Role of Na/K-ATPase α1 caveolin-binding motif in adipogenesis.

Deficiencies in mice and in humans have brought to the fore the importance of the caveolar network in key aspects of adipocyte biology. The conserved N-terminal caveolin-binding motif (CBM) of the ubiquitous Na/K-ATPase (NKA) α1 isoform, which allows NKA/caveolin-1 (Cav1) interaction, influences NKA signaling and caveolar distribution. It has been shown to be critical for animal development and ontogenesis, as well as lineage-specific differentiation of human induced pluripotent stem cells (hiPSCs). However, its role in postnatal adipogenesis has not been fully examined. Using a genetic approach to alter CBM in hiPSC-derived adipocytes (iAdi-mCBM) and in mice (mCBM), we investigated the regulatory function of NKA CBM signaling in adipogenesis. Seahorse XF cell metabolism analyses revealed impaired glycolysis and decreased ATP synthesis-coupled respiration in iAdi-mCBM. These metabolic dysfunctions were accompanied by evidence of extensive remodeling of the extracellular matrix (ECM), including increased collagen staining, overexpression of ECM marker genes, and heightened TGF-ß signaling uncovered by RNAseq analysis. Rescue of mCBM by lentiviral delivery of WT NKA α1 or treatment of mCBM hiPSCs with the TGF-ß inhibitor SB431542 normalized ECM, suggesting that NKA CBM signaling integrity is required for adequate control of TGF-ß signaling and ECM stiffness during adipogenesis. The physiological impact was revealed in mCBM male mice with reduced fat mass accompanied by histological and transcriptional evidence of elevated adipose fibrosis and decreased adipocyte size. Based on these findings, we propose that the genetic alteration of the NKA/Cav1 regulatory path uncovered in human iAdi leads to lipodystrophy in mice.NEW & NOTEWORTHY A Na/K-ATPase α1 caveolin-binding motif regulates adipogenesis. Mutation of this binding motif in the mouse leads to reduced fat with increased extracellular matrix production and inflammation. RNA-seq analysis and pharmacological interventions in human iPSC-derived adipocytes revealed that TGF-ß signal, rather than Na/K-ATPase-mediated ion transport, is a key mediator of NKA regulation of adipogenesis.

The Na/K-ATPase α1/Src interaction regulates metabolic reserve and Western diet intolerance.

AIM: Highly prevalent diseases such as insulin resistance and heart failure are characterized by reduced metabolic flexibility and reserve. We tested whether Na/K-ATPase (NKA)-mediated regulation of Src kinase, which requires two NKA sequences specific to the α1 isoform, is a regulator of metabolic capacity that can be targeted pharmacologically. METHODS: Metabolic capacity was challenged functionally by Seahorse metabolic flux analyses and glucose deprivation in LLC-PK1-derived cells expressing Src binding rat NKA α1, non-Src-binding rat NKA α2 (the most abundant NKA isoform in the skeletal muscle), and Src binding gain-of-function mutant rat NKA α2. Mice with skeletal muscle-specific ablation of NKA α1 (skα1-/-) were generated using a MyoD:Cre-Lox approach and were subjected to treadmill testing and Western diet. C57/Bl6 mice were subjected to Western diet with or without pharmacological inhibition of NKA α1/Src modulation by treatment with pNaKtide, a cell-permeable peptide designed by mapping one of the sites of NKA α1/Src interaction. RESULTS: Metabolic studies in mutant cell lines revealed that the Src binding regions of NKA α1 are required to maintain metabolic reserve and flexibility. Skα1-/- mice had decreased exercise endurance and mitochondrial Complex I dysfunction. However, skα1-/- mice were resistant to Western diet-induced insulin resistance and glucose intolerance, a protection phenocopied by pharmacological inhibition of NKA α1-mediated Src regulation with pNaKtide. CONCLUSIONS: These results suggest that NKA α1/Src regulatory function may be targeted in metabolic diseases. Because Src regulatory capability by NKA α1 is exclusive to endotherms, it may link the aerobic scope hypothesis of endothermy evolution to metabolic dysfunction.