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.

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

OBJECTIVE: To identify glucocorticoid receptor (GR)-associated chromatin sequences and target genes in primary human abdominal subcutaneous fat. METHODS: GR chromatin immunoprecipitation (ChIP)-sequencing (seq) methodology in subcutaneous human adipocytes treated ex vivo with dexamethasone (dex) was optimized to identify genome-wide dex-dependent GR-binding regions (GBRs). Gene expression analyses were performed in parallel ± dex treatment. RESULTS: Fat was obtained from four female surgical patients without obesity with a median age of 50.5 years. ChIP-seq analysis revealed 219 dex-associated GBRs. Of these, 136 GBRs were located within 100 kb of the transcriptional start site and associated with 123 genes. Combining these data with dex-induced gene expression, 70 of the 123 putative direct target genes were significantly up- or downregulated following 4 hours of dex treatment. Gene expression analysis demonstrated that the top 10 pathways reflected regulation of cellular metabolism and inflammation. DEPTOR, an inhibitor of mTOR, was identified as a potential direct GR target gene. CONCLUSIONS: This is the first report of genome-wide GR ChIP-seq and gene expression analysis in human fat. The results implicate regulation of key GR target genes that are involved in dampening inflammation and promoting cellular metabolism.

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.

Serum- and glucocorticoid-induced protein kinase 1 (SGK1) is regulated by store-operated Ca2+ entry and mediates cytoprotection against necrotic cell death.

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 Ca(2+) concentration ([Ca(2+)]c) is required for increased SGK1 expression, but the subcellular source of Ca(2+) 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 Ca(2+) imaging revealed that store-operated Ca(2+) entry played a prominent role in SGK1 induction by TG. Neither ERS nor release of Ca(2+) from the ER was sufficient to activate SGK1. Prolonged elevation of intracellular Ca(2+) 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 Ca(2+) 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 Ca(2+) overloading on mitochondrial membrane potential were significantly reduced in cells expressing SGK1 compared with SGK1-depleted cells. Our findings indicate that store-operated Ca(2+) entry regulates SGK1 expression in epithelial cells and suggest that SGK1-dependent cytoprotective signaling involves effects on maintaining mitochondrial function.

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.

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.

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 individual's 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 individual's 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.

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 20(th) 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.

Escherichia coli O157:H7 requires colonizing partner to adhere and persist in a capillary flow cell.

UNLABELLED: The ability of a strain of waterborne Escherichia coli O157:H7 to colonize a glass flow cell and develop microcolonies when grown alone and with Pseudomonas aeruginosa PAO1 was examined. When introduced alone, planktonic E. coil were unable to attach to the glass surface. When introduced simultaneously with P. aeruginosa (co-inoculation), the two species coadhered to the surface. When E. coliwere introduced into a flow cell precolonized with a P. aeruginosa biofilm (precolonized), 10-fold more cells were retained than in the co-inoculated case. Both species were monitored nondestructively by time-lapse confocal microscopy, direct microscopy of the filtered effluent, and effluent plate counts. While more E. coli initially adhered in the precolonized system, E. coli microcolony formation occurred only in the co-inoculated system, where E. coil comprised 1% of the total surface-associated biovolume but greater than 50% of the biovolume near the edges of the flow cell. The hydrodynamics in the flow cell were evaluated using the finite volume analysis program CFX, revealing that shear stress was likely important in both initial attachment and steady-state colonization patterns. This research elucidates key factors which promote retention and subsequent biofilm development of E. coli 0157:H7. INTRODUCTION: Bacteria exist in nature primarily in communities known as biofilms. These biofilms are usually characterized by differentiated structures, exhibit a different phenotype than their planktonic counterparts, and in nature most often consist of multispecies consortia (1, 2). An important process in shaping the formation and structure of some multispecies biofilms is the ability of certain species to coaggregate. In this process, planktonic cells adhere to genetically distinct cells in a biofilm or to other planktonic cells (3), thereby increasing biofilm formation. This process is growth-phase-dependent and is turned on and off by cells, suggestive that it may also play a role in dispersal and dissemination (4). Due to these and other complexities of the biofilm mode of growth, multiple species can coexist despite one organism having a much higher growth rate than another (5-7). In many cases, bacteria have been shown to gain a fitness advantage when residing in a mixed-species versus single-