the up-
regulation induced by Forskolin was partially decreased aer incubated with metformin. (f) In melanocytes,
treated with 20 µM Forskolin for 72h, TYR and TYRP1 were increased, while the up-regulation induced by
Forskolin was partially decreased aer incubated with metformin (the gels/blots was from the same protein
sample which extracted from the same batch of cells). Results were presented as mean ± SD. *p<0.05, **p < 0.01,
***p < 0.001; ns, not signicant.
◂
Fig. 3. Metformin inhibits melanogenesis partly dependent on the cAMP-Nrf2 pathway. (a) Treated with 10
mM metformin for 72h in melanocytes, the expression of phosphorylated Nrf2 (p-Nrf2) was decreased. (b) In
melanocytes, treated with 20 µM Nrf2 activator (Oltipraz) for 72h, p-Nrf2, TYRP1 and TYR were increased,
while the up-regulation induced by Oltipraz was partially decreased aer incubated with metformin (the gels/
blots was from the same protein sample which extracted from the same batch of cells). (c) In melanocytes,
treated with 20 µM Oltipraz for 72h, cAMP content was increased, whereas the up-regulation induced
by Oltipraz was partially decreased aer incubated with metformin. (d) In melanocytes, treated with 20
µM Forskolin for 72h, the p-Nrf2 expression was increased, while the up-regulation induced by Forskolin
was partially decreased aer incubated with metformin. Results were presented as mean ± SD. **p < 0.01,
***p < 0.001, ****p < 0.0001.
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cells were thinner and shorter, and the number of dendrites was reduced (Figure S3). e results were the same
aer treatment with 5 mM or 10 mM metformin for 96h in melanocytes (Fig.4a). e percentage of cells
with fewer than 3 dendrites was signicantly increased (Fig.4b), while the percentage of cells with more than
3 dendrites was signicantly decreased (Fig.4c). e percentage of arbutin-treated melanocytes with more
or fewer than 3 dendrites did not signicantly dier from that of the control group (Fig.4b,c). Additionally,
scanning electron microscopy revealed that the number of dendrites and lopodia-like structures in 10 mM
metformin-treated melanocytes was lower than that in control melanocytes (Fig.4d). In the coculture system
of primary melanocytes and HaCaT cells, melanosomes with gp100 labelling coupled with FITC (green) in
melanocytes and cytokeratin-positive HaCaT cells coupled with Alexa Fluor® 647 (red) were visualized. A
reduction in the number of green uorescence spots was observed aer treatment with metformin and arbutin
for 48h (Fig.4e), and the number of HaCaT cells positive for both gp100 and cytokeratin was signicantly
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reduced aer metformin or arbutin intervention (Fig.4f). To further determine the eect of metformin on
melanosome transfer, we examined its eects on key molecules involved in melanosome transfer, including
MLPH, Rab27a and Myo5a. In melanocytes, treatment with 5 mM and 10 mM metformin for 12h reduced the
mRNA levels of melanophilin (MLPH) and Rab27a, whereas the mRNA levels of myosin Va (Myo5a) did not
signicantly change. e mRNA expression of these genes did not change in the arbutin-treated melanocytes
(Fig.4g). e levels of MLPH, Rab27a and Myo5a were obviously decreased at the protein level in melanocytes
treated with metformin or arbutin for 72h (Fig.4h).
Metformin may inhibit melanosome transfer by altering the cytoskeleton and Rho small
GTPases
Changes in cell morphology involve proteins that comprise the cytoskeleton. To investigate the mechanism by
which metformin induces the dendritic changes in melanocytes, we observed the expression of F-actin aer
metformin treatment via immunouorescence. Figure5a shows that the expression of phalloides-binding
F-actin was signicantly increased and that F-actin was polymerized in metformin-treated melanocytes.
RhoA and Rac1, members of the Rho small GTPases, are key molecules in cytoskeletal regulatory pathways.
e protein levels of RhoA and downstream ROCK1 were increased, and Rac1 was decreased aer metformin
treatment (Fig.5b). In melanocytes, 30 µM RhoA partially abrogated the inhibitory eect of 10 mM metformin
on dendrite formation (Fig.5c,d). Moreover, metformin partially abrogated the increase in MLPH, Rab27a, and
Rac1 expression and the decrease in RhoA expression induced by Forskolin (Fig.5e).
Discussion
Metformin, which is a type of biguanide, is the most frequently prescribed drug for type 2 diabetes mellitus
(T2DM)26. Moreover, it has been found to be eective in the treatment of other disorders. Previous studies have
veried that the antidiabetic mechanism of metformin involves reducing the production of cAMP17, and cAMP
signalling is a well-known regulator of melanogenesis27, which supports the potential application of metformin
in the treatment of melanin production disorders.
Zebrash serves as a reliable model for screening and evaluating the eects of depigmentation agents on
melanin production and transfer28. As shown in Fig.1, metformin obviously inhibited melanogenesis in vivo.
In primary melanocytes, MNT1 cells, and B16F10 cells, melanin content and tyrosinase activity were obviously
reduced aer 5 mM, 10 mM metformin, or 1 mM arbutin treatment regardless of health status and α-MSH-
induced pigmentation. Overall, metformin may aect melanin synthesis and can serve as a candidate skin-
whitening agent.
In addition to melanin synthesis, melanosome transfer also plays an important role. Several studies using
reectance confocal microscopy (RCM) have shown that the number of dendrites in melanocytes in the basal
layer of melasma patient skin is increased, which is considered a sign of disease activity29–31. Dendrites are
pivotal morphological markers of melanocytes and are involved in diverse modes of melanosome transfer to
surrounding keratinocytes32,33. In response to metformin treatment, the cellular morphology apparently changed,
and the number of dendrites and lopodia-like structures markedly decreased in melanocytes. Furthermore,
melanosome transfer was also decreased. ese ndings demonstrated that metformin, rather than arbutin,
inhibited the formation of dendrites and subsequently decreased melanosome transfer.
To elucidate the mechanisms of metformin, we rst compared the cAMP level in metformin-treated
melanocytes with that in control melanocytes. Figure2a shows the reduction in cAMP levels aer treatment with
metformin. Moreover, cAMP-PKA-MITF serves as the primary pathway in the regulation of melanogenesis, and
we illustrated the downregulation of MITF and its downstream pigmentation enzymes, including TYR, TYRP1
aer metformin treatment. ese ndings reveal that metformin inhibits melanogenesis via downregulation of
members in the cAMP-MITF pathway. Moreover, in B16F10 cells or melanocytes, metformin partially abrogated
the increases in melanin content, tyrosinase activity, and TYR and TYRP1 expression induced by the cAMP
activator Forskolin. In addition, one recent study demonstrated that Nrf2 was one of the targets of metformin22.
Our results showed that metformin reduced the p-Nrf2 expression, and a Nrf2 activator could enhanced the
expression of TYR and TYRP1, whereas metformin partially abrogated the upregulation induced by Oltipraz.
Furthermore, we found that cAMP and Nrf2 interact with each other, Nrf2 activator increased the cAMP
Fig. 4. Metformin alters cellular morphology and inhibits melanosome transfer. (a–c) Treated with metformin
and arbutin for 96h, the cellular morphology of melanocytes was photographed under microscopy (a, scale
bar = 100μm), and the dendrites of melanocytes were counted. the number of bipolar melanocytes was
notably increased (b) and the number of multi-polar melanocytes was prominently decreased (c) aer being
treated with metformin; the dendrites of melanocytes were not changed aer the eect of arbutin. (d) In
incubation with metformin for 72h in melanocytes, the number of melanocytes and lopodia-like structures
were both reduced under scanning electron microscopy (SEM). (e,f) In the co-culture system of HaCaT cells
and melanocytes, the melanosomes (gp100 labeling, green) transferred to keratinocytes (cytokeratin, red)
were reduced aer treatment with metformin and arbutin (presented as white arrow) (scale bar = 50μm), the
number of HaCaT cells positive for both gp100 and cytokeratin was signicantly reduced aer metformin or
arbutin intervention. (g,h) e mRNA and protein expression levels of melanosome transfer-related genes,
MLPH, Rab27a, and Myo5a, were determined by RT-qPCR (g, 12h) and western blot (h, 72h; the gels/blots
was from the same protein sample which extracted from the same batch of cells). Results were presented as
mean ± SD. *p<0.05, **p < 0.01, ***p < 0.001; ns, not signicant.
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content, and vice versa. So to conclude, the cAMP-Nrf2 pathway is at least partially involved in the regulation of
melanin synthesis by metformin.
Additionally, changes in cell morphology are mainly caused by alterations in the cytoskeleton. In this
study, aer metformin treatment, the cytoskeleton and the expression of Rho GTPases, including RhoA and
Rac1, changed. Studies have reported that Rac1 promotes dendrite formation, whereas RhoA inhibits dendrite
formation34,35, which is consistent with our results and the changes in dendrites observed in this study. A RhoA
inhibitor can partially abrogate the inhibitory eect of metformin on dendritic formation, suggesting that the
inhibitory eect is at least partially mediated by Rho GTPases. Moreover, the expression and activity of Rho
and Rac are regulated by cAMP, and a previous study demonstrated that cAMP mediates dendrite formation
in melanocytes by increasing Rac activity and decreasing Rho activity36. In melanocytes, a cAMP activator can
increase the expression of MLPH and Rac1 and decrease the expression of RhoA. Moreover, metformin treatment
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partially reversed the eects of the cAMP activator on melanin transfer-related genes and Rho GTPases. ese
ndings suggest that metformin regulates melanosome transport at least in part through cAMP-Rho GTPases.
e oral dose of metformin for T2DM is 500–2000mg, and the peak plasma concentration is about 2µg/ml,
and rarely exceeds 4µg/ml37. So it is unlikely to reach mM concentration of metformin within skin lesion for
oral application. However, topical preparations give metformin a chance for the treatment of hyperpigmentary
disorders. e results of two published studies indicate that topical metformin is eective in alleviating
melasma19,20. e development of topical metformin needs further research, so that it can be well absorbed into
lesion with signicant eects on skin whitening.
Conclusion
Collectively, these results demonstrated that metformin not only reduced melanin production but also markedly
reduced melanosome transfer, at least partially through the cAMP signalling pathway. In the future, metformin
may serve as an eective depigmentation compound for the treatment and prevention of hyperpigmentation
disorders.
Methods
All methods were carried out in accordance with relevant guidelines and regulations, and all methods are
reported in accordance with ARRIVE guidelines. is study was approved by the institutional ethics committee
of the Institute of Dermatology, Peking Union Medical College, Chinese Academy of Medical Sciences(2022-
KY-071).
Zebrash culture and treatments
e wild-type zebrash embryos were obtained from the China Zebrash Resource Center (CZRC). is
research was approved by the institutional ethics committee of the Institute of Dermatology, Peking Union
Medical College, Chinese Academy of Medical Sciences(2022-KY-071). Embryos were cultured at 28℃ with a
photoperiod (14h light/10h darkness), and the fresh medium was changed daily. At 2h post fertilization (hpf),
they were randomly divided into dierent groups. ey were photoed at 48hpf, 72hpf, and 96hpf, and the area
of melanin granules in the head regions was measured by Image J soware. e pigmentation area (%) was
determined as the area of melanin granules as a percentage of the area of the whole head. At 96hpf, the zebrash
were collected to conduct the experiments on melanin content, tyrosinase activity and extraction of RNA.
Cell culture
All methods were carried out in accordance with relevant guidelines and regulations. Primary melanocytes were
separated from remanent human foreskin tissue aer circumcision, which was approved by the institutional
ethics committee of the Institute of Dermatology, Peking Union Medical College, Chinese Academy of Medical
Sciences (2022-KY-071). All subjects were over 18 years of age and their informed consent was obtained. Cells
was cultured in MelM (Melanocyte Medium, ScienCell, USA), passage 2 to passage 8 were used. B16F10 and
HaCaT cells were cultured in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS,
Newzerum, New Zealand) and 1% penicillin-streptomycin (Gibco, USA), while MNT1 cells were cultured in
DMEM medium supplemented with 20% FBS and 100U/mL penicillin-streptomycin. All cells were incubated
at 37℃ and 5% CO2.
Cell viability assay
Cells were seeded in a 96-well Culture Plate (CORNING, New York, USA) at 4000 cells/well. e next day,
dierent concentrations of metformin (Sigma, USA) was added and cultured for 24–48h, the wells were replaced
with 100µl culture medium plus 10µl CCK-8 (Cell Counting Kit-8, Apex) and then measured at 450nm.
Relative cell viability was calculated as a percent of the control group.
Measurement of melanin contents
Cells (2 × 105) were cultured in a 6-well plate for 24h and treated with 5 mM metformin hydrochloride (metformin
for short), 10 mM metformin or 1 mM arbutin (VangBrand, China) with or without 1 µM of α-melanocyte-
stimulating hormone (α-MSH, Sigma, USA). ereaer, the cells were washed twice with phosphate-buered
saline (PBS, Gibco, USA) and harvested in 1.5ml tubes. Cells were lysed by incubating in 200µl of 1N NaOH,
at 80℃ for 2h. e cell lysates were centrifuged at 12000xg for 10min and the supernatants were transferred to
Fig. 5. Metformin inhibits melanosome transfer by altering the cytoskeleton and Rho small GTPases. (a)
Treated with metformin (5 and 10 mM) for 72h in melanocytes, the cytoskeleton (red) was stained with
TRITC-Phalloidin and cell nuclei were stained with DAPI (blue) through laser confocal microscopy (LCM),
the cytoskeleton was obviously changed (scale bar = 50μm). (b) Treated with metformin (5 and 10 mM) for
72h in melanocytes, the Rho GTPases (ROCK1, RhoA, and Rac1) were determined by Western blot (the gels/
blots was from the same protein sample which extracted from the same batch of cells). (c,d) In melanocytes,
treated with 30 µM RhoA inhibitor and/or metformin for 96h, the number of dendrites was counted and
compared. RhoA inhibitor partially reversed the inhibition eect on dendrite formation by metformin. (e) In
melanocytes, treated with metformin and/or Forskolin for 72h, the protein levels of melanosome transfer-
related genes (MLPH, Rab27a) and Rho GTPases (RhoA, Rac1) were detected by western blot (the gels/blots
was from the same protein sample which extracted from the same batch of cells). Results were presented as
mean ± SD. *p<0.05, ***p < 0.001.
◂
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a 96-well plate (50µl/well, 3 wells each group), then measured at 450nm and 405nm. Melanin contents were
analyzed as the percent of the control group in cells.
Tyrosinase activity assay
e cell culture, treatment and collection were the same as the above melanin contents assay. Cells were lysed
by 300µl of cell lysis buer (includes 1 mM PMSF and 1 mM Triton-X 100) at -80℃ for 2h, melted at room
temperature for about 30min, and then centrifuged at 10000xg for 5min. 80µl supernatants plus 20µl 0.01%
L-DOPA were added to each well of a 96-well plate. Aer incubation at 37℃ for 2h, the dopachrome was
measured at 475nm and 490nm. e results were analyzed as the percent of the control group.
The dendrites counting of melanocytes
Aer treatment with the presence or absence of 5 mM metformin, 10 mM metformin or 1 mM arbutin for 96h,
melanocytes were taken photographs under optical microscopy (200×) that were used to count dendrites. A total
of 300 cells were randomly selected for each group, the number of cells with less than or no less than 3 dendrites
was recorded and the averages were taken for comparison and analysis.
Scanning electron microscopy
Aer treatment with metformin for 72h in melanocytes, the cell slivers were prepared and underwent the steps of
cleaning, xation, dehydration, drying and spraying, and nally observed under scanning electron microscopy.
Melanosome transfer assay using immunouorescence staining
Melanocyte and HaCaT cells were co-cultured in confocal dishes (NEST, China) with a ratio of 1:2, the mixed
medium was prepared with MelM and HaCaT medium with a ratio of 1:2. Mixed medium containing metformin
or arbutin was separately added into each group for 72h. All the cells were rinsed with PBS, xed with 4%
paraformaldehyde, permeated with Triton-X 100, blocked with 5% BSA and incubated in mixed primary
antibodies of rabbit polyclonal anti-wide spectrum cytokeratin (1:200, Abcam, USA) and mouse monoclonal
anti-melanoma gp100 (1:50, Abcam, USA) overnight at 4℃. Followed by mixed secondary antibodies of donkey
anti-rabbit IgG H&L coupling Alexa Fluor® 647 (1:100, Abcam, USA) and goat anti-mouse IgG H&L coupling
FITC (1:100, Abcam, USA), the cells in dishes were lastly stained with DAPI (1:1000, Beyotime, China) and
melanosome transfer can be observed using Confocal Laser Scanning Microscopy (CLSM). Photographs were
randomly selected for each group, the number of HaCaT cells positive for both gp100 and cytokeratin was
recorded and the averages were taken for comparison and analysis.
RNA extraction and RT-qPCR
Total RNA was extracted with RNAiso Plus (Takara, Japan), dissolved in DEPC-treated water (Beyotime,
China) and quantied spectrophotometrically. cDNA was synthesized with HiScript®III RT SuperMix for qPCR
(Vazyme, China) and quantitative real-time PCR was conducted with ChamQ Universal SYBR qPCR Master Mix
(Vazyme, China) according to the manufacturer’s instructions. e expression levels of genes were normalized
against β-actin, and folding change was calculated by comparing 2−ΔΔCT. e primers are listed below (Table1).
Western blot
Melanocytes were seeded in a 6-well plate at the density of 2 × 105cells/well, and then stimulated with or without
metformin, arbutin, Forskolin (MCE, USA) or Oltipraz (MCE, USA) for 72 h. e protein was harvested
by rinsing with cold PBS followed by scraping from the asks and lysis with RIPA buer (Beyotime, China)
containing protease inhibitors. All proteins were quantied using a BCA protein assay kit (KeyGEN BioTECH,
Gene Forward sequence Reverse sequence
MITF (zebrash) C A T T G A A C G A A G A A G G C G G T G C A G G A G A T G T C T G T T T G C G
TYR (zebrash) C T G T C A G G T G T G C A C G G A T A C C C G T C A C A C A A A G C C T C
TYRP1a (zebrash) C T C C T A T G A G G T G C A G T G G C G A A C G A G A G C G A A C G A C A C A
DCT (zebrash) A A A G C C A T C G A C T T C T C G C A G A T T C G G G A T G G G T C A C T G G
MLPHa (zebrash) A A A G T G A T G C G G T C C C T G T A G C G A G A G T G A A C G G C A T A G T
Rab27a (zebrash) C G A G G A G A T C A T C A G G C G T T C C C A G A G C G A G G A A C T T G A T
Myo5a (zebrash) G G A G A A A G C C A C A A G T C C C A C C T C T T G G G G T C A A A C G T G A
β-actin (zebrash) T T G A C A A C G G C T C C G G T A T G T C C C A T G C C A A C C A T C A C T C
MITF (human) A A A T A C G T T G C C T G T C T C G G T G T T G G G A A G G T T G G C T G G A
TYR (human) T C A G C C C A G C A T C A T T C T T C G G C A T C C G C T A T C C C A G T A A
TYRP1 (human) A C C A G A G G G T T C T C A T A G T C A G T T C T C A A A T T G T G G C G T G T T
MLPH (human) T G C C C A T C T G A A C G A G A C C G A G C C G A T C T T C A C G A C T C T G
Rab27a (human) A C A A C A G T G G G C A T T G A T T T C A A A G C T A C G A A A C C T C T C C T G C
Myo5a (human) C A G A G T C C G C T T T A T T G A T T C C A A T C A C C C A T G T T C T G A C C A C T
β-actin (human) G T G G C C G A G G A C T T T G A T T G C C T G T A A C A A C G C A T C T C A T A T T
Tab le 1. Primers used for real-time quantitative PCR in zebrash and cells.
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China) according to the steps of the manual. Equal mass and volume of proteins were resolved by SDS-PAGE and
transferred to nitrocellulose membranes by electro-blotting. e PVDF membrane (Millipore, USA) containing
protein was incubated in diluted primary antibody (MITF, Proteintech, China, 1:1000; TYR, ABclonal, China,
1:1000; TYRP1, Santa Cruz, USA, 1:1000; β-actin, ABclonal, China, 1:1000; MLPH, Proteintech, China, 1:1000;
Rab27a, Proteintech, China, 1:1000; Myo5a, ABclonal, China, 1:1000; RhoA, Proteintech, China, 1:1000; Rac1,
Proteintech, China, 1:750; ROCK1, Proteintech, China, 1:1000; Phospho-Nrf2, ABclonal, China, 1:1000) for
about 14–18h at 4℃. en aer incubation of secondary antibody with HRP-labeled (ABclonal, China, 1:2000),
the membrane was displayed using the ECL chromogenic system.
Cytoskeleton staining
Cells (2 × 105) were cultured in confocal dishes and treated with or without metformin for 48–72h. Cells were
rinsed with PBS, xed with 4% paraformaldehyde, permeated with Triton-X100 and then incubated in TRITC-
Phalloidin (1:400, Servicebio, China) for 90min, following stained with DAPI, nally observed in CLSM.
Determination of cAMP content
Extracellular and intracellular cyclic adenosine monophosphate (cAMP) was measured using an enzyme-linked
immunosorbent assay (ELISA) (Elabscience®, China). e preparation of cell supernatant and cell lysates as well
as procedure of cAMP detecting were carried out according to the instructions.
Statistical analysis
Each experiment was performed in triplicate and values were presented as mean ± standard deviation (SD).
GraphPad Prism 9 (GraphPad Soware Inc., San Diego, CA, USA) was used for data analysis and graphing
formation. p<0.05 was dened as statistically signicant (*p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Data availability
Data and materials supporting the ndings of this study are available in the article and its Supplementary Fig-
ures.
Received: 5 August 2024; Accepted: 19 March 2025
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Acknowledgements
We would like to thank Prof. Xu Chen for his expert assistance, thank Prof. Tong Lin and Yin Yang for their
funding support and experimental project.
Author contributions
Xing Liu: Writing - original dra, Writing - review & editing, Investigation. Xiaojie Sun: Data curation, Formal
analysis, Validation. Yunyao Liu: Methodology, Supervision. Wenzhu Wang: Formal analysis. Hedan Yang: Vis-
ualization, Soware. Yiping Ge: Resources. Yin Yang: Funding acquisition, Conceptualization, Project adminis-
tration. Xu Chen: Project administration, Writing - review & editing. Tong Lin: Conceptualization, Supervision,
Funding acquisition.
Funding
is research was supported by the National Natural Science Foundation of China (82103705), CAMS Innova-
tion Fund for Medical Sciences (CIFMS-2021-I2M-1-001) and Funds for Science and Technology Plan Projects
in Jiangsu Province (BE2023675).
Declarations
Competing interests
e authors declare no competing interests.
Ethical statement
is research was approved by the institutional ethics committee of the Institute of Dermatology, Peking Union
Medical College, Chinese Academy of Medical Sciences(2022-KY-071).
Additional information
Supplementary Information e online version contains supplementary material available at h t t p s : / / d o i . o r g / 1
0 . 1 0 3 8 / s 4 1 5 9 8 - 0 2 5 - 9 5 2 4 5 - x .
Correspondence and requests for materials should be addressed to Y.Y., X.C. or T.L.
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