The inhibitory effects of PKCθ on adiponectin expression is mediated by ERK in 3T3-L1 adipocytes

Abstract

The research suggests that adiponectin plays an important role in sensitizing insulin action. It is interesting to find that the lower levels of adiponectin exist in the plasma of obese and Type 2 diabetes subjects and in the adipose tissue of obese, db/db mice, and insulin-resistant individuals. However, the underlying mechanism by which adiponectin expression is inhibited remains largely unknown. In this study, we reported that adipogenesis was inhibited by the stable over-expression of protein kinase C θ (PKCθ) in 3T3-L1 pre - adipocytes. The prolonged treatment of mature 3T3-L1 adipocytes with palmitate, a kind of saturated free fatty acid, reduced adiponectin expression at both mRNA level and protein level, accompanied with the enhanced phosphorylation of PKCθ and extracellular signal-regulated kinase (ERK), and the impaired expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA. Either PD98059, an ERK inhibitor or PKCθ pseudosubstrate, a specific PKCθ inhibitor, restored palmiate-inhibited PPARγ2 mRNA expression and subsequent adiponectin expression. In addition, the over-expression or activation of PKCθ resulted in the enhanced phosphorylation of ERK in the mature 3T3-L1 adipocytes. PKCθ pseudosubstrate significantly reduced the phorbol 3-myristate 12-acetate (PMA)-induced phosphorylation of ERK. The data suggested that PKCθ-dependent activity of ERK resulted in the impaired expression of PPARγ2 mRNA leading to the reduction of adiponectin expression in the mature 3T3-L1 adipocytes.

Wogonin suppresses osteopontin expression in adipocytes by activating PPARα

“…To convert 3T3-L1 fibroblasts to adipocytes, the cells were maintained in complete medium until they reached 100% confluence. Three days following confluence, the cells were incubated in complete medium supplemented with 1 μg/mL insulin, 0.25 μmol/L dexamethasone, and 0.5 mmol/L 3-isobutyl-1-methylxanthine (IBMX) for 3 d, then transferred to complete medium containing 1 μg/mL insulin for another 3 d, and finally maintained in complete medium for an additional 4 d. At that time, approximately 90% of cells were considered to be mature adipocytes as confirmed by Oil Red O staining [19] .…”

“…RT-PCR was performed as described previously [19] . Briefly, total RNA was isolated from mouse adipose tissues or 3T3-L1 adipocytes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).…”

“…Wogonin downregulated OPN expression and secretion in 3T3-L1 adipocytes To further confirm the effects of wogonin on OPN expression and secretion, 3T3-L1 pre-adipocytes were converted to 3T3- npg L1 adipocytes as previously described by our laboratory [19] . Then, 3T3-L1 adipocytes were incubated in complete medium containing 25 mmol/L glucose to mimic hyperglycemia.…”

Wogonin enhances intracellular adiponectin levels and suppresses adiponectin secretion in 3T3-L1 adipocytes

“…3T3-L1 differentiation was performed as our previously described [28,29]. In To quantify different molecular weight fractions of adiponectin, the cell lysates were subjected to 2-15% SDS-PAGE under non-reducing and non-heat-denaturing conditions.…”

“…RT-PCR was performed as described previously [29]. Briefly, total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA).…”