Role of Na/K-ATPase a1 caveolin binding motif in adipocyte differentiation and metabolism

Abstract

[Hypothesis] In addition to its ion-transporting properties, the signaling functions of Na/K-ATPase (NKA) α1 controls animal development and stem cell differentiation. Here we used a signaling null mutation known to impair NKA α1/caveolin interaction and NKA signaling to test the hypothesis that NKA α1 signaling is involved in adipocyte differentiation and metabolism. [Method] The F97A: F100A mutation (mCBM) of NKA α1 was introduced in mice and hiPSC (human induced pluripotent stem cells). Heterozygous (het) mCBM males and control littermates at six months of age were studied by EchoMRI, followed by histology analysis in the visceral adipose tissues. Metabolism and fibrosis in mCBM and WT adipocytes differentiated from hiPSC (iAdi) were studied using RNA-seq, real-time RT-PCR, Seahorse analysis, immunofluorescence, and flow cytometry. [Result] Compared to WT littermates, het mCBM mice had a reduced fat mass (p<0.05, n=4), and increased adipose fibrosis and inflammation (two-fold induction of crown-like structures and F4/80 staining (p<0.001, n=4-6). This was further supported by increased mRNA expression of tissue fibrosis marker genes such as Col1a1 (collagen, type I, alpha 1, p<0.01, n=6-9) and Fn1 (fibronectin 1, p<0.05) and proinflammatory markers Tnfa (tumor necrosis factor-alpha, p<0.001) and Il6 (interleukin 6, p<0.05). iAdi-mCBM also had increased mRNA expression of fibrosis marker genes COL1a1 (p<0.05), FN1 (p<0.001) and proinflammatory marker gene IL6 (p<0.01, n=4). Seahorse XF cell metabolism analyses revealed impaired glycolysis (ECAR, p<0.01, n=9) and decreased ATP synthesis-coupled respiration (OCAR, p<0.001, n=12-18) in iAdi-mCBM. Increased insulin resistance and mitochondrial reactive oxygen species (ROS) production were observed by glucose uptake assay (p<0.01 in Adi-WT, vs. p>0.05 in Adi-mCBM, n=3-4) and MitoNeoD assay (p<0.001, n=3), respectively. These metabolic dysfunctions were accompanied by extensive remodeling of the extracellular matrix (ECM), reflected by increased expression of ECM component maker genes in iAdi-mCBM (immunostaining for collagen type I, RT-qPCR for COL1A1, FN1), as well as TGF-β signaling (RNA-seq and RT-PCR for TGFB1, p<0.05). Treatment of mCBM hiPSC with the TGF-β inhibitor SB431542 (10 mM) rescued adipogenesis and ECM stiffness, evidenced by increased oil red O staining (p<0.001, n=6) and reduced mRNA expression of COL1A1 (p<0.01, n=6) and FN1 (p<0.001, n=6) in iAdi-mCBM. [Conclusion] These genetic approaches reveal the role of NKA caveolar signaling in adipogenesis. Mechanistically, pharmacological studies in human iAdi-mCBM indicate that NKA signaling integrity is required for adequate control of TGF-β signaling and ECM stiffness during adipogenesis. Funding Sources: National Institutes of Health grant HL-109015; American Heart Association grant 17SDG33661117; NIH P20GM103434 grant to the West Virginia IDeA Network of Biomedical Research Excellence. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.