Chronic high dose of insulin stimulates lipolysis in porcine adipocytes / 生物工程学报

To explore the effect of chronic high dose of insulin on lipolysis in porcine adipocytes and the underlying molecular regulation mechanisms, we cultured primary porcine adipocytes and incubated them with different concentrations of insulin (0, 200, 400, 800, 1600 nmol/L) for 24-96 h in the absence or presence of specific protein kinase A (PKA) inhibitor or extracellular signal-related kinase (ERK) inhibitor. Then, we measured the glycerol release into the culture media as an indicator of the lipolysis, and observed the lipid accumulation morphology by phase-contrast microscopy. Further, we analyzed the gene expressions of perilipin A and peroxisome proliferator-activated receptor-gamma 2 (PPAR gamma 2) with semi-quantitative RT-PCR and Western blotting, respectively. The results showed that chronic high dose of insulin stimulated lipolysis in differentiated porcine adipocytes in a dose- and time-dependent manner, and significantly attenuated the lipolytic response to isoprenaline. Meanwhile, the protein and mRNA expressions of PPAR gamma 2 and perilipin A were significantly reduced. In addition, both PKA and ERK inhibitors significantly suppressed insulin-stimulated lipolysis, however, only ERK inhibitor reversed the insulin-induced down-regulation of perilipin A. These findings imply that chronic high dose of insulin stimulates lipolysis in porcine adipocytes by repressing perilipin A, which is involved in ERK pathway.

Induction of very low density lipoprotein receptor (VLDLR) transcription by VLDL is mediated by the extracellular signal-regulated kinase signaling pathway / 华中科技大学学报（医学）（英德文版）

To elucidate the intracellular signaling pathways for VLDL-induced VLDLR transcription, Western blot analysis was used to examine phosphorylated ERK1/2 protein. It was found that that VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators it was observed that the effect of VLDL-induced VLDL receptor transcription, which is monitored by RTPCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but completely abolished by pretreatment of the cells with PD 98059, an inhibitor of MEK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC/ERK1/2 cascade is the essential signaling pathway by which VLDL activates VLDL receptor mRNA expression.