Annurca Apple Polyphenols Ignite Keratin Production in Hair Follicles by Inhibiting the Pentose Phosphate Pathway and Amino Acid Oxidation

Abstract

Patterned hair loss (PHL) affects around 50% of the adult population worldwide. The negative impact that this condition exerts on people's life quality has boosted the appearance of over-the-counter products endowed with hair-promoting activity. Nutraceuticals enriched in polyphenols have been recently shown to promote hair growth and counteract PHL. Malus pumila Miller cv. Annurca is an apple native to Southern Italy presenting one of the highest contents of Procyanidin B2. We have recently shown that oral consumption of Annurca polyphenolic extracts (AAE) stimulates hair growth, hair number, hair weight and keratin content in healthy human subjects. Despite its activity, the analysis of the molecular mechanism behind its hair promoting effect is still partially unclear. In this work we performed an unprecedented metabolite analysis of hair follicles (HFs) in mice topically treated with AAE. The metabolomic profile, based on a high-resolution mass spectrometry approach, revealed that AAE re-programs murine HF metabolism. AAE acts by inhibiting several NADPH dependent reactions. Glutaminolysis, pentose phosphate pathway, glutathione, citrulline and nucleotide synthesis are all halted in vivo by the treatment of HFs with AAE. On the contrary, mitochondrial respiration, β-oxidation and keratin production are stimulated by the treatment with AAE. The metabolic shift induced by AAE spares amino acids from being oxidized, ultimately keeping them available for keratin biosynthesis.

Keywords: Procyanidin B2; anti-oxidants; apple polyphenols; hair growth; nutraceuticals.

Figure 1

AAE induces early exit from telogen and increases keratin content of hair shafts in murine HFs. C57BL/6 mice were treated topically with a foam supplemented either with AAE or with a placebo. Foams were applied on the dorsal skin of 7-week-old mice. After 4 weeks of treatment, mice were sacrificed and HFs classified following morphological criteria. Haematoxylin-eosin staining of HFs of mice treated with placebo (a–b) showing HFs mostly in Telogen/AnagenI phase. HFs of mice treated with AAE appeared in a later stage of Anagen, mostly Anagen II (c–e). (f–h) SEM (SEM-EDX) analysis of hairs extracted from mice treated with placebo (f) or with AAE (g). SEM quantitative analysis indicates (h) an increase in Sulfur concentration (cystine, methionine, cysteine and cysteic acid all abundant amino acids of hair keratins) in hairs of mice treated with AAE. Values in € and (h) are reported as mean ± SEM (n = 8, *** p < 0.001). Scale bars in a-d correspond to 200 μm. Scale bars in (f) and (g) correspond to 10 μm.

Figure 2

AAE diverts HF metabolic pathways from PPP and amino acid oxidation. Metabolomic analysis (Carbohydrates (a); nucleotides (b); amino acids (c); carnitine derivatives (d)) of HF cells extracted from C57BL/6 mice treated topically for 4 weeks with a foam containing AAE (red dots, ANNURCA) or a Placebo (black dots PLACEBO). Each point represents the normalized intensity of the metabolite (n = 15 measurements, mean ± SEM. Two way ANOVA and Bonferroni post-test analysis were performed; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; n.s.: non statically different).

Figure 3

AAE increases mitochondrial membrane potential in murine HFs. Skin biopsies collected from mice treated with AAE or placebo foam were incubated ex-vivo with Mitotracker CMX-ROS. UPPER PANEL: A faint fluorescence emission of the probe (red channel) is detectable in Keratin 14 positive and Keratin 6 HF cells (green channel) in placebo biopsies. LOWER PANEL: Increased fluorescence emission of Mitotracker CMX-ROS in Keratin 14 and Keratin 6 positive HF cells and hair bulges of mice treated with AAE. Asterisks indicate HFs. Arrows indicate bulge regions. Scale bars correspond to 200 μm in (a), (c) and (d), 50 μm in (b).

Figure 3

AAE increases mitochondrial membrane potential in murine HFs. Skin biopsies collected from mice treated with AAE or placebo foam were incubated ex-vivo with Mitotracker CMX-ROS. UPPER PANEL: A faint fluorescence emission of the probe (red channel) is detectable in Keratin 14 positive and Keratin 6 HF cells (green channel) in placebo biopsies. LOWER PANEL: Increased fluorescence emission of Mitotracker CMX-ROS in Keratin 14 and Keratin 6 positive HF cells and hair bulges of mice treated with AAE. Asterisks indicate HFs. Arrows indicate bulge regions. Scale bars correspond to 200 μm in (a), (c) and (d), 50 μm in (b).

Figure 4

Topical treatment with AAE alters the murine HF metabolome. Schematic cartoon depicting some of the metabolic reactions positively (green box) or negatively (red boxes) affected by AAE in murine HFs. Red and green arrowheads indicate reactions halted or stimulated by AAE, respectively. Red and green dots indicate metabolites whose intracellular concentration resulted decreased or increased by treatment with AAE, respectively.