Phycojuvenine and skin cell respiration
Pierre-Yves Morvan and Romuald VallTe - Life Science Research Department, Codif International, France
Marine brown algae could play an important role in the cellular metabolism thanks to specific sugars and polyols naturally produced or induced after stress.
We have developed a seaweed extract (Phycojuvenine) obtained from the brown algae Laminaria digitata by lixiviation and concentration, and we have studied its effect on oxygen consumption of two types of human skin cells: epidermal keratinocytes and dermal fibroblasts isolated from normal human skin. The effect on respiration was evaluated at the level of the entire cells (basal cellular respiration) and at the level of the mitochondria in fixed cells. Mitochondria are subcellular units, extensively studied since a clear link between their activity and ageing was highlighted: an accelerated ageing is detected when mitochondria activity decreases and their circular DNA (mtDNA) is fragmented. It was observed that an accumulation of mtDNA mutations is associated with premature ageing phenotypes and a reduced lifespan, and more recently, it was proposed that respiratory chain dysfunction per se is the primary inducer of premature ageing in mtDNA mutator mice.1
We have compared two methods for measuring cellular and mitochondrial respiration, a classical method using an oxygraph and a Clark oxygen electrode, and an original method using a fluorescent probe. In parallel, we used another fluorescent probe for labelling mitochondria and visualised their activation and shape by microscopic observation.
Materials and methods
A Laminaria digitata extract was obtained by a short-time extraction made at room temperature with demineralised water using freeze-dried seaweed, followed by a filtration with 0.22 µm filter and a concentration by reverse osmosis in order to obtain a concentration of approximately 5% (w/w) dry matter.
Primary cultures of human dermal fibroblasts and epidermal keratinocytes were obtained starting from skin fragments after cosmetic surgery. Cells of 3T3-L1 lines were obtained from ATCC (American Type Culture Collection).
Basal cellular and mitochondrial Respiration
Basal cellular and mitochondrial respiration have been studied on human keratinocytes thanks to two different methods: the first one was the classical method using an oxygraph and a Clark oxygen electrode; the second method used a fluorescent probe (BD Oxygen Biosensor System). These assays were performed under two different experimental conditions: study of the basal cellular respiration rate on skin cells in the presence of glucose at 1.25 mM, and study of the mitochondrial respiration rate of permeabilised cells, in the presence of pyruvate at 625 µM and malate at 500 µM, as respiratory substrates.
Oxygen consumption assay
This study was conducted on human keratinocytes in culture dissociated with trypsin and permeabilised or not with digitonin, and maintained in suspension with HBSS medium at 30°C containing either glucose or pyruvate-malate. The respiration was monitored in real time and expressed in picoatoms of oxygen consumed/minute/106cells. The addition of the test product into the oxygraph vessel detected any stimulation or inhibition of respiration. The assay was conducted after leaving the test product to stand with the cells for 20 minutes. Each experimental condition was produced in quadruplicate.
Human keratinocytes in monolayer were suspended after dissociation with trypsin, then permeabilised or not with saponin at 50 µg/ml and cultured in 96-well plates with medium without (control) or with the tested products. The respiration rate, expressed in arbitrary units (A.U.)/minute, was calculated on the linear part of the fluorescence emission curve. Each experimental condition was performed in hexaplicate.
The morphology of mitochondria was studied using the fluorescence probe MitoTracker Green (molecular probes), which specifically integrates the membranes of mitochondria. Fibroblasts were cultivated in complete culture medium (DMEM medium with fetal calf serum (FCS) at 10%). At confluency, mediums were removed and replaced by assay medium (DMEM medium with 2% FCS) containing or not (control) the tested product. Each experimental condition was produced in triplicate. Cells were cultured during 24 hours at 37°C, 5% CO2. Media were replaced by media without FCS containing the fluorescence probe MitoTracker Green. After 45 minutes of incubation, cells were washed and observed thanks to an automated cell imager “InCell Analyzer 1000” (Amersham Biosciences), that delivers the flexibility required for cell-based research while maximising image and data quality through its combination of modular design, breadth of analysis options, and proven biological applications. Thus, globular mitochondria were counted, thanks to a morphological filter, in the optical field of the microscope.
Measure of ATP level
The effect of the products on the cellular and mitochondrial ATP synthesis rate of the 3T3-L1 cells in culture was measured by bioluminescence using the luciferin/luciferase kit. The cells were suspended in the oxygraph vessel at 30°C and stirred. To measure the mitochondrial ATP synthesis, the cells were permeabilised using digitonin in the presence of pyruvate-malate. The test product was applied directly at the wanted concentration on the cells in suspension in the oxygraph vessel. The speed of ATP synthesis is expressed in nmoles/min/106cells. A control batch receiving no product was produced. Each experimental condition was produced in quadruplicate.
Incorporation of tritiated proline
The effect of the test product on collagen synthesis was assessed by measuring the incorporation of tritiated proline by human skin fibroblasts in culture. The test is based on measuring the incorporation of proline in “proline-rich” proteins of fibroblast cultures (this incorporation mainly takes place in essentially type I collagen). The analysis was carried out on total proteins (pool of secreted and intracellular proteins) synthesised by two different fibroblast models: normal (young) human skin fibroblasts (used for the 8th passage) and senescent human skin fibroblasts (pre-senescent, having undergone a high number of passages, used for the 16th passage). Normal and senescent fibroblasts were seeded in 96-well plates in complete culture medium (DMEM medium with FCS at 10%).
At 80% confluence, the medium was replaced by new medium (10% FCS) both with and without (control) the test compounds. Each test condition was carried out in triplicate. The cells were then incubated at 37°C for 72 hours with the addition of L-[2,3-3H]-proline (1,7Tbq/mmol, 46Ci/mmol), 24 hours before the end of incubation. Proline incorporated in total proteins (soluble protein fractions and intracellular and deposited proteins) was analysed by addition of 1 V of buffer (Tris/HCl 50mM, guanidine 4 M, EDTA 5 mM, pH 8.0), trichloracetic acid precipitation (TCA), collection on filters, washing with TCA and then with 70% ethanol and liquid scintillation counting.
The unprocessed data was transferred and processed in Excel and analysed in PRISM (Graph Pad Software). The between-group comparisons were conducted by means of an analysis of variance (ANOVA) using the Dunnet multiple comparison test.
Effect of Phycojuvenine on basal cellular and mitochondrial respiration
Laminaria digitata extract tested at 1% and 5% significantly (p<0.01) increased basal cellular and mitochondrial respiration of human keratinocytes using the oxygraph method: +17% and +24% at 1% and 5% respectively for the basal respiration, and + 14% and +21% at the same concentrations for the mitochondrial respiration (Fig. 1). This effect was confirmed using the fluorescence method: Laminaria digitata extract tested at 5% increased basal cellular and mitochondrial respiration by +37% and +33% respectively (Fig. 2).
Effect of Phycojuvenine on mitochondrial morphology
Human fibroblasts in culture presented elongated mitochondria, i.e. slightly active (Fig. 3A). After addition of TGF beta at 10 ng/ml, mitochondria presented a globular form (Fig. 3B). These globular mitochondria were grouped mainly around the cellular nuclei. TGF beta increased significantly by 26% the number of globular mitochondria (Fig. 4). Addition of the new Laminaria digitata extract induced the same type of modification: globular mitochondria were visualised mainly around the cellular nucleus, thus mitochondria were activated (Fig. 3C).
Effect of Phycojuvenine on ATP level
The stimulation of respiration by the novel Laminaria digitata extract at 5% was well correlated to an elevated ATP production measured in 3T3-L1 cells. Laminaria digitata at 5% significantly (p<0.05, Dunnett’s test) increased the basal ATP synthesis by 17% and the mitochondrial ATP synthesis by 13% (Fig. 5). There is a good correlation between mitochondrial activation and stimulation of ATP level.
Effect of Phycojuvenine on protein synthesis
Aged fibroblasts synthesise much less collagen than young fibroblasts: the tritiated proline incorporation decreased by 33%. Laminaria digitata at 5% did not significantly modify the incorporation of proline in total proteins by young fibroblasts, whereas it significantly stimulated the incorporation of proline in aged fibroblasts (137% of control, p<0.01). Thanks to Laminaria digitata extract, aged fibroblasts synthesise as much collagen as young fibroblasts (Fig. 6).
Phycojuvenine, a seaweed extract of Laminaria digitata, increased basal cellular and mitochondrial respiration in skin cells. These results obtained using the fluorescent probe were confirmed with the classical method using an oxygraph and a Clark oxygen electrode. The increase of mitochondrial respiration was well correlated with an activation of mitochondria, which results in a modification from the elongated form (fusion) to a globular form (fission), and a grouping of mitochondria around the cellular nuclei in fibroblasts. This modification of the shape and localisation of mitochondria was previously described for yeasts.2 It is linked to the expression of specific adhesion proteins.
The stimulation of the respiration observed in human keratinocytes was associated with an increase of the metabolism, in particular to an elevated ATP production.
In the skin cells, stimulation of respiration leads to a higher energetic level and thus to a stimulation of the cellular proliferation and an increase of the protein synthesis. In the case of the new Laminaria digitata composition, it increases basal cellular and mitochondrial respiration, activates mitochondria, increases ATP level, and restores the protein synthesis by the ageing cells. Laminaria digitata could therefore be considered as an anti-ageing ingredient for cosmetics.
1 Trifunovic A., Hansson A., Wredenberg A., Rovio A.T., Dufour E., Khvorostov I., Spelbrink J.N., Wibom R., Jacobs H.T., Larsson N.G. Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production. Proc Natl Acad Sci USA. 102(50): 17993–17998, 2005.
2 Pelloquin L., Ducommun B., Belenguer P. Interaction between the fission yeast nim1/cdr1 protein kinase and a dynamin-related protein. FEBS Lett. 443: 71-74, 1999.