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Retrovirus insertion-mediated random mutagenesis was applied in 3T3-L1 preadipocyte cells to better understand the molecular basis of obesity (the expansion of individual adipocytes). We found that tryptophan hydroxylase-1, a rate-limiting enzyme for the synthesis of serotonin (5-HT), is expressed in adipocytes and is required for their differentiation. A 5-HT type 2A receptor (5-HT2AR) antagonist, ketanserin, and a 5-HT2cR antagonist, SB-242084, inhibited adipocyte differentiation. Because 5-HT2cR mRNA levels are up-regulated during adipocyte differentiation and micro-RNA (miR)-448 is located in the fourth intron of 5-HT2cR, we also studied the role of miR-448 in 3T3-L1 cells. Through a bioinformatics approach, Krüppel-like factor 5 (KLF5) was identified as a potential target of miR-448. Using a luciferase reporter assay, we confirmed that miR-448 targets the KLF5 3′-intranslated region. Overexpression of miR-448 reduced the expression of KLF5 and adipocyte differentiation, which was confirmed by the reduced expression of adipogenic genes and triglyceride accumulation. To examine the loss of miR-448 function, we constructed a decoy gene that had tandem complementary sequences for miR-448 in the 3′-untranslated region of a luciferase gene under the control of a cytomegalovirus promoter. When the miR-448 decoy gene was introduced into 3T3-L1 preadipocytes, KLF5 was up-regulated and triglyceride concentration was increased. In this study, we identified the regulation of adipocyte differentiation by 5-HT, 5-HT2AR, and 5-HT2CR. miR-448-mediated repression of KLF5 was identified as a negative regulator for adipocyte differentiation.

Issue Section:
Translational Highlights from Molecular Endocrinology
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