Deletion of CD44 promotes adipogenesis and insulin signalling in adipocytes

Background and aims: Growing evidence suggest a close link between adipose fibrosis, inflammation, and insulin resistance in obesity. Hyaluronan, one of the main components of the extracellular matrix is increased in adipose tissue of obese and diabetic mice. CD44, the main hyaluronan receptor is associated with Type 2 diabetes from expression-based genome-wide association studies and its expression level in adipose tissue is positively correlated with adipose inflammation and insulin resistance. This study is to determine the role of CD44 in adipose function and insulin resistance.

Materials and methods: Stable, CD44-deficient 3T3-L1 cells were generated by Crispr Cas 9 technology using guide RNAs targeting the exon 3 of cd44 gene. The CD44 knockout (KO) cells were confirmed by Western blot and site mutations were determined by biallelic sequencing. Cells that were transfected but maintained normal level of CD44 protein were used as Crispr wildtype (WT) controls. Differentiation of 3T3-L1 cells to adipocytes was included by a cocktail of isobutylmethylxanthine, insulin, and dexamethosone. Mouse primary adipocytes were derived from stromal cells of the subcutaneous adipose tissue in 10 week old C57BL/6 WT and global CD44 KO mice. Adipogenesis was measured by Oil Red O staining and insulin sensitivity was measured by phosphorylation of Akt. Insulin resistance was induced by treating the cells with 250μM palmitic acid for 24 hours.

Results: CD44 gene expression decreased by 81% after differentiation in WT 3T3-L1 cells (P<0.05). Deletion of CD44 in 3T3-L1 cells increased adipogenesis as assessed by Oil Red O staining (3.24±0.86 arbitrary units vs 2.52±0.67 in 3T3-L1 naïve cells and 1.98±0.86 in Crispr WT cells) (P<0.05). Gene expression of the adipogenic markers PPARɣ and CEBPα were also consistently increased in the CD44 KO cells when compared with the control cells. Upon insulin stimulation, knocking out CD44 enhanced phosphorylation of AKT at S473 in differentiated 3T3-L1 adipocytes (2.1±0.8-fold increase, P<0.05). Palmitate acid induced a blunted response of Akt phosphorylation and P38 dephosphorylation in 3T3-L1 WT adipocytes, which was reversed in CD44 KO 3T3-L1 adipocytes (P<0.01). Consistent with the results in 3T3-L1 cells, primary adipose stromal cells isolated from CD44 KO mice displayed an enhanced adipogenic capacity and increased phosphorylation of Akt after insulin stimulation compared to those from the WT mice.

Conclusion: Deletion of CD44 promoted adipogenesis and improved insulin signalling in vitro in 3T3-L1 cells and mouse primary adipocytes. This study extends our knowledge of the role of CD44 in regulating adipocyte function, representing a potential target for mitigating adipose dysfunction in metabolic disorders.