Byounghoon Hwang

Pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) deficiency attenuates the long-term negative effects of a high-saturated fat diet.

The hypothesis that PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) has potential as a target for the treatment of type 2 diabetes was tested by feeding wild-type and PDHK4 knockout mice a high saturated fat diet that induces hyperglycemia, hyperinsulinaemia, glucose intolerance, hepatic steatosis and obesity. Previous studies have shown that PDHK4 deficiency lowers blood glucose by limiting the supply of three carbon gluconeogenic substrates to the liver. There is concern, however, that the increase in glucose oxidation caused by less inhibition of the pyruvate dehydrogenase complex by phosphorylation will inhibit fatty acid oxidation, promote ectopic fat accumulation and worsen insulin sensitivity. This was examined by feeding wild-type and PDHK4 knockout mice a high saturated fat diet for 8 months. Fasting blood glucose levels increased gradually in both groups but remained significantly lower in the PDHK4 knockout mice. Hyperinsulinaemia developed in both groups, but glucose tolerance was better and body weight was lower in the PDHK4 knockout mice. At termination, less fat was present in the liver and skeletal muscle of the PDHK4 knockout mice. Higher amounts of PGC-1alpha [PPARgamma (peroxisome proliferator-activated receptor gamma) coactivator 1alpha] and PPARalpha and lower amounts of fatty acid synthase and acetyl-CoA carboxylase isoenzyme 1 were present in the liver of the PDHK4 knockout mice. These findings suggest PDHK4 deficiency creates conditions that alter upstream signalling components involved in the regulation of lipid metabolism. The findings support the hypothesis that PDHK4 is a viable target for the treatment of type 2 diabetes.

Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high saturated fat diet

Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may prove beneficial in the treatment of type 2 diabetes or diet‐induced obesity, it may have detrimental effects by inhibiting fatty acid oxidation. Peroxisome proliferator‐activated receptor α (PPARα) agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment using a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of a PDK4 inhibitor because PPARα agonists induce PDK4 expression. In the present study, the effects of clofibric acid, a PPARα agonist, on blood and liver lipids were determined in wild‐type and PDK4 knockout mice fed a high‐fat diet. As expected, treatment of wild‐type mice with clofibric acid resulted in less body weight gain, smaller epididymal fat pads, greater insulin sensitivity, and lower levels of serum and liver triacylglycerol. Surprisingly, rather than decreasing the effectiveness of clofibric acid, PDK4 deficiency enhanced the beneficial effects of clofibric acid on hepatic steatosis, reduced blood glucose levels, and did not prevent the positive effects of clofibric acid on serum triacylglycerols and free fatty acids. The metabolic effects of clofibric acid are therefore independent of the induction of PDK4 expression. The additive beneficial effects on hepatic steatosis may be due to induction of increased capacity for fatty acid oxidation and partial uncoupling of oxidative phosphorylation by clofibric acid, and a reduction in the capacity for fatty acid synthesis as a result of PDK4 deficiency.

IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation

Background: Nuclear import of NEMO is critical for DNA damage-dependent NF-κB signaling, but the mechanism remains unknown. Results: IPO3 binds NEMO, promotes its nuclear import, and is critical for DNA damage-dependent NF-κB activation. Conclusion: IPO3 is a nonclassical nuclear import receptor for NEMO. Significance: IPO3 is a new player in DNA damage-dependent NF-κB signaling with implications in cancer therapy. Activation of IκB kinase (IKK) and NF-κB by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-κB essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-κB signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-κB activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin α and β family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin β family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-κB activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-κB signaling arm of the mammalian DNA damage response.

Nuclear Import of JAK1 is Mediated by a Classical NLS and is Required for Survival of Diffuse Large B-cell Lymphoma

JAKs are non-receptor tyrosine kinases that are generally found in association with cytokine receptors. In the canonical pathway, roles of JAKs have well been established in activating STATs in response to cytokine stimulation to modulate gene transcription. In contrast, a noncanonical role of JAK2 has recently been discovered, in which JAK2 in the nucleus imparts the epigenetic regulation of gene transcription through phosphorylation of tyrosine 41 on the histone protein H3. Recent work further demonstrated that this noncanonical mechanism is conserved with JAK1, which is activated by the autocrine cytokines IL6 and IL10 in activated B-cell–like diffuse large B-cell lymphoma (ABC DLBCL), a cancer type that is particularly difficult to treat and has poor prognosis. However, how JAK1 gains access to the nucleus to enable epigenetic regulation remains undefined. Here, we investigated this question and revealed that JAK1 has a classical nuclear localization signal toward the N-terminal region, which can be recognized by multiple importin α isoforms. Moreover, the nuclear import of JAK1 is independent of its kinase activity but is required for the optimal expansion of ABC DLBCL cells in vitro. Implications: This study demonstrates that the nuclear import of JAK1 is essential for the optimal fitness of ABC DLBCL cells, and targeting JAK1 nuclear localization is a potential therapeutic strategy for ABC DLBCL. Mol Cancer Res; 15(3); 348–57. ©2016 AACR.

Quantification of cellular NEMO content and its impact on NF-κB activation by genotoxic stress.

NF-κB essential modulator, NEMO, plays a key role in canonical NF-κB signaling induced by a variety of stimuli, including cytokines and genotoxic agents. To dissect the different biochemical and functional roles of NEMO in NF-κB signaling, various mutant forms of NEMO have been previously analyzed. However, transient or stable overexpression of wild-type NEMO can significantly inhibit NF-κB activation, thereby confounding the analysis of NEMO mutant phenotypes. What levels of NEMO overexpression lead to such an artifact and what levels are tolerated with no significant impact on NEMO function in NF-κB activation are currently unknown. Here we purified full-length recombinant human NEMO protein and used it as a standard to quantify the average number of NEMO molecules per cell in a 1.3E2 NEMO-deficient murine pre-B cell clone stably reconstituted with full-length human NEMO (C5). We determined that the C5 cell clone has an average of 4 x 105 molecules of NEMO per cell. Stable reconstitution of 1.3E2 cells with different numbers of NEMO molecules per cell has demonstrated that a 10-fold range of NEMO expression (0.6–6x105 molecules per cell) yields statistically equivalent NF-κB activation in response to the DNA damaging agent etoposide. Using the C5 cell line, we also quantified the number of NEMO molecules per cell in several commonly employed human cell lines. These results establish baseline numbers of endogenous NEMO per cell and highlight surprisingly normal functionality of NEMO in the DNA damage pathway over a wide range of expression levels that can provide a guideline for future NEMO reconstitution studies.