Paul A. Volden

Transcription factor GATA4 inhibits doxorubicin-induced autophagy and cardiomyocyte death

Doxorubicin (DOX) is a potent anti-tumor drug known to cause heart failure. The transcription factor GATA4 antagonizes DOX-induced cardiotoxicity. However, the protective mechanism remains obscure. Autophagy is the primary cellular pathway for lysosomal degradation of long-lived proteins and organelles, and its activation could be either protective or detrimental depending on specific pathophysiological conditions. Here we investigated the ability of GATA4 to inhibit autophagy as a potential mechanism underlying its protection against DOX toxicity in cultured neonatal rat cardiomyocytes. DOX markedly increased autophagic flux in cardiomyocytes as indicated by the difference in protein levels of LC3-II (microtubule-associated protein light chain 3 form 2) or numbers of autophagic vacuoles in the absence and presence of the lysosomal inhibitor bafilomycin A1. DOX-induced cardiomyocyte death determined by multiple assays was aggravated by a drug or genetic approach that activates autophagy, but it was attenuated by manipulations that inhibit autophagy, suggesting that autophagy contributes to DOX cardiotoxicity. DOX treatment depleted GATA4 protein levels, which predisposed cardiomyocytes to DOX toxicity. Indeed, GATA4 gene silencing triggered autophagy that rendered DOX more toxic, whereas GATA4 overexpression inhibited DOX-induced autophagy, reducing cardiomyocyte death. Mechanistically, GATA4 up-regulated gene expression of the survival factor Bcl2 and suppressed DOX-induced activation of autophagy-related genes, which may likely be responsible for the anti-apoptotic and anti-autophagic effects of GATA4. Together, these findings suggest that activation of autophagy mediates DOX cardiotoxicity, and preservation of GATA4 attenuates DOX cardiotoxicity by inhibiting autophagy through modulation of the expression of Bcl2 and autophagy-related genes.

Diminished Autophagy Limits Cardiac Injury in Mouse Models of Type 1 Diabetes

Background: Autophagy activity is reduced in type 1 diabetic heart, but the functional role remains unclear. Results: Further reduction in autophagy protects against diabetic heart injury, whereas restoration of autophagy exacerbates cardiac damage. Conclusion: The diminished autophagy limits cardiac dysfunction in type 1 diabetes. Significance: Understanding the functional role of autophagy will facilitate drug design to fight diabetic heart disease. Cardiac autophagy is inhibited in type 1 diabetes. However, it remains unknown if the reduced autophagy contributes to the pathogenesis of diabetic cardiomyopathy. We addressed this question using mouse models with gain- and loss-of-autophagy. Autophagic flux was inhibited in diabetic hearts when measured at multiple time points after diabetes induction by streptozotocin as assessed by protein levels of microtubule-associated protein light chain 3 form 2 (LC3-II) or GFP-LC3 puncta in the absence and presence of the lysosome inhibitor bafilomycin A1. Autophagy in diabetic hearts was further reduced in beclin 1- or Atg16-deficient mice but was restored partially or completely by overexpression of beclin 1 to different levels. Surprisingly, diabetes-induced cardiac damage was substantially attenuated in beclin 1- and Atg16-deficient mice as shown by improved cardiac function as well as reduced levels of oxidative stress, interstitial fibrosis, and myocyte apoptosis. In contrast, diabetic cardiac damage was dose-dependently exacerbated by beclin 1 overexpression. The cardioprotective effects of autophagy deficiency were reproduced in OVE26 diabetic mice. These effects were associated with partially restored mitophagy and increased expression and mitochondrial localization of Rab9, an essential regulator of a non-canonical alternative autophagic pathway. Together, these findings demonstrate that the diminished autophagy is an adaptive response that limits cardiac dysfunction in type 1 diabetes, presumably through up-regulation of alternative autophagy and mitophagy.

PCB 126 and other dioxin-like PCBs specifically suppress hepatic PEPCK expression via the aryl hydrocarbon receptor.

Dioxins and dioxin-like compounds encompass a group of structurally related heterocyclic compounds that bind to and activate the aryl hydrocarbon receptor (AhR). The prototypical dioxin is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a highly toxic industrial byproduct that incites numerous adverse physiological effects. Global commercial production of the structurally similar polychlorinated biphenyls (PCBs), however, commenced early in the 20th century and continued for decades; dioxin-like PCBs therefore contribute significantly to total dioxin-associated toxicity. In this study, PCB 126, the most potent dioxin-like PCB, was evaluated with respect to its direct effects on hepatic glucose metabolism using primary mouse hepatocytes. Overnight treatment with PCB 126 reduced hepatic glycogen stores in a dose-dependent manner. Additionally, PCB 126 suppressed forskolin-stimulated gluconeogenesis from lactate. These effects were independent of acute toxicity, as PCB 126 did not increase lactate dehydrogenase release nor affect lipid metabolism or total intracellular ATP. Interestingly, provision of cells with glycerol instead of lactate as the carbon source completely restored hepatic glucose production, indicating specific impairment in the distal arm of gluconeogenesis. In concordance with this finding, PCB 126 blunted the forskolin-stimulated increase in phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels without affecting glucose-6-phosphatase expression. Myricetin, a putative competitive AhR antagonist, reversed the suppression of PEPCK induction by PCB 126. Furthermore, other dioxin-like PCBs demonstrated similar effects on PEPCK expression in parallel with their ability to activate AhR. It therefore appears that AhR activation mediates the suppression of PEPCK expression by dioxin-like PCBs, suggesting a role for these pollutants as disruptors of energy metabolism.

The influence of glucocorticoid signaling on tumor progression.

The diagnosis of cancer elicits a broad range of well-characterized stress-related biobehavioral responses. Recent studies also suggest that an individuals neuroendocrine stress response can influence tumor biology. One of the major physiological pathways altered by the response to unrelenting social stressors is the hypothalamic-pituitary-adrenal or HPA axis. Initially following acute stress exposure, an increased glucocorticoid response is observed; eventually, chronic stress exposure can lead to a blunting of the normal diurnal cortisol pattern. Interestingly, recent evidence also links high primary tumor glucocorticoid receptor expression (and associated increased glucocorticoid-mediated gene expression) to more rapid estrogen-independent breast cancer progression. Furthermore, animal models of human breast cancer suggest that glucocorticoids inhibit tumor cell apoptosis. These findings provide a conceptual basis for understanding the molecular mechanisms underlying the influence of the individuals stress response, and specifically glucocorticoid action, on breast cancer and other solid tumor biology. How this increased glucocorticoid signaling might contribute to cancer progression is the subject of this review.

Chronic Social Isolation Is Associated with Metabolic Gene Expression Changes Specific to Mammary Adipose Tissue

Chronic social isolation is linked to increased mammary tumor growth in rodent models of breast cancer. In the C3(1)/SV40 T-antigen FVB/N (TAg) mouse model of “triple-negative” breast cancer, the heightened stress response elicited by social isolation has been associated with increased expression of metabolic genes in the mammary gland before invasive tumors develop (i.e., during the in situ carcinoma stage). To further understand the mechanisms underlying how accelerated mammary tumor growth is associated with social isolation, we separated the mammary gland adipose tissue from adjacent ductal epithelial cells and analyzed individual cell types for changes in metabolic gene expression. Specifically, increased expression of the key metabolic genes Acaca, Hk2, and Acly was found in the adipocyte, rather than the epithelial fraction. Surprisingly, metabolic gene expression was not significantly increased in visceral adipose depots of socially isolated female mice. As expected, increased metabolic gene expression in the mammary adipocytes of socially isolated mice coincided with increased glucose metabolism, lipid synthesis, and leptin secretion from this adipose depot. Furthermore, application of media that had been cultured with isolated mouse mammary adipose tissue (conditioned media) resulted in increased proliferation of mammary cancer cells relative to group-housed–conditioned media. These results suggest that exposure to a chronic stressor (social isolation) results in specific metabolic reprogramming in mammary gland adipocytes that in turn contributes to increased proliferation of adjacent preinvasive malignant epithelial cells. Metabolites and/or tumor growth-promoting proteins secreted from adipose tissue could identify biomarkers and/or targets for preventive intervention in breast cancer. Cancer Prev Res; 6(7); 634–45. ©2013 AACR.

Mammary Adipose Tissue-Derived Lysophospholipids Promote Estrogen Receptor–Negative Mammary Epithelial Cell Proliferation

Lysophosphatidic acid (LPA), acting in an autocrine or paracrine fashion through G protein–coupled receptors, has been implicated in many physiologic and pathologic processes, including cancer. LPA is converted from lysophosphatidylcholine (LPC) by the secreted phospholipase autotaxin (ATX). Although various cell types can produce ATX, adipocyte-derived ATX is believed to be the major source of circulating ATX and also to be the major regulator of plasma LPA levels. In addition to ATX, adipocytes secrete numerous other factors (adipokines); although several adipokines have been implicated in breast cancer biology, the contribution of mammary adipose tissue–derived LPC/ATX/LPA (LPA axis) signaling to breast cancer is poorly understood. Using murine mammary fat-conditioned medium, we investigated the contribution of LPA signaling to mammary epithelial cancer cell biology and identified LPA signaling as a significant contributor to the oncogenic effects of the mammary adipose tissue secretome. To interrogate the role of mammary fat in the LPA axis during breast cancer progression, we exposed mammary adipose tissue to secreted factors from estrogen receptor–negative mammary epithelial cell lines and monitored changes in the mammary fat pad LPA axis. Our data indicate that bidirectional interactions between mammary cancer cells and mammary adipocytes alter the local LPA axis and increase ATX expression in the mammary fat pad during breast cancer progression. Thus, the LPC/ATX/LPA axis may be a useful target for prevention in patients at risk of ER-negative breast cancer. Cancer Prev Res; 9(5); 367–78. ©2016 AACR.

Serum- and Glucocorticoid-induced Protein Kinase 1 (SGK1) is Regulated by Store-Operated Ca2+ Entry and Mediates Cytoprotection Against Necrotic Cell Death

Background: Mechanisms underlying SGK1 activation are incompletely understood in epithelial cells. Results: Store-operated Ca2+ entry up-regulates SGK1, thereby modulating the lethal effects of Ca2+ overloading on mitochondrial membrane potential. Conclusion: Ca2+-induced SGK1 activates cytoprotective signaling and modifies mitochondrial function in epithelial cells. Significance: This work reveals a cytoprotective role for SGK1 in necrosis and has potential relevance for epithelial cell protection and cancer treatment. Serum and glucocorticoid-regulated kinase 1 (SGK1) encodes a phosphatidylinositol 3-kinase-dependent serine/threonine kinase that is rapidly induced in response to cellular stressors and is an important cell survival signal. Previous studies have suggested that an increase in cytoplasmic Ca2+ concentration ([Ca2+]c) is required for increased SGK1 expression, but the subcellular source of Ca2+ regulating SGK1 transcription remains uncertain. Activation of endoplasmic reticulum stress (ERS) with thapsigargin (TG) increased SGK1 mRNA and protein expression in MDA-MB-231 cells. Intracellular Ca2+ imaging revealed that store-operated Ca2+ entry played a prominent role in SGK1 induction by TG. Neither ERS nor release of Ca2+ from the ER was sufficient to activate SGK1. Prolonged elevation of intracellular Ca2+ levels, however, triggered cell death with a much greater proportion of the cells undergoing necrosis rather than apoptosis. A relative increase in the percentage of cells undergoing necrosis was observed in cells expressing a short hairpin RNA targeted to the SGK1 gene. Necrotic cell death evoked by cytoplasmic Ca2+ overloading was associated with persistent hyperpolarization of the inner mitochondrial membrane and a modest increase in calpain activation, but did not involve detectable caspase 3 or caspase 7 activation. The effects of cytoplasmic Ca2+ overloading on mitochondrial membrane potential were significantly reduced in cells expressing SGK1 compared with SGK1-depleted cells. Our findings indicate that store-operated Ca2+ entry regulates SGK1 expression in epithelial cells and suggest that SGK1-dependent cytoprotective signaling involves effects on maintaining mitochondrial function.

Glucocorticoid receptor ChIP-sequencing of subcutaneous fat reveals modulation of inflammatory pathways.

To identify glucocorticoid receptor (GR)‐associated chromatin sequences and target genes in primary human abdominal subcutaneous fat.

Aliphatic chain length by isotropic mixing (ALCHIM): determining composition of complex lipid samples by 1H NMR spectroscopy

Quantifying the amounts and types of lipids present in mixtures is important in fields as diverse as medicine, food science, and biochemistry. Nuclear magnetic resonance (NMR) spectroscopy can quantify the total amounts of saturated and unsaturated fatty acids in mixtures, but identifying the length of saturated fatty acid or the position of unsaturation by NMR is a daunting challenge. We have developed an NMR technique, aliphatic chain length by isotropic mixing, to address this problem. Using a selective total correlation spectroscopy technique to excite and transfer magnetization from a resolved resonance, we demonstrate that the time dependence of this transfer to another resolved site depends linearly on the number of aliphatic carbons separating the two sites. This technique is applied to complex natural mixtures allowing the identification and quantification of the constituent fatty acids. The method has been applied to whole adipocytes demonstrating that it will be of great use in studies of whole tissues.

Refeeding-Induced Brown Adipose Tissue Glycogen Hyper-Accumulation in Mice Is Mediated by Insulin and Catecholamines

Brown adipose tissue (BAT) generates heat during adaptive thermogenesis through a combination of oxidative metabolism and uncoupling protein 1-mediated electron transport chain uncoupling, using both free-fatty acids and glucose as substrate. Previous rat-based work in 1942 showed that prolonged partial fasting followed by refeeding led to a dramatic, transient increase in glycogen stores in multiple fat depots. In the present study, the protocol was replicated in male CD1 mice, resulting in a 2000-fold increase in interscapular BAT (IBAT) glycogen levels within 4–12 hours (hr) of refeeding, with IBAT glycogen stores reaching levels comparable to fed liver glycogen. Lesser effects occurred in white adipose tissues (WAT). Over the next 36 hr, glycogen levels dissipated and histological analysis revealed an over-accumulation of lipid droplets, suggesting a potential metabolic connection between glycogenolysis and lipid synthesis. 24 hr of total starvation followed by refeeding induced a robust and consistent glycogen over-accumulation similar in magnitude and time course to the prolonged partial fast. Experimentation demonstrated that hyperglycemia was not sufficient to drive glycogen accumulation in IBAT, but that elevated circulating insulin was sufficient. Additionally, pharmacological inhibition of catecholamine production reduced refeeding-induced IBAT glycogen storage, providing evidence of a contribution from the central nervous system. These findings highlight IBAT as a tissue that integrates both canonically-anabolic and catabolic stimulation for the promotion of glycogen storage during recovery from caloric deficit. The preservation of this robust response through many generations of animals not subjected to food deprivation suggests that the over-accumulation phenomenon plays a critical role in IBAT physiology.