Fetuin-A, a hepatic secretory protein, has recently been implicated in insulin resistance and Type 2 diabetes. It is an endogenous inhibitor of insulin receptor tyrosine kinase. However, regulation of fetuin-A synthesis in relation to insulin resistance is unclear. In the present paper, we report that both non-esterified ('free') fatty acids and fetuin-A coexist at high levels in the serum of db/db mice, indicating an association between them. For an in-depth study, we incubated palmitate with HepG2 cells and rat primary hepatocytes, and found enhanced fetuin-A secretion to more than 4-fold over the control. Interestingly, cell lysates from these incubations showed overexpression and activity of NF-kappaB (nuclear factor kappaB). In NF-kappaB-knockout HepG2 cells, palmitate failed to increase fetuin-A secretion, whereas forced expression of NF-kappaB released fetuin-A massively in the absence of palmitate. Moreover, palmitate stimulated NF-kappaB binding to the fetuin-A promoter resulting in increased reporter activity. These results suggest NF-kappaB to be the mediator of the palmitate effect. Palmitate-induced robust expression of fetuin-A indicates the occurrence of additional targets, and we found that fetuin-A severely impaired adipocyte function leading to insulin resistance. Our results reveal a new dimension of lipid-induced insulin resistance and open another contemporary target for therapeutic intervention in Type 2 diabetes.

Protein post-translational modification by ubiquitin represents a complex signaling system that regulates many cellular events including proteostasis to intercellular communications. Deubiquitinating enzymes (DUBs) that specifically disassemble Ub-chains or regulate ubiquitin homeostasis reside as a central component in ubiquitin signaling. Human genome encodes almost 100 DUBs and majority of them are not well characterized. Considerable progress has been made in the understanding of enzymatic mechanism; however, their cellular substrate specificity and regulation are largely unknown. Involvement of DUBs in disease regulation has been depicted since its discovery and several attempts have been made for evaluating DUBs as a drug target. In this review, we have updated briefly a new insight of DUBs activity, their cellular role, disease regulation, and therapeutic potential. V C 2015 IUBMB Life, 67(7): [544][545][546][547][548][549][550][551][552][553][554][555] 2015

Lipid induced NF-kappaB activation is known to be associated with insulin resistance and type2 diabetes. Here we show that incubation of L6 skeletal muscle cells with palmitate significantly increased NF-kappaB p65 and NF-kappaB p50 expression along with their phosphorylation. NF-kappaB p65 siRNA inhibited palmitate induced overexpression of NF-kappaB p65 indicating palmitate effect on transcriptional activation. RT-PCR and real time PCR experiments also showed a significant increase in NF-kappaB p65 gene expression due to palmitate. Overexpression of NF-kappaB p65 by palmitate was linked to impairment of insulin activity. Palmitate effect on NF-kappaB gene and protein expression was found to be mediated by phospho-PKCepsilon as calphostin C (an inhibitor of PKC) and epsilonV1 (PKCepsilon translocation inhibitor) significantly reduced NF-kappaB expression. To understand the underlying mechanism, we purified NF-kappaB and pPKCepsilon from palmitate incubated skeletal muscle cells and their interaction in cell free system demonstrated the transfer of phosphate from PKCepsilon to NF-kappaB. This prompted us to transduct pPKCepsilon to the skeletal muscle cells. These cells showed increased amount of pNF-kappaB and NF-kappaB. Excess of NF-kappaB p65 pool thus created in the cells made them insulin resistant. Addition of NF-kappaB p65 siRNA and SN50 inhibited palmitate induced NF-kappaB p65 expression indicating NF-kappaB regulation of its gene expression. Increase of NF-kappaB did not affect the activation of IKK/IkappaB indicating NF-kappaB p65 expression to be a distinct effect of palmitate. Since NF-kappaB p65 is linked to several diseases, including type2 diabetes, this report may be important in understanding the pathogenicity of these diseases.

BRCA1 associated protein 1 (BAP1) is a nuclear deubiquitinase that regulates tumor suppressor activity and widely involves many cellular processes ranging from cell cycle regulation to gluconeogenesis. Impairment of enzymatic activity and nuclear localization induce abnormal cell proliferation. It is considered to be an important driver gene, which undergoes frequent mutations in several cancers. However the role of mutation and oncogenic gain of function of BAP1 are poorly understood. Here, we investigated cellular localization, enzymatic activity and structural changes for four missense mutants of the catalytic domain of BAP1, which are prevalent in different types of cancer. These mutations triggered cytoplasmic/perinuclear accumulation in BAP1 deficient cells, which has been observed in proteins that undergo aggregation in cellular condition. Amyloidogenic activity of mutant BAP1 was revealed from its reactivity towards anti oligomeric antibody in HEK293T cells. We have also noted structural destabilization in the catalytic domain mutants, which eventually produced beta amyloid structure as indicated in atomic force microscopy study. The cancer associated mutants up-regulate heat shock response and activates transcription of genes normally co-repressed by BAP1. Overall, our results unambiguously demonstrate that structural destabilization and subsequent aggregation abrogate its cellular mechanism leading to adverse outcome.

Non-small cell lung carcinoma (NSCLC) is a major killer in cancer related human death. Its therapeutic intervention requires superior efficient molecule(s) as it often becomes resistant to present chemotherapy options. Here we report that vapor of volatile oil compounds obtained from Litsea cubeba seeds killed human NSCLC cells, A549, through the induction of apoptosis and cell cycle arrest. Vapor generated from the combined oils (VCO) deactivated Akt, a key player in cancer cell survival and proliferation. Interestingly VCO dephosphorylated Akt at both Ser473 and Thr308; through the suppression of mTOR and pPDK1 respectively. As a consequence of this, diminished phosphorylation of Bad occurred along with the decreased Bcl-xL expression. This subsequently enhanced Bax levels permitting the release of mitochondrial cytochrome c into the cytosol which concomitantly activated caspase 9 and caspase 3 resulting apoptotic cell death. Impairment of Akt activation by VCO also deactivated Mdm2 that effected overexpression of p53 which in turn upregulated p21 expression. This causes enhanced p21 binding to cyclin D1 that halted G1 to S phase progression. Taken together, VCO produces two prong effects on lung cancer cells, it induces apoptosis and blocked cancer cell proliferation, both occurred due to the deactivation of Akt. In addition, it has another crucial advantage: VCO could be directly delivered to lung cancer tissue through inhalation.

Fatty acids (FAs) are known to impair insulin signaling in target cells. Accumulating evidences suggest that one of the major sites of FAs adverse effect is insulin receptor (IR). However, the underlying mechanism is yet unclear. An important clue was indicated in leptin receptor deficient (db/db) diabetic mice where increased circulatory FAs was coincided with phosphorylated PKCε and reduced IR expression. We report here that central to this mechanism is the phosphorylation of PKCε by FAs. Kinase dead mutant of PKCε did not augment FA induced IRβ downregulation indicating phosphorylation of PKCε is crucial for FA induced IRβ reduction. Investigation with insulin target cells showed that kinase independent phosphorylation of PKCε by FA occurred through palmitoylation. Mutation at cysteine 276 and 474 residues in PKCε suppressed this process indicating participation of these two residues in palmitoylation. Phosphorylation of PKCε endowed it the ability to migrate to the nuclear region of insulin target cells. It was intriguing to search about how translocation of phosphorylated PKCε occurred without having canonical nuclear localization signal (NLS). We found that F-actin recognized phospho-form of PKCε and chaperoned it to the nuclear region where it interact with HMGA1 and Sp1, the transcription regulator of IR and HMGA1 gene respectively and impaired HMGA1 function. This resulted in the attenuation of HMGA1 driven IR transcription that compromised insulin signaling and sensitivity.

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It is well known that free fatty acids (FFAs) play a key role in implementing insulin resistance and type 2 diabetes. Resources of chemical compounds that intervene the derogatory effect of FFAs are indeed very limited. We have isolated mahanine, a carbazole alkaloid, from the leaves of Murraya koenegii that prevented palmitate-induced inhibition of insulin-stimulated phosphorylation of IRbeta, PI3K, PDK1, and Akt in L6 myotubes. This was also reflected in the palmitate-induced inhibition of insulin-stimulated [(3)H] 2-DOG uptake by L6 myotubes, where palmitate adverse effect was significantly blocked by mahanine. Previous reports indicated that one of the major targets of lipid-induced damage in insulin signaling pathway resulting impairment of insulin sensitivity is insulin receptor (IR). Here, we have observed that palmitate significantly increased pPKCepsilon in both cytosol and nuclear region of L6 myotubes in comparison to control. Translocation of pPKCepsilon to the nucleus was associated with the impairment of HMGA1, the architectural transcription factor of IR gene and all these were reversed by mahanine. Palmitate-induced activation of IKK/IkappaBeta/NF-kappaBeta pathway was also attenuated by mahanine. Taken together, mahanine showed encouraging possibility to deal with lipid induced insulin resistance. In order to examine it further, mahanine was administered on nutritionally induced type 2 diabetic golden hamsters; it significantly improved hyperglycemia in all the treated animals. Our results, therefore, suggest that mahanine acts on two important sites of lipid induced insulin resistance (i) impairment of IR gene expression and (ii) activation of NF-kappaBeta pathway, thus, showing promise for its therapeutic choice for type 2 diabetes.