ESPE2023 Rapid Free Communications Fat, metabolism and obesity 2 (6 abstracts)
Effects of leptin knockdown on a human preadipocyte model
Lasse Fuchs 1 , Mariami Jasaszwili 1 , Sandy Richter 1 , Anna Kirstein 1,2 , Felipe Engelberger 3 , Georg Künze 3 , Jens Meiler 3,4 , Johannes Lemke 5 , Wieland Kiess 1 , Diana Le Duc 5 & Antje Garten 1
1Pediatric Research Center, University Hospital for Children and Adolescents, Leipzig University, Leipzig, Germany. 2Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark. 3Institute for Drug Development, Medical Faculty, Leipzig University, Leipzig, Germany. 4Department of Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN, USA. 5Institute of Human Genetics, University Medical Center Leipzig, Leipzig, Germany
Obesity presents a major worldwide challenge, due to its numerous, severe adverse effects on health. This leads to a necessity to further investigate the mechanisms underlying lipid accumulation. The adipocytokine leptin may contribute to this process. While there already has been thorough research into central leptin action, deepening our understanding of leptin’s effects on whole-body energy homeostasis, relatively little is known about its auto- and paracrine effects. Ingrained in the larger effort of understanding the pathomechanisms underlying obesity and lipid accumulation, the aim of this study is to further elucidate possible auto- and paracrine functions of leptin on human adipocyte progenitor cells. Our group identified a somatic leptin variant (c.250C>A) present in a spontaneous lipoma of a lean male person. Computer modeling predicted a reduced leptin - leptin receptor interaction and potential destabilization of the protein for this genetic variant. This suggests that this variant is likely pathogenic and may play a role in the increased lipid accumulation in lipoma. To simulate this potential loss of function of leptin we successfully performed leptin knockdown using siRNA in a LipPD1 cell model. Differentiation was studied by staining lipids with fluorescent Nile-Red dye and Oil Red O staining. Implications of leptin knockdown on adipogenesis marker expression were analyzed using qPCR. Proliferation was assessed by fluorescent Hoechst and proliferation marker Ki-67 staining. Effects on lipolysis were examined by measuring basal glycerol in the culture medium of LipPD1 cells, which were differentiated into adipocytes. The experiments were conducted at least three times independently. Leptin knockdown resulted in a reduction in the accumulation of lipid droplets, evidenced by an approximately 0.8 fold decrease in Nile Red and 0.7 fold decrease in Oil Red O staining, compared to the control group. This was accompanied by a decreased expression of the adipogenesis markers adiponectin, fatty acid synthase (FASN) and proliferator-activated receptor γ (PPARγ). Furthermore, leptin knockdown led to an 1.1 fold increase in cell number, compared to the control group, after 7 days of incubation. Collectively, our preliminary results suggest that adipogenesis is impacted by leptin deficiency. It attenuates differentiation and expression of adipogenesis markers but stimulates proliferation in LipPD1 cells.
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