Jan-Inge Bjune1, Laurence Dyer2, Gro V Røsland3, Karl Johan Tronstad4, Pål R Njølstad5, Jørn V Sagen2, Simon N Dankel6, Gunnar Mellgren7. 1. Center for Diabetes Research, Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway; Mohn Nutrition Research Laboratory, Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway; Hormone Laboratory, Haukeland University Hospital, N-5021 Bergen, Norway. 2. Center for Diabetes Research, Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway; Hormone Laboratory, Haukeland University Hospital, N-5021 Bergen, Norway. 3. Department of Biomedicine, University of Bergen, N-5020 Bergen, Norway. 4. Department of Biomedicine, University of Bergen, N-5020 Bergen, Norway; Department of Oncology and Medical Physics, Haukeland University Hospital, N-5021 Bergen, Norway. 5. Center for Diabetes Research, Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway; Department of Pediatrics and Adolescents, Haukeland University Hospital, N-5021 Bergen, Norway. 6. Center for Diabetes Research, Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway; Mohn Nutrition Research Laboratory, Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway; Hormone Laboratory, Haukeland University Hospital, N-5021 Bergen, Norway. Electronic address: simon.dankel@uib.no. 7. Center for Diabetes Research, Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway; Mohn Nutrition Research Laboratory, Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway; Hormone Laboratory, Haukeland University Hospital, N-5021 Bergen, Norway. Electronic address: gunnar.mellgren@uib.no.
Abstract
BACKGROUND: Inhibition of Irx3 and Irx5 has been shown to reduce body weight and white adipose tissue (WAT) mass through cell-autonomous and sympathetic-induced increases in adipocyte beiging and thermogenesis in mice and humans. However, the underlying mechanisms of the Irx control over beiging are still largely unknown, as illustrated by recent reports showing divergent effects of Irx3 on adipocyte metabolism and function. Here, we investigated the role of Irx3 in controlling beige preadipocyte function and differentiation. METHODS: Stable knock out of Irx3 in ME3 mouse preadipocytes capable of beiging was performed using a CRISPR-Cas9 system, and the effect on cell differentiation was assessed by qPCR, RNA-seq, Oil-red-O lipid staining and Alcian Blue staining of proteoglycans. Changes in cell identities were validated using cell type enrichment analysis from RNA-seq data. Proliferation and cell cycle progression in undifferentiated cells were measured by WST-1 and flow cytometry, reactive oxygen species (ROS) generation was determined by fluorescence spectrometry and mitochondrial respiration was investigated by Seahorse assay. RESULTS: Irx3 was found to be essential for the identity, function and adipogenic differentiation of beige adipocyte precursors. Irx3-KO impaired proliferation, ROS generation and mitochondrial respiration in the preadipocytes. We further observed profound changes in numerous genes during both early and late stages of adipogenic differentiation, including genes important for adipocyte differentiation, cell cycle progression, oxidative phosphorylation (OXPHOS) and morphogenesis. Irx3-KO cells failed to accumulate lipids following adipogenic stimuli, and cell enrichment analysis revealed a loss of preadipocyte identity and a gain of chondrocyte-like identity in Irx3-KO cells during early differentiation. Finally, unlike the control cells, the Irx3-KO cells readily responded to chondrogenic stimuli. CONCLUSIONS: Irx3 is required for preadipocyte identity and differentiation capacity. Our findings suggest that, while inhibition of Irx3 may be beneficial during later developmental stages to modulate adipogenesis in the beige direction, constitutive and complete absence of Irx3 in the embryonic fibroblast stage leads to detrimental loss of adipogenic differentiation capacity.
BACKGROUND: Inhibition of Irx3 and Irx5 has been shown to reduce body weight and white adipose tissue (WAT) mass through cell-autonomous and sympathetic-induced increases in adipocyte beiging and thermogenesis in mice and humans. However, the underlying mechanisms of the Irx control over beiging are still largely unknown, as illustrated by recent reports showing divergent effects of Irx3 on adipocyte metabolism and function. Here, we investigated the role of Irx3 in controlling beige preadipocyte function and differentiation. METHODS: Stable knock out of Irx3 in ME3mouse preadipocytes capable of beiging was performed using a CRISPR-Cas9 system, and the effect on cell differentiation was assessed by qPCR, RNA-seq, Oil-red-O lipid staining and Alcian Blue staining of proteoglycans. Changes in cell identities were validated using cell type enrichment analysis from RNA-seq data. Proliferation and cell cycle progression in undifferentiated cells were measured by WST-1 and flow cytometry, reactive oxygen species (ROS) generation was determined by fluorescence spectrometry and mitochondrial respiration was investigated by Seahorse assay. RESULTS:Irx3 was found to be essential for the identity, function and adipogenic differentiation of beige adipocyte precursors. Irx3-KO impaired proliferation, ROS generation and mitochondrial respiration in the preadipocytes. We further observed profound changes in numerous genes during both early and late stages of adipogenic differentiation, including genes important for adipocyte differentiation, cell cycle progression, oxidative phosphorylation (OXPHOS) and morphogenesis. Irx3-KO cells failed to accumulate lipids following adipogenic stimuli, and cell enrichment analysis revealed a loss of preadipocyte identity and a gain of chondrocyte-like identity in Irx3-KO cells during early differentiation. Finally, unlike the control cells, the Irx3-KO cells readily responded to chondrogenic stimuli. CONCLUSIONS:Irx3 is required for preadipocyte identity and differentiation capacity. Our findings suggest that, while inhibition of Irx3 may be beneficial during later developmental stages to modulate adipogenesis in the beige direction, constitutive and complete absence of Irx3 in the embryonic fibroblast stage leads to detrimental loss of adipogenic differentiation capacity.
Authors: Anastasiia S Boiko; Ivan V Pozhidaev; Diana Z Paderina; Anna V Bocharova; Irina A Mednova; Olga Yu Fedorenko; Elena G Kornetova; Anton J M Loonen; Arkadiy V Semke; Nikolay A Bokhan; Svetlana A Ivanova Journal: Pharmgenomics Pers Med Date: 2021-09-07