Adipose tissue, which is excessively stored in obesity, not only accumulates lipids, but secretes numerous adipocytokines. One of them is leptin, a peptide hormone produced mainly by white adipose tissue and encoded by the leptin gene (LEP). The main role of leptin is energy balance regulation by acting on the hypothalamus. Additionally, leptin contributes to cardiovascular function, immune system activation and regulates the reproductive system through leptin receptor (LEPR) and endocrine mode of action [1,2]. However, the autocrine and paracrine effects of leptin on adipocytes are not completely understood, and the current state of the art provides contradictory data. Nevertheless, it is known that leptin may play an important role in lipid accumulation and metabolism.
Our group identified a somatic variant in the leptin gene (c.250C>A), p.(Gln84Lys) in a spontaneous lipoma. In silico analysis indicated that this variant may result in reduced stability of the protein. Therefore, we aimed to evaluate the effects of leptin knockdown as a model for leptin loss of function and leptin stimulation on adipose progenitor cells – LipPD1 [3].
To access cell viability we used water-soluble tetrazolium salt (WST-1), cell proliferation was examined by fluorescent staining (Hoechst). Next, we studied adipocyte differentiation by staining lipids with Oil Red O (ORO) and fluorescent stain (Nile Red). The effects of leptin knockdown on adipogenesis marker expression was evaluated by real-time PCR. Data was presented as the fold change normalized to controls (±SEM), and analyzed using Student’s t-test or one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. Experiments were repeated independently three times, n=6-8, p≤0.05 was considered to indicate a statistically significant difference.
We found that leptin knockdown increased cell viability [1.3180(±0.0543) fold, p=0.0001], and cell number [1.1460(±0.0209) fold, p<0.0001]. Moreover, leptin knockdown decreased intracellular lipid droplet accumulation – shown after ORO [0.6588(±0.0458) fold, p<0.0001] and Nile Red staining [0.8254(±0.0444) fold, p=0.0200]. These changes were associated with reduced expression of adipogenesis markers – proliferator-activated receptor γ (PPARγ) [0.5655(±0.2005) fold, p=0.0500] and fatty acid synthase (FASN) [0.2607(±0.0796) fold, p=0.0007]. Efficiency of leptin knockdown was confirmed based on reduced leptin expression [0.1700(±0.0685) fold, p=0.0003]. Next, we showed that treatment with recombinant leptin (10, 100 nM) attenuated viability of adipose progenitors cultured in 10% FCS containing medium [0.7981(±0.0462), p=0.0300 and 0.8018(±0.0419), p=0.0400 fold, respectively], but did not affect adipocyte differentiation [0.9199(±0.0436), p=0.4247 and 0.9813(±0.0420), p=0.9922]. Furthermore, leptin treatment (1 nM) reversed the phenotype observed after leptin knockdown by stimulating adipocyte differentiation [1.5900(±0.0922) fold, p=0.0084].
To sum up, our data indicates that leptin knockdown affected adipogenesis by stimulating preadipocyte viability and proliferation, and inhibiting adipocyte differentiation. Leptin treatment attenuated preadipocyte viability without affecting their maturation and reversed the phenotype observed after leptin knockdown by restoring adipocyte differentiation. In the nearest future, we aim to study the effects of the newly discovered leptin variant on adipogenesis.
These findings may contribute to implementing leptin treatment in patients with lipoma, and obese patients with leptin gene variants. Our conclusions may lead to the explanation of potential effects of leptin on adipocyte physiology.