Persistent ER stress, mitochondrial dysfunction and failure of the heat shock response (HSR) are fundamental hallmarks of insulin resistance (IR); one of the early core metabolic aberrations that leads to type 2 diabetes (T2D). The antioxidant α-lipoic acid (ALA) has been shown to attenuate metabolic stress and improve insulin sensitivity in part through activation of the heat shock response (HSR). However, these studies have been focused on a subset of heat shock proteins (HSPs). In the current investigation, we assessed whether ALA has an effect on modulating the expression of DNAJB3/HSP40 cochaperone; a potential therapeutic target with a novel role in mitigating metabolic stress and promoting insulin signaling. Treatment of C2C12 cells with 0.3 mM of ALA triggers a significant increase in the expression of DNAJB3 mRNA and protein. A similar increase in DNAJB3 mRNA was also observed in HepG2 cells. We next investigated the significance of such activation on endoplasmic reticulum (ER) stress and glucose uptake. ALA pre-treatment significantly reduced the expression of ER stress markers namely, GRP78, XBP1, sXBP1 and ATF4 in response to tunicamycin. In functional assays, ALA treatment abrogated significantly the tunicamycin-mediated transcriptional activation of ATF6 while it enhanced the insulin-stimulated glucose uptake and Glut4 translocation. Silencing the expression of DNAJB3 but not HSP72 abolished the protective effect of ALA on tunicamycin-induced ER stress, suggesting thus that DNAJB3 is a key mediator of ALA-alleviated tunicamycin-induced ER stress. Furthermore, the effect of ALA on insulin-stimulated glucose uptake is significantly reduced in C2C12 and HepG2 cells transfected with DNAJB3 siRNA. In summary, our results are supportive of an essential role of DNAJB3 as a molecular target through which ALA alleviates ER stress and improves glucose uptake.

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.

Metabolic stress involved in several dysregulation disorders such as type 2 diabetes mellitus (T2DM) results in down regulation of several heat shock proteins (HSPs) including DNAJB3. This down regulation of HSPs is associated with insulin resistance (IR) and interventions which induce the heat shock response (HSR) help to increase the insulin sensitivity. Metabolic stress leads to changes in signaling pathways through increased activation of both c-jun N-terminal kinase-1 (JNK1) and the inhibitor of κB inflammatory kinase (IKKβ) which in turn leads to inactivation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2). DNAJB3 interacts with both JNK1 and IKKβ kinases to mitigate metabolic stress. In addition DNAJB3 also activates the PI3K-PKB/AKT pathway through increased phosphorylation of AKT1 and its substrate AS160, a Rab GTPase-activating protein, which results in mobilization of GLUT4 transporter protein and improved glucose uptake. We show through pull down that AK T1 is an interacting partner of DNAJB3, further confirmed by isothermal titration calorimetry (ITC) which quantified the avidity of AKT1 for DNAJB3. The binding interface was identified by combining protein modelling with docking of the AKT1-DNAJB3 complex. DNAJB3 is localized in the cytoplasm and ER, where it interacts directly with AKT1 and mobilizes AS160 for glucose transport. Inhibition of AKT1 resulted in loss of GLUT4 translocation activity mediated by DNAJB3 and also abolished the protective effect of DNAJB3 on tunicamycin-induced ER stress. Taken together, our findings provide evidence for a direct protein-protein interaction between DNAJB3 and AKT1 upon which DNAJB3 alleviates ER stress and promotes GLUT4 translocation.

Obesity is a major risk factor for a myriad of disorders such as insulin resistance and diabetes. The mechanisms underlying these chronic conditions are complex but low grade inflammation and alteration of the endogenous stress defense system are well established. Previous studies indicated that impairment of HSP-25 and HSP-72 was linked to obesity, insulin resistance and diabetes in humans and animals while their induction was associated with improved clinical outcomes. In an attempt to identify additional components of the heat shock response that may be dysregulated by obesity, we used the RT(2)-Profiler PCR heat shock array, complemented with RT-PCR and validated by Western blot and immunohistochemistry. Using adipose tissue biopsies and PBMC of non-diabetic lean and obese subjects, we report the downregulation of DNAJB3 cochaperone mRNA and protein in obese that negatively correlated with percent body fat (P = 0.0001), triglycerides (P = 0.035) and the inflammatory chemokines IP-10 and RANTES (P = 0.036 and P = 0.02, respectively). DNAJB positively correlated with maximum oxygen consumption (P = 0.031). Based on the beneficial effect of physical exercise, we investigated its possible impact on DNAJB3 expression and indeed, we found that exercise restored the expression of DNAJB3 in obese subjects with a concomitant decrease of phosphorylated JNK. Using cell lines, DNAJB3 protein was reduced following treatment with palmitate and tunicamycin which is suggestive of the link between the expression of DNAJB3 and the activation of the endoplasmic reticulum stress. DNAJB3 was also shown to coimmunoprecipiate with JNK and IKKβ stress kinases along with HSP-72 and thus, suggesting its potential role in modulating their activities. Taken together, these data suggest that DNAJB3 can potentially play a protective role against obesity.