DNAJB3 attenuates metabolic stress and promotes glucose uptake by eliciting Glut4 translocation

Abstract

Failure of the heat shock response is a key event that leads to insulin resistance and type 2 diabetes. We recently showed that DNAJB3 co-chaperone is downregulated in obese and diabetic patients and that physical exercise restores its normal expression with a significant improvement of the clinical outcomes. In 3T3-L1 adipocytes, DNAJB3 has a role in improving the sensitivity to insulin and glucose uptake. In co-immunoprecipitation assays, DNAJB3 interacts with both JNK1 and IKKβ kinases. However, the functional impact of such interaction on their activities has not been investigated. Here, we assessed the effect of DNAJB3 on the respective activity of JNK1 and IKKβ in cell-based assays. Using JNK1- and IKKβ-dependent luciferase reporters, we show a marked decrease in luciferase activity by DNAJB3 in response to PMA and TNF-α that was consistent with a decrease in the translocation of p65/NF-κB to the nucleus in response to LPS. Furthermore, TNF-α-mediated IL-6 promoter activation and endogenous mRNA expression are significantly abrogated by DNAJB3 both in 3T3-L1 and C2C12 cells. The ability of DNAJB3 to mitigate ER stress and oxidative stress was also investigated and our data show a significant improvement of both forms of stress. Finally, we examined the effect of overexpressing and knocking down the expression of DNAJB3 on glucose uptake in C2C12 as well as the molecular determinants. Accordingly, we provide evidence for a role of DNAJB3 in promoting both basal and insulin-stimulated glucose uptake. Our finding reveals also a novel role of DNAJB3 in eliciting Glut4 translocation to the plasma membrane. These results suggest a physiological role of DNAJB3 in mitigating metabolic stress and improving glucose homeostasis and could therefore represent a novel therapeutic target for type 2 diabetes.

Alpha lipoic acid attenuates ER stress and improves glucose uptake through DNAJB3 cochaperone

“…Fluorescently labeled d-glucose analog (2-NBDG) was purchased from Cayman (Cayman, Ann Arbor, MI). pHA-Glut4-GFP was a gift from Dr. MacGraw (Weill Cornell University, New York, NY) and consists of an exofacial HA epitope and a GFP tag located at the N-terminal and C-terminal of Glut4, respectively 30 . Reporter plasmid carrying five copies of ATF6 binding site upstream of the Luc2P reporter gene was described previously 30 .…”

“…On next day, they were plated on 96-well plates at 1 × 10 4 cells/well in complete DMEM media containing 0.3 mM ALA or vehicle. After 8 h of incubation, cells were stimulated with 0.5 µg/ml tunicamycin or vehicle and incubated at 37 °C for overnight and then, harvested for luciferase assays using the Bright Glo Luciferase Assay kit as we described previously 30 . To investigate if the effect of ALA on ER stress is mediated through DNAJB3, knocking down DNAJB3 in C2C12 cells was carried out with DNAJB3 siRNA or scrambled siRNA using lipofectamine RNAiMAX protocol.…”

“…GAPDH and actin genes were used as internal controls. The primers corresponding to genes of the heat shock response, ER stress and oxidative stress have been published previously 30,32 . The ones used for mitochondria were self-designed using Primer 3 software version 4.0 (http:// bioin fo.ut.ee/prime r3-0.4.0).…”

“…Cells were then washed with Cell-Based Assay Buffer and transferred to 96-well assay plate at 1 × 10 4 cells/well. Finally, fluorescence was quantified on Glomax Multi + Microplate Multimode Reader (Promega, Madison, WI) as previously described 29,30 . Monitoring glucose uptake in C2C12 and HepG2 cells where the expression of DNAJB3 has been silenced was done by transfecting cells with DNAJB3-siRNA or scrambled control and after 2 days, cells were pre-treated with either 0.3 mM ALA or vehicle for 8 h and the media was changed to glucose free media with either ALA or vehicle for starvation and glucose uptake was carried out as described above.…”

“…We further demonstrated the positive effect of a 3-month physical exercise in restoring the normal expression of DNAJB3 with the concomitant improvement of various physical, biochemical and clinical parameters, suggesting thus a protective role of DNAJB3 against metabolic diseases associated with increased IR 28 . More recently, we provided evidence for a novel role of DNAJB3 in attenuating various forms of metabolic stress as well as in promoting insulin action and glucose uptake in 3T3-L1 adipocytes and C2C12 skeletal muscle cells 29,30 . Given the similarities in metabolic actions elicited by DNAJB3 overexpression and upon ALA treatment, we hypothesized that DNAJB3 might represent a molecular intermediate through which ALA mediates its beneficial actions.…”