PROJECT SUMMARY Brown and beige adipocytes play key roles in mediating adaptive thermogenesis and controlling energy metabolism. In response to cold exposure, beige adipocytes develop within subcutaneous white adipose tissue (sWAT), and together with brown adipose tissue (BAT), contribute to the thermogenic response. As a result, there is intense interest in elucidating the mechanism by which cold induces ?beiging? or further ?browning? of adipocytes, and identifying putative downstream targets that mediate thermogenesis and energy expenditure as a therapeutic strategy for addressing metabolic diseases such as obesity and diabetes. In preliminary studies, we found that exposure of mice to cold (4oC) induces the rapid activation of ROCK2; increases beiging of sWAT; and upregulates the thermogenic gene program consisting of Pgc-1?, Ucp1, and Prdm16. Mutant mice lacking Rock2 in adipocytes (adipo-Rock2?/? mice) failed to develop cold-induced beiging of sWAT; exhibited reduced thermogenic activity; and have cold intolerance. Interestingly, we also found that BAT of adipo-Rock2?/? mice have less mitochondria content. The mechanism may be due, in part, to decreased mitochondrial biogenesis through the loss of ROCK2-induced myocyte enhancer factor (MEF)-2A phosphorylation and activation. The overall aim of this proposal, therefore, is to determine how adipocyte Rock2 could regulate energy metabolism and obesity through the induction of beiging and thermogenesis. Specific aim 1 will test the hypothesis that adipocyte Rock2 mediates thermogenesis through the induction of beiging and the thermogenic gene program in sWAT. Single-cell transcriptome and proteome analyses will be performed on adipocytes from sWAT to help delineate Rock2-mediated signaling pathways that could regulate cold-induced beiging and thermogenesis. Specific aim 2 will test the hypothesis that Rock2 mediates BAT thermogenesis through MEF2A- dependent mitochondrial biogenesis. Using reconstituted Mef2a?/? mouse embryonic fibroblasts (MEFs) or BA, we will determine the role of MEF2A in Pgc-1? gene transcription. We will also perform ATAC/ChIP/RNA-seq to assess for potential enrichment of phosphorylated MEF2A-binding regions in promoters of genes that are involved in mitochondrial biogenesis. Specific aim 3 will test the hypothesis that adipocyte Rock2-Mef2a pathway is critical for energy metabolism and diet-induced obesity (DIO). We will profile metabolic changes in adipo-Rock2?/? (loss-of- function) and adipo-caRock (gain-of-function) mice on high fat diet (HFD). We will also generate mice with mutations in Rock2-dependent phosphorylation sites on MEF2A using CRISPR-Cas9 gene editing or Cre/loxP technology. These Mef2a mutant mice, if viable, will be studied to determine their response to cold and HFD with regards to beiging, mitochondrial biogenesis, and thermogenesis.

R56DK129644

2021-08-19

2022-08-18