Redefining the Body Minimizing Aging

As skin gets thinner and more flaccid with aging, there is a loss of subcutanous fatty tissue (which normally acts as a supporting element), which makes the skin loose and saggy, causing unsought visible changes.¹ Additionally, aging induces fatty tissue redistribution from subcutaneous stores to visceral depots, diminishing local volume in areas such as breasts or hands. The breast is a key element of the feminine silhouette. A high percentage of the total composition of this gland is formed by adipose tissue, so a volume change in this tissue provides a substantial effect on the gland as a whole, determining breast shape and size.

On the other hand, fat depots can abnormally expand due to several factors such as genetics or an imbalance between high caloric intake and energy expenditure. Extra lipid content augments volume and pushes and distorts the connective tissue; at the same time, it obstructs the lymphatic drainage system, impairing the removal of waste materials (like toxins or proteins) that together with collagen fibers create an immobile network that retains fat cells. These facts result in a longer junction line between dermis and hypodermis (dermo-hypodermal junction line), local volume increase and an uneven skin surface, with visible fat nodules and irregularities on the skin surface (cellulite).²

Adipose tissue is a specialized connective tissue located beneath the skin, which can experience rapid and extensive size changes as it has no anatomic constraints to growth and it accumulates lipids from the diet, augmenting cell volume and local volume as a result. Therefore, the amount of subcutaneous fat and its body distribution plays a key role in physical appearance, and depends on diet, age, hormones, gender and sport practice. In women, fat is basically located in buttocks, thighs and hips, differently from men and menopausal women, who primarily store fat in the abdomen and waist.

Figure 1. PGC-1α effect in adipogenesis.

White adipose tissue (WAT) is the prevalent type in adults functioning as the major storage site for the lipids from daily intake (triglycerides and cholesterol esters), which are burned whenever body cells require energy. Although adipocytes are the main cells in WAT, this tissue also contains preadipocytes (adipocytes precursor cells, 15-50%), macrophages, fibroblasts, leukocytes and many collagen fibers, which provide structural support.³ Adipocytes contain a single large lipid droplet (80% of the cell content), which forces the nucleus and cytoplasm to remain in the periphery of the cell. One of the most relevant features of mature adipocytes is their capacity to accumulate triglycerides, while preadipocytes cannot.

Figure 2. PGC-1α mRNA expression relative quantity in human subcutaneous adipocytes after incubation with acetyl hexapeptide-38 or acetyl hexapeptide-39.

WAT volume can vary depending on the number of adipocytes and their size, parameter that increases along with lipid accumulation. The number of mature adipocytes with capacity to store lipids rises due to the differentiation process, which transforms preadipocytes into mature adipocytes. This maturation is a complex process known as adipogenesis, in which Peroxisome Proliferator-Activated Receptor-Gamma (PPAR-γ) and Peroxisome proliferator-activated receptor-Gamma Coactivator 1 alpha (PGC-1α) are both indispensable.

PPAR-γ belongs to a family of nuclear receptor proteins that function as transcriptional factors and regulate gene expression in cellular differentiation. This receptor is mainly expressed in the adipose tissue and it forms heterodimers with Retinoid X Receptors, which bind to specific regions of the DNA of target genes and regulate their expression. PPAR-γ is strictly necessary but not sufficient for the differentiation of preadipocytes.

Figure 3. Lipid accumulation in human adipocytes after different treatments.

PGC-1α is a transcriptional coactivator that interacts with a broad range of transcriptional factors and nuclear receptors, increasing the probability of certain genes related to adipogenesis of being transcribed.⁴ A clear induction of PGC-1α expression was observed during ex vivo human subcutaneous preadipocyte differentiation in WAT, being its level as high as in mature adipocytes.⁵ In WAT, PGC-1α coactivates PPAR-γ and, subsequently, they positively regulate the necessary gene expression which transforms preadipocytes into mature adipocytes, with capacity to accumulate lipids.
Thus, stimulating or reducing PGC-1α expression would increase or diminish adipocyte maturation and, consequently, local volume.

Lipotec developed two innovative ingredients for a complete remodelling treatment trough PGC-1α expression in WAT. Adifyline (INCI: Acetyl hexapeptide-38) induces PGC-1α expression, stimulating adipogenesis and leading to lipid storage and volume increase. On the contrary, Silusyne is a cosmetic ingredient that contains a hexapeptide (acetyl hexapeptide-39) in a novel delivery system that decreases PGC-1α expression, diminishing adipogenesis and lipid accumulation as a consequence. Both peptides were identified by a combinatorial chemistry approach from a library of 49,521,980 hexapeptides, which was screened using a reporter gene assay in a stably transfected cell line where luciferase expression was controlled by PGC-1α promoter activity. Several in vitro and in vivo assays were performed in order to study the effects of these peptides on PGC-1α expression and volume changes in key areas such as thighs and breasts.

Expression of Adipogenesis Key Elements
The efficacy of both peptides (acetyl hexapeptide-38 and acetyl hexapeptide-39) was studied separately by quantitative real-time PCR to determine the effects on PGC-1α expression. Human subcutaneous preadipocytes were incubated at 37°C for 24 hours in the preadipocyte growth medium (PGM-2), which was used as the basal control (non-treated non-differentiated cells). Differentiation was induced by changing this medium into preadipocyte differentiation medium (PDM-2), which was used as a control for non-treated differentiated cells. During the differentiation process, either one peptide or the other was added and all samples were incubated at 37°C for 10 days.

Afterwards, cells were lysed, RNA was extracted and reverse transcription was carried out. The resulting cDNA was analysed by R-T PCR, which can relate cDNA to the original mRNA levels present in samples using an endogenous expression control (eukaryotic 18S ribosome subunit RNA). Fluorescence was analyzed and the original levels of PGC-1α mRNA were quantified.

Effect on Lipid Accumulation
Human subcutaneous preadipocytes were incubated at 37°C for 24 hours in PGM-2, which was subsequently changed to PDM-2 to induce adipogenesis. Cells were then incubated for 10 days in presence of different treatments, including the peptides at two different concentrations. PDM-2 was used as a negative control and PGM-2 as a basal control. After 10 days of incubation, the accumulation of intracellular triglycerides using the AdipoRed assay reagent was measured. The supernatants were removed, wells were washed and 5µL of AdipoRed reagent was added. After 15 minutes at room temperature, fluorescence was measured with an automated multiplate fluorescence reader set for excitation at 485nm and detection at 535nm.

Measuring Breast Volume
For the evaluation of the in vivo efficacy of acetyl hexapeptide-38 in increasing breast local volume, a panel of 22 females (25-40 years old) with a European bra cup size between 80 and 90 was selected.
They applied the placebo cream in a bust and the cream containing 2% of a solution with acetyl hexapeptide-38 in the other, twice a day for 56 days. 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), which allows reconstructing the surface and the volume of the breast based on the principle of optical interferometry. The relative volume of the area and different 3D images were obtained, calculating the differences in mm³ versus the initial time, as well as the evolution of the breast volume.


























Figure 4. Real 3D images of a volunteer at the initial time (left) and at day 56 (right),
after applying a cream with acetyl hexapeptide-38.

The anti-cellulite and slimming efficacy of acetyl hexapeptide-39 was determined in vivo, evaluating its effects on the dermo-hypodermal junction line by ultrasound ecography in B-scan mode (Ultrasound Scanner Dermascan C). A panel of 20 female volunteers between 25 and 45 years old, 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% Silusyne on the other, twice a day for three weeks.

Results and Discussion
Results showed that preadipocytes treated with acetyl hexapeptide-38 increased PGC-1α expression by 25.6% and 61.1%, while acetyl hexapeptide-39 reduced its expression by 16.7% and 36.5% versus non-treated differentiated cells. Therefore, both peptides had a clear effect on the expression of this key adipogenesis element.

The AdipoRed reagent facilitates the detection of intracellular lipid droplets as it is a hydrophilic solution that turns into fluorescent in hydrophobic environments. According to test results, acetyl hexapeptide-38 demonstrated increased lipid accumulation by 27.9% and 32.4%, compared to non-treated differentiated cells in adipocytes coming from preadipocytes treated with this peptide. On the contrary, lipid accumulation was reduced by 45.8% and 67.2% in adipocytes coming from preadipocytes treated with acetyl hexapeptide-39.

Results at the end of the study showed that acetyl hexapeptide-38 clearly generated a positive growing tendency in breast volume, while the placebo did not. The volume increase caused by this peptide was 30 times higher than the placebo at the end of the study.

In the presence of fat nodules, the inner disorder of the tissue is higher and the dermo-hypodermal junction line is more irregular and wavier, becoming longer and stimulating irregularities in the skin surface. A shorter dermo-hypodermal union line would mean a more regular junction line and a better inner organisation of the subcutaneous tissue.
















Figure 5. Images from the dermo-hypodermal junction line of the thigh of a
volunteer at the initial time (left) and after 3 weeks of treatment with a cream
containing the slimming peptide (right).


The areas treated with acetyl hexapeptide-39 showed a significant 21% reduction of the dermo-hypodermal junction line at the end of the study; it improved the uniformity of the skin by 5-fold compared to placebo. Therefore, this peptide ameliorated the homogeneity of the subcutaneous tissue and helped to reduce skin irregularities.

Conclusion
The desire to modulate and improve silhouette is usual in both men and women. As subcutaneous fatty tissue is a relevant factor in this matter, controlling the maturation of the cells in charge of storing fat is essential. Lipotec designed two peptides either to enhance or decrease local fat accumulation, resulting in desired volume changes.

Acetyl hexapeptide-38 proved to enhance PGC-1α expression (61.1%) and lipid accumulation (32.4%), suggesting a stimulation of the adipogenesis in WAT. Its volume replenishing properties were confirmed in vivo by enhancing breast volume by 30-fold versus placebo effect.

On the other hand, acetyl hexapeptide-39 clearly decreased the expression of PGC-1α (36.5%) and lipid accumulation (67.2%) in WAT. As lipid accumulation and volume increase became more difficult, skin irregularities and cellulite appearance were also diminished. In addition, the cream containing this peptide proved to reduce the dermo-hypodermal junction line by 21%, which implied an improvement of skin uniformity and adipose tissue inner organization.

Considering the results of both peptides, they can be confirmed as excellent active ingredients to modulate body shape enhancing or reducing local volume where it is required, via modulating PGC-1α expression in WAT. Adifyline demonstrated to be a volume booster ingredient that can increase local volume and revert aging-induced volume losses, making it essential in replenishing formulations and anti-aging treatments. Conversely, Silusyne proved to be effective in reducing fat stores and skin irregularities, so it is a splendid option for slimming and anti-cellulite formulations.

References

1. Petrofsky J, Prowse M, Lohman E. The influence of ageing and diabetes on skin and subcutaneous fat thickness in different regions of the body. The Journal of Applied Research. 8 (1): 55-61, 2008.
2. Quatresooz P, Xhauflaire-Uhoda E, Piérard-Franchimont C, et al. Cellulite histopathology and related mechanobiology. International Journal of Cosmetic Science. 28: 207-210, 2006.
3. Tchkonia T, Morbeck DE, Zglinicki T., et al. Fat tissue, aging, and cellular senescence. Aging Cell. 9: 667-684, 2010.
4. Liang H, Ward WF. PGC-1α: a key regulator of energy metabolism. Adv Physiol Educ. 30: 145-151, 2006.
5. Semple RK, Crowley VC, Sewter CP, et al. Expression of the thermogenic nuclear hormone receptor coactivator PGC-1α is reduced in the adipose tissue of morbidly obese subjects. International Journal of Obesity. 28: 176-179, 2004.

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