Browsing by Subject "palmitate"

Now showing 1 - 3 of 3
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    FASN Negatively Regulates NF-kB/P65 Expression in Breast Cancer Cells by Disrupting Its Stability
    (2020-02) Barlow, Lincoln James; Lu, Tao; Zhang, Jian-Ting; Fehrenbacher, Jill; Herbert, Brittney-Shea; Safa, Ahmad
    The overexpression of the multi-domain enzyme fatty acid synthase (FASN) has long been associated with poor clinical prognosis and treatment outcome in various cancers. Previous research in the Zhang lab has determined a role for FASN in mediating increases in non-homologous end-joining (NHEJ) DNA double-strand break repair activity allowing for increased cancer cell survival, and this mechanism was found to involve inhibition of NF-kB/p65. The mechanism responsible for the regulation of NF-kB/p65 by FASN in cancer cells, however, remains unknown. To this end, I was able to determine that FASN negatively regulates both the expression and activity of NF-kB/p65 in breast cancer cells, and that this effect was likely mediated by the 16-carbon saturated fatty acid palmitate, the end product of FASN catalytic activity. Specifically, FASN was found to negatively regulate p65 expression by disrupting its protein stability as a result of an increase in poly-ubiquitination of p65 protein and subsequent proteasomal degradation. Further, I found that the phosphorylation site Thr254 of p65 is involved in the regulation of p65 protein stability by FASN, in that mutation of this residue resulted in a disruption in p65 stability. Finally, I was able to determine that FASN likely inhibits the ability of the peptidyl-prolyl cis/trans isomerase Pin1 to assist in maintaining p65 stability, in that both siRNA knockdown and pharmacological inhibition of Pin1 resulted in a reduction of p65 expression in FASN shRNA knockdown cells. The determination of this signaling mechanism serves to expand our understanding of the role of FASN in breast cancer cells and has the potential to assist in uncovering more effective ways to target the oncogenic FASN pathway to kill breast tumor cells and to overcome resistance to drug treatment.
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    Novel Roles of p21 in Apoptosis During Beta-Cell Stress in Diabetes
    (2014) Hernández-Carretero, Angelina M.; Fueger, Patrick T.; Sturek, Michael Stephen; Wek, Ronald C.; Evans-Molina, Carmella; Elmendorf, Jeffrey S.
    Type 2 diabetes manifests from peripheral insulin resistance and a loss of functional beta cell mass due to decreased beta cell function, survival, and/or proliferation. Beta cell stressors impair each of these factors by activating stress response mechanisms, including endoplasmic reticulum (ER) stress. The glucolipotoxic environment of the diabetic milieu also activates a stress response in beta cells, resulting in death and decreased survival. Whereas the cell cycle machinery (comprised of cyclins, kinases, and inhibitors) regulates proliferation, its involvement during beta cell stress in the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two independent pharmacologic inducers of beta cell stress - dexamethasone and thapsigargin. In addition, glucolipotoxic stress mimicking the diabetic milieu also induced p21. To further investigate p21’s role in the beta cell, p21 was adenovirally overexpressed in 832/13 cells and rat islets. As expected given p21’s role as a cell cycle inhibitor, p21 overexpression decreased [3H]-thymidine incorporation and blocked the G1/S and G2/M transitions as quantified by flow cytometry. Interestingly, p21 overexpression activated apoptosis, demonstrated by increased annexin- and propidium iodide-double-positive cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the anti-apoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the pro-apoptotic proteins Bax and Bak. Therefore, the intrinsic apoptotic pathway is central for p21-mediated cell death. Like glucolipotoxicity, p21 overexpression inhibited the insulin cell survival signaling pathway while also impairing glucose-stimulated insulin secretion, an index of beta cell function. Under both conditions, phosphorylation of insulin receptor substrate-1, Akt, and Forkhead box protein-O1 was reduced. p21 overexpression increased Bim and c-Jun N-terminal Kinase, however, siRNA-mediated reduction or inhibition of either protein, respectively, did not alter p21-mediated cell death. Importantly, islets of p21-knockout mice treated with the ER stress inducer thapsigargin displayed a blunted apoptotic response. In summary, our findings indicate that p21 decreases proliferation, activates apoptosis, and impairs beta cell function, thus being a potential target to inhibit for the protection of functional beta cell mass.
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    SIRT6 protects against palmitate-induced pancreatic β-cell dysfunction and apoptosis
    (BioScientifica, 2016-11) Xiong, Xiwen; Sun, Xupeng; Wang, Qingzhi; Qian, Xinlai; Zhang, Yang; Pan, Xiaoyan; Dong, X. Charlie; Biochemistry and Molecular Biology, School of Medicine
    Chronic exposure of pancreatic β-cells to abnormally elevated levels of free fatty acids can lead to β-cell dysfunction and even apoptosis, contributing to type 2 diabetes pathogenesis. In pancreatic β-cells, SIRT6 has been shown to regulate insulin secretion in response to glucose stimulation. However, what roles SIRT6 play in β-cells in response to lipotoxicity remain poorly understood. Our data indicated that SIRT6 protein and mRNA levels were reduced in islets from diabetic and aged mice. High concentrations of palmitate also led to a decrease in SIRT6 expression in MIN6 β-cells and resulted in cell dysfunction and apoptosis. Knockdown of Sirt6 caused an increase in cell apoptosis and impairment in insulin secretion in response to glucose in MIN6 cells even in the absence of high palmitate. Furthermore, overexpression of SIRT6 alleviated the palmitate-induced lipotoxicity with improved cell viability and increased glucose-stimulated insulin secretion. In summary, our data suggest that SIRT6 can protect against palmitate-induced β-cell dysfunction and apoptosis.