Modelling body shape is a common concern for both women and men in modern society, and the amount of subcutaneous fat and its distribution greatly influences physical appearance.
Lipid storage and arrangement in the body depends on eating habits, exercise, age and hormone levels. In women, fat is mainly stored in buttocks, thighs, and hips due to the influence of oestrogens. However, in men and menopausal women fat accumulates in the waist and the abdominal area. On the other hand, the amount of fat decreases and is redistributed from the subcutaneous stores to visceral depots with age. Consequently, the volume of several regions of the body (such as cheeks, breasts or hands) diminishes, which results in a visibly older appearance.1 Stimulating adipose tissue growth in some areas could help to recover the volume lost as a consequence of ageing. The relevance of the breast in the silhouette becomes an aesthetical concern for all women. The adipose tissue represents a high percentage of the total gland composition, so a variation in adipose tissue volume provides a substantial effect on the gland as a whole, determining breast shape and size. There have been described two types of adipose tissue according to its main function and their kind of adipocytes. The brown adipose tissue (BAT) is necessary in the early stages of human life and is found at a low percentage (around 5%). Human BAT is characterised by a high expression of mitochondrial genes and polygonal adipocytes. It contains a great number of mitochondria in the cytoplasm and several small lipid droplets. The main function of brown adipocytes is to dissipate energy in the form of heat. White adipose tissue (WAT) is the prevalent type in adults and the small levels of brown tissue decrease with age. WAT functions as the major storage site for the lipids coming from daily food intake. Lipids contained in these cells are burned whenever the cells of the body require energy for its normal function. This tissue basically contains round adipocytes, responsible for lipid storage, but is also composed of macrophages, fibroblasts, leukocytes and many collagen fibres, which act as a support for the rest of the cells. Preadipocytes are also present in the fat tissue and are the precursors of adipocytes. Preadipocytes are distinct cell subtypes that mainly reside in different fat depots and comprise 15% to 50% of cells in fat. Their main role is to give rise to new fat cells.2 Adipocytes contain a large lipid droplet that represents 80% of the cell content, which forces the nucleus and cytoplasm to remain in the periphery of the cell. One of the most important features of adipocytes is the ability to store triglycerides, while preadipocytes cannot. Due to several factors such as genetics or high caloric intake, fat deposits of human WAT can expand to such a great extent that they push and distort the connective tissue. This increase of local volume may induce irregularities in the junction line between the dermis and hypodermis, increasing its length and making the skin surface appear uneven and with visible fat nodules.3 This extra lipid volume can also obstruct the lymphatic drainage system impairing the removal of waste materials like toxins or proteins, which together with the collagen fibres create an immobile network, where fat cells are trapped. This situation inevitably leads to an increase of local volume and to the appearance of unsightly irregularities on the skin surface (cellulite). WAT volume depends on both adipocyte number and size, so its enlargement can be caused either by an increase in the number of adipocytes or in lipid content. Cellulite and being overweight are related to a rise in the adipose depots, which is the result of an imbalance between food intake and energy expenditure. Cellulite can appear as a consequence of an extra local fat storage and it is known that overweight individuals produce more white adipocytes per year and have a greater number of mature adipocytes than thin individuals.4,5 Mature white adipocytes have the capacity to store fat, so that acting on their differentiation cycle could help to locally diminish or increase lipid storage in WAT. The differentiation process from preadipocytes to mature adipocytes is a complex process known as adipogenesis in which many factors and genes interfere. Some genes must be expressed as they are distinctive of mature adipocytes while the typical genes of preadipocytes need to be downregulated to finally lead to the adipocyte phenotype.6 The Peroxisome proliferator-activated receptor-Gamma Coactivator 1 alpha (PGC-1?) is a transcriptional coactivator that interacts with a broad range of transcriptional factors and nuclear receptors (including PPAR?).7 Peroxisome Proliferator-Activated Receptor-??(PPAR?) belongs to a family of nuclear receptor proteins that functions as transcriptional factors and regulates gene expression in cellular differentiation among other processes. This receptor forms heterodimers with Retinoid X Receptors which bind to specific regions on the DNA of target genes and regulate their expression. PPAR??is predominantly expressed in the adipose tissue and it is strictly necesary but not sufficient for the differentiation of preadipocytes. PGC-1??is a transcriptional coactivator that interacts with a broad range of transcriptional factors and nuclear receptors (including PPAR?),7 increasing the probability of certain genes related to adipocyte differentiation being transcribed. It was observed that a robust induction of PGC-1??expression during ex vivo human subcutaneous preadipocyte differentiation in WAT, had a level as high as in mature adipocytes.8 It was confirmed that in WAT, PGC-1??interacts with PPAR??potentiating the expression of relevant genes related to adipocyte differentiation, thus stimulating adipogenesis. Thus, stimulating or reducing PGC-1??expression would increase or diminish adipocyte maturation and, therefore, the number of adipocytes capable of storing lipids. The total number of white adipocytes is constant throughout adult life. Thus, a modulation of the differentiation process would alter the equilibrium between adipocyte maturation and death rate, and hence the amount of mature white adipocytes.2 Cosmetic ingredients targeting adipogenesis would become a key solution for increasing or reducing fat tissue volume in localised areas. Two new products have been developed in order to modulate PGC-1??expression and so influence adipogenesis and lipid accumulation to get the desired effect. Adifyline (INCI Name: Acetyl Hexapeptide- 38) is a new hexapeptide that induces PGC-1??expression, and thus, stimulates adipogenesis, leading to an increase in lipid storage which boosts fat tissue volume, for both anti-ageing and volume enhancing formulations. The novel ingredient, Silusyne, contains a hexapeptide (INCI Name: Acetyl Hexapeptide-39) included in a novel delivery system, which decreases PGC-1??expression, obtaining the opposite effect and shortens the dermo-hypodermal junction line for slimming and anti-cellulite treatments. In vitro and in vivo assays were performed on both ingredients in order to study its effect in PGC-1??expression and lipid accumulation, and the macroscopic effects.
Materials and methods
PGC-1??expression in human adipocytes
The efficacy of the peptides was studied by RT-PCR in order to determine its effect on the expression of PGC-1?. Human subcutaneous preadipocytes were incubated for 24 hours in the Preadipocyte Growth Medium (PGM-2), which was used as the basal control (non-treated nondifferentiated cells). Differentiation was induced by changing this medium to the Preadipocyte Differentiation Medium (PDM-2), which was used as a control for non-treated differentiated cells. During the differentiation process, different concentrations of the peptides were added to the cultures and all the samples (controls included) were incubated for 10 days. Then, cells were lysed, RNA was extracted and reverse transcription was carried out. The resulting cDNA was analysed by quantitative real-time PCR. This method can relate cDNA to the original mRNA levels present in samples using an endogenous expression control, which in this case was the eukaryotic 18S ribosome subunit RNA. Fluorescence was analysed and the original levels of PGC-1??mRNA were quantified. The results were corrected from the basal levels of PGC-1??(PGM-2) and normalised regarding the PGC-1??levels of a control experiment (PDM-2-without test items).
Effect on lipid accumulation
Human subcutaneous preadipocytes were incubated for 24 hours in the Preadipocyte Growth Medium (PGM-2). Differentiation was induced by changing this medium to the Preadipocyte Differentiation Medium (PDM-2) and incubating the cells for 10 days in the presence of different treatments. The peptides were tested at two different concentrations, caffeine (200 ?g/mL) was used as positive control, PGM-2 as a basal control (non-treated non-differentiated cells), and PDM-2 as negative control (non-treated differentiated cells). After 10 days of incubation with the different treatments, plates were analysed by measuring the accumulation of intracellular triglycerides using the AdipoRed assay reagent. The culture plate was removed from the incubator and allowed to cool at room temperature. The supernatants were carefully removed and wells were washed with PBS. Then, each well was filled with PBS and 5 ?L of AdipoRed reagent. Assay plates were incubated for 15 minutes at room temperature before fluorescence was measured with an automated multiplate fluorescence reader set for excitation at 485 nm and detection at 535 nm. The results were corrected from the basal fluorescence and normalised with respect to the fluorescence of the differentiation medium.
Instrumental evaluation of dermo-hypodermal junction
The efficacy of the novel slimming ingredient was determined in vivo, evaluating its effects on the dermohypodermal junction by ultrasound ecography in B-scan mode (Ultrasound Scanner Dermascan C). A panel of 20 female volunteers aged between 2 5 and 45 years, with cellulite on their thighs (Pinch test stage I-III) was selected for the study. They applied the placebo cream on one thigh and the cream containing 2% of the slimming product on the other, twice a day for three weeks.
Instrumental evaluation of facial and breast volume increase
In order to evaluate the in vivo efficacy of acetyl hexapeptide-38 in increasing skin volume, two different panels of 22 female volunteers aged between 50 and 60 years, and between 25 and 40 years applied two creams with 2% of a solution containing the peptide on a cheek (the first group) and on a breast (the second group) twice a day for 14 and 56 days respectively. Cheek volume was analysed by Fringe projection at the beginning and at the end of the study. Measurements of breast volume were taken at the beginning and at day 14, 28 and 56 using the Fast Optical In vivo Topometry Technique (FOITS). The relative volume of the area and different 3D images were obtained. The differences in mm3 versus initial time were measured as well as the evolution of the breast volume compared to initial time and normalised with respect to placebo results.
Results and discussion
PGC-1??expression in human adipocytes
Results showed that preadipocytes treated with acetyl hexapeptide-39 had a lower expression of PGC-1??mRNA and those treated with acetyl hexapeptide-38 had a higher expression. Compared to non-treated differentiated cells, acetyl hexapeptide-39 reduced PGC-1??transcription by 36.5% and 16.7% when tested at 100 ?g/mL and 25 ?g/mL, respectively. According to these results, acetyl hexapeptide-39 efficiently reduces the expression of PGC-1??mRNA, which has been demonstrated to be linked to adipogenesis and lipid accumulation. Preadipocytes treated with acetyl hexapeptide-38 increased their expression by 25.6% at 0.1 mg/mL and by 61.1% at 0.5 mg/mL versus non-treated differentiated cells.
Effect on lipid accumulation
The AdipoRed reagent is a hydrophilic solution that turns into fluorescent in hydrophobic environments, and the fluorescent emission is different whether it is bound to phospholipids or partitioned in droplets of triglyceride. This facilitates the detection of the levels of intracellular lipid droplets accumulated during preadipocyte differentiation. Results demonstrated that adipocytes coming from preadipocytes treated with acetyl hexapeptide-39 had a lower lipid accumulation compared to non-treated differentiated cells or even to cells treated with caffeine. Acetyl hexapeptide-39 reduced lipid accumulation by 67.2% and 45.8% at a concentration of 100 ?g/mL and 25 ?g/mL, respectively. Adipocytes coming from preadipocytes treated with acetyl hexapeptide-38 enhanced lipid accumulation by 27.9% at 0.1 mg/mL and by 32.4% at 0.5 mg/mL versus non-treated differentiated cells.
Instrumental evaluation of dermo-hypodermal junction line
In the presence of fat nodules and cellulite, the inner disorder of the tissue increases and the dermo-hypodermal junction line becomes more irregular and wavy. In order to surround all cells with extra volume, this junction line becomes longer and induces irregularities on the skin surface. A decrease in the length of the dermohypodermal union would translate in a more regular junction line and a better organisation of the tissue, which would imply an improvement of the subcutaneous tissue uniformity. It was observed that the thighs treated with the cream containing the active product showed a significant reduction of their dermo-hypodermal junction line by 21% at the end of the study. The areas treated with the placebo cream presented no significant diminishing effect. Moreover, at the end of the treatment the slimming product improved by five-fold the uniformity of the dermohypodermal junction line compared to placebo. The new active ingredient reduced the dermo-hypodermal junction line and its irregularities, making the subcutaneous tissue more homogeneous and regular, which facilitates the skin surface to become flatter.
Instrumental evaluation of facial and breast volume increase
After 14 days, areas treated with the cream containing acetyl hexapeptide-38 had a significant volume growth of 11.9% versus initial time, and 79% of volunteers experienced a visible volume enhancement. Moreover, acetyl hexapeptide-38 clearly generated a growing tendency in breast volume, increasing breast volume of the treated area by 30-fold compared to placebo at the end of the study.
Conclusion
Acetyl hexapeptide-39 clearly reduced PGC-1??mRNA decreasing the expression of PGC-1??and lowering preadipocyte differentiation in white adipose tissue. Moreover, it diminished lipid accumulation, obtaining even better reductions than caffeine, demonstrating to effectively reduce the lipid content. Therefore, as lipid accumulation and volume increase becomes more difficult, irregularities and cellulite appearance would be reduced. In addition, the slimming product proved to reduce the length of the dermo-hypodermal junction line by 21%, improving skin uniformity and adipose tissue inner organisation. It can be considered an essential solution to be taken into account in slimming and anti-cellulite formulations. The volume booster acetyl hexapeptide- 38 proved to enhance PGC-1??levels and lipid accumulation, suggesting to stimulate adipogenesis. Its volume replenishing properties were confirmed in vivo by increasing the fatty tissue lost in the cheeks, and enhancing breast volume by 30-fold respect to placebo. The peptide turns out to be an excellent ingredient for cosmetic treatments which seek a replenishing effect in specified areas such as cheeks, breasts, buttocks, hands, lips and cleavage. In the light of the results, two peptides modulating PGC-1??mRNA expression can be good strategies for either recovering the fat lost with ageing or helping to improve cellulite appearance and reduce lipids depots. Two solutions for two opposite effects.
References
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