Phorbol ester-regulated oligomerization of diacylglycerol kinase delta linked to its phosphorylation and translocation

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. In yeast two-hybrid screening, we unexpectedly found a self-association of the C-terminal part of DGKdelta containing a sterile alpha-motif (SAM) domain. We then bacterially expressed the SAM domain fused with maltose-binding protein and confirmed the formation of dimeric and tetrameric structures. Moreover, gel filtration and co-immunoprecipitation analyses demonstrated that DGKdelta formed homo-oligomeric structures in intact cells and that the SAM domain was critically involved in the oligomerization. Interestingly, phorbol ester stimulation induced dissociation of the oligomeric structures with concomitant phosphorylation of DGKdelta. Furthermore, we found that DGKdelta was translocated from cytoplasmic vesicles to the plasma membrane upon phorbol ester stimulation. In this case, DGKdelta mutants lacking the ability of self-association were localized at the plasma membranes even in the absence of phorbol ester. A protein kinase C inhibitor, staurosporine, blocked all of the effects of phorbol ester, i.e. oligomer dissociation, phosphorylation, and translocation. We confirmed that tumor-promoting phorbol esters did not directly bind to DGKdelta. The present studies demonstrated that the formation and dissociation of oligomers serve as the regulatory mechanisms of DGKdelta and that DGKdelta is a novel downstream effector of phorbol ester/protein kinase C signaling pathway.     

Regulation of clathrin-dependent endocytosis by diacylglycerol kinase delta: importance of kinase activity and binding to AP2alpha

DGKdelta (diacylglycerol kinase delta), which phosphorylates DAG (diacylglycerol) and converts it into PA (phosphatidic acid), has an important role in signal transduction. In the present study, we have demonstrated the molecular mechanism of DGKdelta-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGKdelta in the regulation of clathrin-dependent endocytosis was previously proposed following genome-wide RNAi (RNA interference) screening. Clathrin-coated pits are mainly formed by clathrin and AP-2 (adaptor protein 2) complex. These proteins assemble a polyhedral lattice at the membrane and gather several endocytic accessory proteins. As the intracellular localization of DGKdelta2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGKdelta2 and its interaction with some endocytosis-regulatory proteins. DGKdelta2 contained the DXF-type binding motifs, and DGKdelta2 bound to AP2alpha, a subunit of the AP-2 complex. DGKdelta2 interacted with the platform subdomain in the AP2alpha ear domain via F369DTFRIL and D746PF sequences in the catalytic domain of DGKdelta2. For further insight into the role for DGKdelta2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptake-expressing wild-type or mutant DGKdelta2 under knockdown of endogenous DGKdelta. Mutants lacking binding ability to AP2alpha as well as kinase-negative mutants could not compensate for the uptake of transferrin inhibited by siRNA (small interfering RNA) treatment, whereas overexpression of wild-type DGKdelta2 completely recovered the transferrin uptake. These results demonstrate that binding between DGKdelta2 and AP2alpha is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.     

Over-expression of AQP7 contributes to improve insulin resistance in adipocytes

OBJECTIVE: Aquaglyceroporin 7 (AQP7) is required for efflux of glycerol from adipocytes. In this study, we aimed to analyze expression profiles of AQP7 in the different differentiation phases of adipocytes and to investigate the role of AQP7 in the insulin resistance of adipocytes. Methods: 3T3-L1 pre-adipocyte cells were induced to be fully differentiated adipocytes and then insulin resistance was induced by Dexamethasone (DXM) or TNF-α. Adenovirus vector with over-expression AQP7 (Ad-AQP7) was constructed and transfected into adipocytes. The expression level of AQP7 and phosphorylated PKB (p-PKB) were measured. The glycerol released from adipocytes and glucose consuming rate were tested too. Results: AQP7 expression was gradually up-regulated along with the differentiation processing of 3T3-L1 preadipocytes, which was consistent with the expression level of p-PKB. Dexamethasone down-regulated the expression of AQP7, p-PKB and the glycerol content in adipocytes. Over-expression of AQP7 by transfecting Ad-AQP7 to insulin resistant adipocytes restored the phosphorylation of PKB and attenuated the glycerol secretion and glucose consuming rate of adipocytes. Conclusions: AQP7 is down-regulated in adipocytes with insulin resistance. The over-expression of AQP7 contributes to improve insulin resistance in adipocytes, which is potentially correlated with the increased phosphorylation of PKB.     

SOCS-3 contributes to bisphenol a exposure-induced insulin resistance in hepatocytes

<正>Dear Editor,Bisphenol A (BPA), a common and widely used monomer in the production of polycarbonate plastics and epoxy resin,(Healy et al., 2015; De Filippis et al., 2018) has been defined as an endocrine disrupting chemical (EDC) associated with the etiology of insulin resistance (IR)(Gioiosa et al., 2015;Wei et al., 2017) and metabolic disorders (Padmanabhan et al., 2010; Fang et al., 2015; Balistrieri et al., 2018). However,     

Coordinated regulation of miR-27 by insulin/CREB/Hippo contributes to insulin resistance

MicroRNA-27 is a critical non-coding metabolic gene that is often aberrantly overexpressed in non-alcoholic fatty livers (NAFLD). However, the pathogenic role of miR-27 in NAFLD remains unknown. In this study, we attempted to identify the mechanism by which miR-27 was regulated in the context of insulin resistance, a predisposed metabolic disorder in NAFLD. Our data from cell culture and animal studies showed that insulin, CREB, and Hippo signalings coordinately regulated miR-27. First, miR-27 was upregulated in palmitate-treated cells and high fat diet-fed mouse livers, which exhibited insulin resistance and CREB overexpression. Second, miR-27 peaked in the mouse liver at the post-absorptive phase when CREB activity was increased. Also, miR-27 was increased rapidly in cell lines when CREB was deactivated by insulin treatment. Third, miR-27 was decreased in cultured cells when CREB was downregulated by siRNA or metformin treatment. In contrast, Forskolin-mediated activation of CREB promoted miR-27 expression. Fourth, Hippo signaling repressed miR-27 in a CREB-independent manner: miR-27 was reduced in cells at full confluence but was inhibited in cells transfected with siRNA against Lats2 and Nf2, which were two positive regulators of Hippo signaling. Lastly, bioinformatics and luciferase assay showed that miR-27 inhibited Akt phosphorylation by targeting Pdpk1 and Pik3r1. Overexpression of miR-27 impaired Akt phosphorylation in cell lines and primary mouse hepatocytes upon insulin stimulation. In conclusion, our data suggest that insulin, CREB, and Hippo signalings contribute to aberrant miR-27 overexpression and eventually lead to insulin resistance in NAFLD.     

Downregulation of Rap1GAP contributes to Ras transformation

Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors.     

Mitochondrial fission contributes to mitochondrial dysfunction and insulin resistance in skeletal muscle

Mitochondrial dysfunction in skeletal muscle has been implicated in the development of insulin resistance and type 2 diabetes. Considering the importance of mitochondrial dynamics in mitochondrial and cellular functions, we hypothesized that obesity and excess energy intake shift the balance of mitochondrial dynamics, further contributing to mitochondrial dysfunction and metabolic deterioration in skeletal muscle. First, we revealed that excess palmitate (PA), but not hyperglycemia, hyperinsulinemia, or elevated tumor necrosis factor alpha, induced mitochondrial fragmentation and increased mitochondrion-associated Drp1 and Fis1 in differentiated C2C12 muscle cells. This fragmentation was associated with increased oxidative stress, mitochondrial depolarization, loss of ATP production, and reduced insulin-stimulated glucose uptake. Both genetic and pharmacological inhibition of Drp1 attenuated PA-induced mitochondrial fragmentation, mitochondrial depolarization, and insulin resistance in C2C12 cells. Furthermore, we found smaller and shorter mitochondria and increased mitochondrial fission machinery in the skeletal muscle of mice with genetic obesity and those with diet-induced obesity. Inhibition of mitochondrial fission improved the muscle insulin signaling and systemic insulin sensitivity of obese mice. Our findings indicated that aberrant mitochondrial fission is causally associated with mitochondrial dysfunction and insulin resistance in skeletal muscle. Thus, disruption of mitochondrial dynamics may underlie the pathogenesis of muscle insulin resistance in obesity and type 2 diabetes.     

Macrophage TNF-alpha contributes to insulin resistance and hepatic steatosis in diet-induced obesity

Obesity is commonly associated with development of insulin resistance and systemic evidence of inflammation. Macrophages contribute to inflammatory amplification in obesity and may contribute directly to insulin resistance and the development of nonalcoholic fatty liver disease through the production of inflammatory cytokines, including tumor necrosis factor (TNF)-alpha. To test this hypothesis, we transplanted male wild-type (WT) and TNF-alpha deficient (KO) mice with either TNF-alpha-sufficient (TNF-alpha(+/+)) or TNF-alpha-deficient (TNF-alpha(-/-)) bone marrow. After consuming a high-fat diet for 26 wk, metabolic and morphometric characteristics of the animals were analyzed. While there were no differences in terms of relative weight gain, body composition analysis yielded a lower relative adipose and higher relative lean mass in mice lacking TNF-alpha, which was partially explained by reduced epididymal fat pad and liver weight. TNF-alpha(-/-) -->KO mice exhibited enhanced insulin sensitivity compared with that observed in TNF-alpha(+/+)-->KO mice; remarkably, no protection against insulin resistance was provided by transplanting TNF-alpha(-/-) bone marrow in WT mice compared with TNF-alpha(+/+)-->WT. The preserved insulin sensitivity seen in TNF-alpha(-/-)-->KO mice provided protection against the development of hepatic steatosis. Taken together, these data indicate that macrophage-derived TNF-alpha contributes to the pattern and extent of fat accumulation and insulin resistance in diet-induced obesity; however, this contribution is negligible in the presence of host-derived TNF-alpha.     

N-acetylcysteine and taurine prevent hyperglycemia-induced insulin resistance in vivo: possible role of oxidative stress

Exposure to high concentrations of glucose and insulin results in insulin resistance of metabolic target tissues, a characteristic feature of type 2 diabetes. High glucose has also been associated with oxidative stress, and increased levels of reactive oxygen species have been proposed to cause insulin resistance. To determine whether oxidative stress contributes to insulin resistance induced by hyperglycemia in vivo, nondiabetic rats were infused with glucose for 6 h to maintain a circulating glucose concentration of 15 mM with and without coinfusion of the antioxidant N-acetylcysteine (NAC), followed by a 2-h hyperinsulinemic-euglycemic clamp. High glucose (HG) induced a significant decrease in insulin-stimulated glucose uptake [tracer-determined disappearance rate (Rd), control 41.2 +/- 1.7 vs. HG 32.4 +/- 1.9 mg. kg-1. min-1, P < 0.05], which was prevented by NAC (HG + NAC 45.9 +/- 3.5 mg. kg-1. min-1). Similar results were obtained with the antioxidant taurine. Neither NAC nor taurine alone altered Rd. HG caused a significant (5-fold) increase in soleus muscle protein carbonyl content, a marker of oxidative stress that was blocked by NAC, as well as elevated levels of malondialdehyde and 4-hydroxynonenal, markers of lipid peroxidation, which were reduced by taurine. In contrast to findings after long-term hyperglycemia, there was no membrane translocation of novel isoforms of protein kinase C in skeletal muscle after 6 h. These data support the concept that oxidative stress contributes to the pathogenesis of hyperglycemia-induced insulin resistance.     

Disruption of diacylglycerol kinase delta (DGKD) associated with seizures in humans and mice

We report a female patient with a de novo balanced translocation, 46,X,t(X;2)(p11.2;q37)dn, who exhibits seizures, capillary abnormality, developmental delay, infantile hypotonia, and obesity. The 2q37 breakpoint observed in association with the seizure phenotype is of particular interest, because it lies near loci implicated in epilepsy in humans and mice. Fluorescence in situ hybridization mapping of the translocation breakpoints showed that no known genes are disrupted at Xp11.2, whereas diacylglycerol kinase delta (DGKD) is disrupted at 2q37. Expression studies in Drosophila and mouse suggest that DGKD is involved in central nervous system development and function. Electroencephalographic assessment of Dgkd mutant mice revealed abnormal epileptic discharges and electrographic seizures in three of six homozygotes. These findings implicate DGKD disruption by the t(X;2)(p11.2;q37)dn in the observed phenotype and support a more general role for DGKD in the etiology of seizures.     

Increased protein synthesis response to insulin in fibroblasts treated with the diacylglycerol kinase inhibitor R59022

Insulin stimulated protein synthesis in quiescent 3T3 fibroblasts. This effect of the hormone was greater in the presence of the diacylglycerol kinase inhibitor R59022 (10(-5) M) over a range of insulin concentrations from 1 microU to 1 mU/ml; R59022 increased the sensitivity of cells to insulin. The amount of radioactive diacylglycerol recovered from cells prelabelled with [3H]glycerol was increased transiently in response to insulin; the response was larger and prolonged in cells given the kinase inhibitor. The results (i) support the hypothesis that diacylglycerol production is part of the signal pathway by which insulin stimulates protein synthesis and (ii) suggest that inhibition of diacylglycerol breakdown leads to increased sensitivity to the hormone.     

Pyruvate dehydrogenase kinase 1 contributes to cisplatin resistance of ovarian cancer through EGFR activation

Patients with ovarian cancer frequently develop acquired drug resistance after the long-term chemotherapy, leading to disease progression. Enhanced epithelial–mesenchymal transition (EMT) has been implicated in chemoresistance of ovarian cancer cells; however, the molecular mechanisms involved are largely undefined. Pyruvate dehydrogenase kinase 1 (PDK1), a key regulatory enzyme in glucose metabolism, has been recognized as a gatekeeper of the Warburg effect, a hallmark of cancer. In this study, the function of PDK1 in cisplatin resistance of ovarian cancer in terms of growth and EMT was investigated. PDK1 was upregulated in cisplatin-resistant ovarian cancer cells. PDK1 knockdown in resistant cells led to increased sensitivity to cisplatin-induced cell death and apoptosis. PDK1 downregulation also reversed the EMT and cell motility in cisplatin-resistant cells. In a mouse xenograft model, tumors derived from PDK1-silenced ovarian cancer cells exhibited decreased tumor growth and EMT compared with control after the cisplatin treatment. Mechanistically, PDK1 overexpression led to increased phosphorylation of EGFR, and blocking EGFR kinase activity by erlotinib reversed cisplatin resistance induced by PDK1 overexpression. Furthermore, in patients with ovarian cancer, higher PDK1 and p-EGFR levels were associated with chemoresistance. These results supported that PDK1 contributes to chemoresistance of ovarian cancer by activating EGFR. Therefore, PDK1 may serve as a promising target to combat chemoresistance of ovarian cancer.     

Up-regulation of Tropomyosin related kinase B contributes to resistance to detachment-induced apoptosis in hepatoma multicellular aggregations

To elucidate the molecular mechanisms of resistance to detachment-induced apoptosis, we cultured BEL7402 hepatoma cells on plates coated with poly (2-hydroxyethyl methacrylate), which blocked access to the extracellular matrix. When BEL7402 hepatoma cells were suspended, they could self-assemble into aggregations and resist to detachment-induced apoptosis. Expression of TrkB on detached cells was much higher than that of attached ones. Protein structure analysis revealed that TrkB contained adhesion domain, which might contribute to the aggregation formation of hepatoma cells. These aggregations had higher proliferation indices with BDNF treatment. These data demonstrate that TrkB may contribute to metastasis by facilitating formation of multicellular aggregations and induce their resistance to detachment-induced apoptosis. Zhiyong Zhang and Lihui Han contributed equally to this work.     

Structure-activity relationship of diacylglycerol kinase theta

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid (PA). Among the nine mammalian isotypes identified, DGKtheta is the only one with three cysteine-rich domains (CRDs) (instead of two) in its N-terminal regulatory region. We previously reported that DGKtheta binds to and is negatively regulated by active RhoA. We now report that RhoA strongly binds to the C-terminal catalytic domain, which would explain its inhibition of DGK activity. To help finding a physiological function of DGKtheta, we further determined its activity in vitro as a function of 15 different truncations and point mutations in the primary structure. Most of these alterations, located throughout the protein, inactivated the enzyme, suggesting that catalytic activity depends on all of its conserved domains. The most C-terminal CRD is elongated with a stretch of 15 amino acids that is highly conserved among DGK isotypes. Mutation analysis revealed a number of residues in this region that were essential for enzyme activity. We suggest that this CRD extension plays an essential role in the correct folding of the protein and/or in substrate presentation to the catalytic region of the protein.     

Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might represent a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent.     

Focal adhesion kinase negatively regulates neuronal insulin resistance

Focal adhesion kinase (FAK), a non-receptor protein kinase, is known to be a phosphatidyl inositol 3-kinase (PI3K) pathway activator and thus widely implicated in regulation of cell survival and cancer. In recent years FAK has also been strongly implicated as a crucial regulator of insulin resistance in peripheral tissues like skeletal muscle and liver, where decrease in its expression/activity has been shown to lead to insulin resistance. However, in the present study we report an altogether different role of FAK in regulation of insulin/PI3K signaling in neurons, the post-mitotic cells. An aberrant increase in FAK tyrosine phosphorylation was observed in insulin resistant Neuro-2a (N2A) cells. Downregulation of FAK expression utilizing RNAi mediated gene silencing in insulin resistant N2A cells completely ameliorated the impaired insulin/PI3K signaling and glucose uptake. FAK silencing in primary cortical neurons also showed marked enhancement in glucose uptake. The results thus suggest that in neurons FAK acts as a negative regulator of insulin/PI3K signaling. Interestingly, the available literature also demonstrates cell-type specific functions of FAK in neurons. FAK that is well known for its cell survival effects has been shown to be involved in neurodegeneration. Along with these previous reports, present findings highlight a novel and critical role of FAK in neurons. Moreover, as this implicates differential regulation of insulin/PI3K pathway by FAK in peripheral tissues and neuronal cells, it strongly suggests precaution while considering FAK modulators as possible therapeutics.