Aging skin typically shows changes such as a loss in tone and elasticity, hyperpigmentation, roughness and wrinkles caused by, among other factors, impaired hydration and barrier function. Wrinkling is associated with progressive atrophy of the dermis, as well as changes in architectural organization.1 Extracellular matrix dermis is mainly composed of proteins, proteoglycans and glycoproteins that produce a protective and supportive environment for cells and allow them to communicate with their surroundings. Collagen is the most abundant protein group in skin, accounting for 70–80% of skin’s dry weight,2 and the vast majority of it comprises types I and III. Fibrillar collagen in particular provides the tensile strength and recoil of skin.3
The most common amino acid sequence in a collagen molecule is Gly-X-Y, where X and Y may be any amino acid residue but often are proline and hydroxyproline (Hyp). This Gly-X-Y triplet facilitates the left-handed helix structure and, subsequently, the creation of the right-handed triple helical structure.4 Collagen synthesis involves many steps,5 as summarized in Figure 1. Each type of collagen is encoded by a specific gene and synthesized as the precursor preprocollagen. Post-translational modification follows, taking place in the lumen of the endoplasmic reticulum. This step involves the hydroxylation of specific prolines and the emergence of Hyp, and is catalyzed by prolyl-4-hydroxylase with ascorbate as a cofactor, and molecular oxygen, iron (II) and α-ketoglutarate, as cosubstrates.6
In collagen I, Hyp accounts for about 10% of all amino acids,7 and its presence is important for the proper arrangement and stability of collagen under physiological temperatures.6 Hydroxylations of prolines and lysines (step two in Figure 1) are also necessary for the rapid secretion of procollagen molecules into the cytoplasm—conversely, fewer hydroxylated molecules are prone to degradation. At this point in the collagen synthesis process, collagen molecules are readily soluble and transportable thanks to C- and N-propeptides. Newly synthesized procollagen chains associate into trimers via their C-propeptides, leading to nucleation and folding of triplehelical redion. Once procollagen is found in the extracellular matrix, the propeptides are removed by C- and N-proteases and the spontaneous self-assembly of collagen molecules into fibers is triggered.8
As stated, qualitative and quantitative changes in collagen in the dermis, and the extent of modification, occur with age.9 This phenomenon is even more pronounced in areas affected by photoaging.10 This reduced quantity and quality of collagen is not solely attributed to a drop in proliferative and synthetic activity in skin cells, or an increase in the activity of degradative enzymes.11 It also occurs due to a fall in the activity of enzymes catalyzing the post-translational modification of proline and lysine12—e.g., prolyl 4-hydroxylase.
In order to address these reductions in collagen, enzyme cofactors in the collagen synthesis process were of interest; in particular, α-ketoglutarate. To better penetrate cell membranes, a hydrophobically modified derivative of α-ketoglutarate (HEαKG) was developed and evaluated both in vitro, for effects on collagen production by dermal fibroblasts, and in vivo, for effects on wrinkles.
Materials and Methods
Test materials: To obtain the hydrophobized α-ketoglutarate (aKG) derivate, a mix of hexylesters of aKG (HEαKG) was prepared. This mix was tested at a concentration of 100 µg/mL. Normal human dermal fibroblasts (NHDF), isolated from skin grafts from healthy donors undergoing facial plastic surgery, and 3T3 lines of murine embryonal fibroblasts were used. The waste skin samples were obtained by informed consent. Both cell types were cultivated in DMEM complemented with 10% FBS, L-glutamine and penicillin/streptomycin.
Picrosirius red assay for collagen: NHDF from younger donors, ages 6–10, and older donors, ages 50–65, were seeded in testing plates. After overnight adhesion, cells were pre-incubated in a serum-free medium with ascorbate and proline. This medium was replaced by a serum-free medium with HEαKG at a concentration of 100 μg/mL. Native aKG and succinate, at a concentration of 50 ug/mL, were used as positive and negative controls, respectively. After 24 hr, the collagen content in cell supernatant was measured by picrosirius red assay according to Walsh.13 Collagen I from calf skin was used as standard.
ATP assay: 3T3 cells were seeded to testing plates and, after adhesion, were treated with HEαKG in a serum-free medium. After 24 hr of treatment, the ATP content was evaluated by a luminescent cell viability assaya according to the production manual.
In vivo test: Two groups of human volunteers were used to measure the effects of HEαKG. The first group (n = 5), ages 28–46, applied a test formulation daily (see Formula 1), including 0.01% HEαKG. The second group (n = 15), ages 24–63, applied the same formulation omitting only the HEαKG. Each subject’s skin was measured by cutometerb and photographedc before the first application, then measured by cutometer again after 14 and 28 days of application
Statistical analysis: All experiments were performed at least in triplicate. All data obtained from experiments was assessed by a student’s t-test for statistical significance; p values lower than 0.05 were considered significant, as signified by an asterisk (*). In vitro
Results and Discussion
To assess the effect of HEαKG on the quality of the extracellular matrix, the content of collagen in the supernatant of cells isolated from two groups of donors was determined by picrosirius red assay. The first group, as noted, comprised donors less than 10 years of age; the second, donors greater than 50 years. Figure 2 shows that HEαKG increased the collagen content of fibroblasts isolated from older donors by up to 200%, with the control unaffected. This effect, which was not observed in cells from younger donors, can be explained by the difference in how α-ketoglutarate is used.
In “young” cells, where there is sufficient production of collagen, externally supplied α-ketoglutarate is probably utilized in the Krebs energy cycle.14 In contrast, in cells having reduced collagen production, the molecule may contribute to increased efficiency in collagen production by influencing the process of proline hydroxylation via prolyl 4-hydroxylase. This difference in the use of the supplied HEαKG is also indicated by the results of the ATP assay (see Figure 3). In this model, the addition of HEαKG to the 3T3 fibroblasts with sufficient synthetic activity, similar to fibroblasts from young donors, resulted in a 20% increase in ATP production.
This theory supporting the hydroxylation and, therefore, more efficient production of collagen is consistent with the literature. Difficulties in the direct stimulation of collagen expression in aging skin, due to reduced procollagen signalling, have been reported.4 Furthermore, these findings were confirmed when the application of HEαKG did not increase the expression of mRNA COL1A2 (data not shown).
Alternatively, another mechanism of the procollagen action of HEαKG could be the effect of similar molecules on the treatment of pseudohypoxia, as previously described.15 Under conditions of normoxia, prolyl hydroxylase 1-3 hydroxylates prolyl residues in the oxygen-dependent degradation domain of hypoxia inducible factor (HIF) 1, facilitating subsequent degradation thereof. With this reaction, molecular oxygen and aKG are also necessary. With pseudohypoxia, aKG in the form of octyl esters was able to increase the hydroxylation of proline and thus the degradation of HIF, returning signalling back to normal.15 HIF itself, under conditions of hypoxia, is an essential factor for the hydroxylation of proline in collagen.16 However, under conditions of normoxia, where aKG contributes to degradation, it is assumed that the effect of HEαKG on collagen content lies more in support of the hydroxylation of proline in collagen than in a change in HIF1 signalling.
In vivo Results and Discussion
After obtaining positive in vitro results, researchers monitored the in vivo effects of HEαKG. For 28 days, five volunteers applied the formulation with or without (placebo) the 0.01% HEαKG, once daily. The extent of the wrinkles on their foreheads was then assessed after 28 days of application. The results are summarized in Figure 4. Application of the active ingredient significantly reduced wrinkle depth by 60%, while the placebo group showed almost no change. This effect was consistent with the observed in vitro results as well as those reported by Son et al.,2 who demonstrated the positive effect of native α-ketoglutarate on the reduction of wrinkles caused by UVB radiation.
Apart from the direct effect on wrinkle depth, this derivative also positively influenced the elasticity of skin. Figure 5 shows that the viscoelastic coefficient of skin on the forehead area of volunteers was reduced; the lower this parameter, the greater the ability of skin to reconstitute itself after stretching. After 28 days of application, test subjects using the active ingredient showed a 20% reduction in this parameter while the placebo group remained unchanged. This effect could also be associated with an increase in collagen in the dermis.
Wrinkles and sagging skin are among the most visible signs of aging. Both are associated with a decrease in the quantity and quality of the collagen produced. The HEαKG tested here was shown, in vitro, to support collagen production in senescent fibroblasts. This appears to occur via an original mechanism that does not directly stimulate the synthesis or inhibit the degradation of collagen, but instead enhances the efficiency of its production through the improved hydroxylation of collagen fiber.
Hydrophobization of otherwise autologous α-ketoglutarate molecules increases the chances of penetration and, therefore, the observed effects. This is particularly true in terms of the in vivo reduction of deep wrinkles and improved skin elasticity. These facts make hexyl ester of α-ketoglutarate a promising tool in preventing or minimizing the external manifestations of skin aging, especially wrinkles.
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