Aberrant levels of preadipocyte differentiation, triggered by adipocyte hyperplasia and hypertrophy, results in the obesogenic phenotype. Obesity is a risk factor for several metabolic disorders. In this paper, dehydroleucodine inhibited the accumulation of lipid droplets and decreased the elevations of triglycerides, and this inhibitory effect occurred during the early stage of adipogenesis. Thus, not only did dehydroleucodine downregulate the expression of C/EBPα and PPARγ, it also strongly blocked the expression of C/EBPβ, an early stage biomarker of early adipogenesis, in a concentration-dependent manner. The proliferation of preadipocytes was dramatically suppressed when dehydroleucodine was added to the medium as early as 24 hr. These results indicate that dehydroleucodine may specifically affect mitotic clonal expansion to inhibit preadipocyte differentiation. Dehydroleucodine arrested the cell cycle at the G /G phase, increased p27 and decreased both cyclins A and D and their partners (e.g., CDK2 and CDK4). Additionally, dehydroleucodine decreased phosphorylation of Erk1/2 and Akt. Furthermore, dehydroleucodine downregulated expression of histone demethylase JMJD2B as well as repressed the expression of histone methyltransferase MLL4, which in turn diminished the expression of C/EBPβ and PPARγ, respectively. Collectively, our results indicate that dehydroleucodine inhibits preadipocyte differentiation by blocking mitotic clonal expansion via cell cycle arrest, which may be mediated by regulation of selective histone methylation/demethylation in transcription activation during the early step of adipogenesis.
Human type I 3 -hydroxysteroid dehydrogenase/ isomerase (3 -HSD/isomerase) is an integral membrane protein of human placental trophoblast and of insect Sf9 cells transfected with recombinant baculovirus containing the cDNA encoding the enzyme. Purified native or wild-type enzyme remains in solution only in the presence of detergent that may prevent crystallization. The membranespanning domain (residues 283-310) of the enzyme protein was deleted in the cDNA using PCR-based mutagenesis. The modified enzyme was expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells. The cytosolic form of 3 -HSD/isomerase was purified using affinity chromatography with Cibacron Blue 1000. The NAD + and NaCl used to elute the enzyme were removed by size-exclusion centrifugation. Hydroxylapatite chromatography yielded a 26-fold purification of the enzyme. SDS-PAGE revealed a single protein band for the purified cytosolic enzyme (monomeric molecular mass 38·8 kDa) that migrated just below the wild-type enzyme (monomeric molecular mass 42·0 kDa). Michaelis-Menten constants measured for 3 -HSD substrate (dehydroepiandrosterone) utilization by the purified cytosolic enzyme (K m =4·5 µM, V max =53 nmol/min per mg) and the pure wild-type enzyme (K m =3·7 µM, V max =43 nmol/min per mg), for isomerase substrate (5-androstene-3,17-dione) conversion by the purified cytosolic (K m =25 µM, V max =576 nmol/min per mg) and wild-type (K m =28 µM, V max =598 nmol/min per mg) enzymes, and for NAD + reduction by the 3 -HSD activities of the cytosolic (K m =35 µM, V max =51 nmol/min per mg) and wild-type (K m =34 µM, V max =46 nmol/min per mg) enzymes are nearly identical. The isomerase activity of the cytosolic enzyme requires allosteric activation by NADH (K m =4·6 µM, V max =538 nmol/min per mg) just like the wild-type enzyme (K m =4·6 µM, V max =536 nmol/min per mg). Crystals of the purified, cytosolic enzyme protein have been obtained. The inability to crystallize the detergentsolubilized, wild-type microsomal enzyme has been overcome by engineering a cytosolic form of this protein. Determining the tertiary structure of 3 -HSD/isomerase will clarify the mechanistic roles of potentially critical amino acids (His 261 , Tyr 253 ) that have been identified in the primary structure.
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