Excessive dietary intake of fat results in its storage in white adipose tissue (WAT). Energy expenditure through lipid oxidation occurs in brown adipose tissue (BAT). Certain WAT depots can undergo a change termed beiging where markers that BAT express are induced. Little is known about signalling pathways inducing beiging. Here, inhibition of a signalling pathway regulating alternative pre‐mRNA splicing is involved in adipocyte beiging. Clk1/2/4 kinases regulate splicing by phosphorylating factors that process pre‐mRNA. Clk1 inhibition by TG003 results in beige‐like adipocytes highly expressing PGC1α and UCP1. SiRNA for Clk1, 2 and 4, demonstrated that Clk1 depletion increased UCP1 and PGC1α expression, whereas Clk2/4 siRNA did not. TG003‐treated adipocytes contained fewer lipid droplets, are smaller, and contain more mitochondria, resulting in proton leak increases. Additionally, inhibition of PKCβII activity, a splice variant regulated by Clk1, increased beiging. PGC1α is a substrate for both Clk1 and PKCβII kinases, and we surmised that inhibition of PGC1α phosphorylation resulted in beiging of adipocytes. We show that TG003 binds Clk1 more than Clk2/4 through direct binding, and PGC1α binds to Clk1 at a site close to TG003. Furthermore, we show that TG003 is highly specific for Clk1 across hundreds of kinases in our activity screen. Hence, Clk1 inhibition becomes a target for induction of beige adipocytes.

Excessive dietary intake of fat results in its storage in white adipose tissue (WAT), whereas energy expenditure by lipid oxidation occurs in brown adipose tissue. A third type of fat is beige, and it is observed in WAT depots. Certain WAT can undergo beiging. Numerous factors have roles in beiging. However, little is known about the signaling pathways inducing pre-adipocytes to become beige adipocytes. Known participants are activators of PPARgamma, PGC1alpha, and PRDM16 that induce UCP1-containing mitochondria upon cold exposure. We found a signaling pathway that regulates alternative pre-mRNA splicing is also involved in beiging when it is inhibited. Clk1 kinase phosphorylates proteins with serine and arginine rich domains. Clk1 inhibition by 50 nM TG003 during differentiation of 3T3-L1 adipocytes (post day 3) results in beige-like adipocytes demonstrating increased PGC1alpha and UCP1 mRNA and proteins. TG003 inhibits Clk1, 2, and 4. We demonstrate using siRNA for Clk1, 2, and 4, that Clk1 inhibition increased UCP1, PGC1alpha and PPARgamma mRNA whereas Clk1 and 4 siRNA did not. TG003-treated adipocytes contained fewer lipid droplets and more mitochondria which resulted in significant proton leak. In addition to TG003, CG53353, a PKCbetaII inhibitor, also increased mitochondria number and decreased lipid droplet size. PKCbetaII is an mRNA splice variant regulated by Clk1. Others previously showed that TG003 treatment of mice on high fat diets (HFD) resulted in weight loss, and PKCbeta(I+II) knockout mice did not gain weight on HFD. Using bioinformatics, we determined that PGC1alpha was a substrate for Clk1 and PKCbetaII. PGC1alpha was shown to be phosphorylated by Clk2 previously. Hence, we believe that inhibition of Clk1 and PKCbetaII phosphorylation of PGC1alpha results in upregulation of UCP1 expression in adipocytes. This newly identified pathway supports a role for Clk1 and PKCbetaII as potential targets for inhibition to treat weight loss in obesity. Disclosure A. Patel: None. T. Dobbins: None. S. Kong: None. N.A. Patel: None. D.R. Cooper: None. Funding U.S. Department of Veterans Affairs (5|01BX003689)