Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G

BACKGROUND: In type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved. METHODS: Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated. RESULTS: The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial β-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid β-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-indpendent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis. CONCLUSIONS: Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA

Excavation of Silenced Voices: (Re)visiting Menka Shivdasani’s Frazil through the Modern Feminist Discourse of Indian Writing in English

The postmodernist phase of Indian English writing is characterized by the voices of many strong women expressing a feminist exploration of alternative discourses in women’s writing which are distinguished from the patriarchal framework of literary discourse. Along with Kamala Das, Meena Alexander, Imtiaz Dharkar, and Eunice de Souza, Menka Shivdasani is an active voice in contemporary Indian English poetry. Shivdasani is a prolific poet who has written poetry on various social, cultural, religious, and personal issues. Her four poetry collections include Nirvana at Ten Rupees (1990), Stet (2001), Safe House (2015), and Frazil (2018). Through her poetry, she has endeavored to deconstruct the constructed nature of women in patriarchal societies. However, in literary criticism, she has not been explored in detail; thus, Shivdasani deserves to be better known. She is truly an unsung voice in Indian English poetry because of her poetic excellence. The present paper will attempt to (re)visit Shivdasani’s work with particular reference to her recent poetry collection Frazil. The themes it depicts are women’s sensibilities, man-woman relationships, domesticity, myth, culture, religion, memory, loss, and the anxiety of city life. Therefore, the paper will analyze the thematic and structural aspects of her poetry, with assistance from the work of Menka Shivdasani, to move Shivdasani onto the center stage in the postmodernist phase of Indian writing in English

(A) Cells were treated with control (Con) or palmitate (Palm) in the presence and absence of the cell permeable caspase 9 inhibitor (C9I, Z-LEHD-FMK) or the caspase 8 inhibitor (C8I, Z-IETD-FMK), both at 40 μM

<p><b>Copyright information:</b></p><p>Taken from "Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G"</p><p>Cardiovascular Diabetology 2005;4():2-2.</p><p>Published online 10 Jan 2005</p><p>PMCID:PMC546189.</p><p>Copyright © 2005 Mishra and Simonson; licensee BioMed Central Ltd.</p> Addition of the caspase inhibitors was concurrent with addition of the FFA. After 24 and 48 h, caspase 9 enzyme activity was measured and expressed as fold-change over control. (B) Palmitate and Z-LEHD-FMK were added to HMC as above, and cleavage of caspase-3 was measured by ELISA as described in Experimental Procedures. (C) The number of pyknotic nuclei was assessed in cells treated with palmitate with and without Z-LEHD-FMK. (D) DNA fragmentation, assessed by ELISA as the number of nucleosomal fragments in the cytosol, was measured in cells treated with palmitate and by Z-LEHD-FMK. For A-D, data are mean ± SEM for 3 independent experiments. **, < 0.01, *, < 0.05 versus control by ANOVA in (A, B and D) or Chi-square test in (C)

(A) HMC were incubated with palmitate (Palm) and palmitate plus the Z-LEHD-FMK caspase 9 inhibitor (C9I, 40 μM) for the times indicated

<p><b>Copyright information:</b></p><p>Taken from "Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G"</p><p>Cardiovascular Diabetology 2005;4():2-2.</p><p>Published online 10 Jan 2005</p><p>PMCID:PMC546189.</p><p>Copyright © 2005 Mishra and Simonson; licensee BioMed Central Ltd.</p> The cells were fractionated and the level of p33 endonuclease G protein was measured by Western blotting in the cytoplasmic fraction. Equivalent amounts of protein were added to each lane, as confirmed by reprobing with β-actin. (B) Densitometry of cytoplasmic p33 kDa endonuclease G protein from 3 independent experiments. **, < 0.01, *, < 0.05 by ANOVA versus 0 time

IL-13 induces periostin and eotaxin expression in human primary alveolar epithelial cells: Comparison with paired airway epithelial cells.

Alveolar epithelial cells are critical to the pathogenesis of pulmonary inflammation and fibrosis, which are associated with overexpression of type 2 cytokine IL-13. IL-13 is known to induce the production of profibrotic (e.g., periostin) and pro-inflammatory (e.g., eotaxin-3) mediators in human airway epithelial cells, but it remains unclear if human primary alveolar epithelial cells increase periostin and eotaxin expression following IL-13 stimulation. The goals of this study are to determine if alveolar epithelial cells increase periostin and eotaxin expression upon IL-13 stimulation, and if alveolar and airway epithelial cells from the same subjects have similar responses to IL-13. Paired alveolar and airway epithelial cells were isolated from donors without any lung disease, and cultured under submerged or air-liquid interface conditions with or without IL-13. Up-regulation of periostin protein and mRNA was observed in IL-13-stimulated alveolar epithelial cells, which was comparable to that in IL-13-stimulated paired airway epithelial cells. IL-13 also increased eotaxin-3 expression in alveolar epithelial cells, but the level of eotaxin mRNA was lower in alveolar epithelial cells than in airway epithelial cells. Our findings demonstrate that human alveolar epithelial cells are able to produce periostin and eotaxin in responses to IL-13 stimulation. This study suggests the need to further determine the contribution of alveolar epithelial cell-derived mediators to pulmonary fibrosis