Using the Synergy between HPLC-MS and MALDI-MS Imaging to Explore the Lipidomics of Clear Cell Renal Cell Carcinoma.

Lipid imaging mass spectrometry (LIMS) has been tested in several pathological contexts, demonstrating its ability to segregate and isolate lipid signatures in complex tissues, thanks to the technique's spatial resolution. However, it cannot yet compete with the superior identification power of high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS), and therefore, very often, the latter is used to refine the assignment of the species detected by LIMS. Also, it is not clear if the differences in sensitivity and spatial resolution between the two techniques lead to a similar panel of biomarkers for a given disease. Here, we explore the capabilities of LIMS and HPLC-MS to produce a panel of lipid biomarkers to screen nephrectomy samples from 40 clear cell renal cell carcinoma patients. The same set of samples was explored by both techniques, and despite the important differences between them in terms of the number of detected and identified species (148 by LIMS and 344 by HPLC-MS in negative-ion mode) and the presence/absence of image capabilities, similar conclusions were reached: using the lipid fingerprint, it is possible to set up classifiers that correctly identify the samples as either healthy or tumor samples. The spatial resolution of LIMS enables extraction of additional information, such as the existence of necrotic areas or the existence of different tumor cell populations, but such information does not seem determinant for the correct classification of the samples, or it may be somehow compensated by the higher analytical power of HPLC-MS. Similar conclusions were reached with two very different techniques, validating their use for the discovery of lipid biomarkers.

Molecular and cellular insights into the role of SND1 in lipid metabolism.

Staphylococcal nuclease and Tudor domain containing 1 (SND1) is an evolutionarily conserved protein present in eukaryotic cells from protozoa to mammals. SND1 has gained importance because it is overexpressed in aggressive cancer cells and diverse primary tumors. Indeed, it is regarded as a marker of cancer malignity. A broad range of molecular functions and the participation in many cellular processes have been attributed to SND1, mostly related to the regulation of gene expression. An increasing body of evidence points to a relevant relationship between SND1 and lipid metabolism. In this review, we summarize the knowledge about SND1 and its molecular and functional relationship with lipid metabolism. We highlight that SND1 plays a direct role in the regulation of cholesterol metabolism by affecting the activation of sterol response element-binding protein 2 (SREBP2) and we propose that that might have implications in the response of lipid homeostasis to stress situations.

Channeling of newly synthesized fatty acids to cholesterol esterification limits triglyceride synthesis in SND1-overexpressing hepatoma cells.

SND1 is a putative oncoprotein whose molecular function remains unclear. Its overexpression in hepatocellular carcinoma impairs cholesterol homeostasis due to the altered activation of the sterol regulatory element-binding protein (SREBP) 2, which results in the accumulation of cellular cholesteryl esters (CE). In this work, we explored whether high cholesterol synthesis and esterification originates changes in glycerolipid metabolism that might affect cell growth, given that acetyl-coenzyme A is required for cholesterogenesis and fatty acids (FA) are the substrates of acyl-coenzyme A:cholesterol acyltransferase (ACAT). SND1-overexpressing hepatoma cells show low triglyceride (TG) synthesis, but phospholipid biosynthesis or cell growth is not affected. Limited TG synthesis is not due to low acetyl-coenzyme A or NADPH availability. We demonstrate that the main factor limiting TG synthesis is the utilization of FAs for cholesterol esterification. These metabolic adaptations are linked to high Scd1 expression, needed for the de novo production of oleic acid, the main FA used by ACAT. We conclude that high cholesterogenesis due to SND1 overexpression might determine the channeling of FAs to CEs.

High-fat diet overfeeding promotes nondetrimental liver steatosis in female mice.

High-fat diet (HFD) feeding or leptin-deficient mice are extensively used as models resembling features of human nonalcoholic fatty liver disease (NAFLD). The concurrence of experimental factors as fat content and source or total caloric intake leads to prominent differences in the development of the hepatic steatosis and related disturbances. In this work, we characterized the hepatic lipid accumulation induced by HFD in wild-type (WT) and ob/ ob mice with the purpose of differentiating adaptations to HFD from those specific of increased overfeeding due to leptin deficiency-associated hyperphagia. Given that most published works have been done in male models, we used female mice with the aim of increasing the body of evidence regarding NAFLD in female subjects. HFD promoted liver lipid accumulation only in the hyperphagic strain. Nevertheless, a decrease of lipid droplet-associated cholesteryl ester (CE) in both WT and obese animals was observed. These changes were accompanied by an improvement in the profile of lipoproteins that transport cholesterol and liver function markers in plasma from ob/ ob mice and a lower hepatic index. Using primary hepatocytes from female mice, overaccumulation of CE induced by 0.4 mM oleic acid reversed in the presence of a specific Takeda G protein-coupled bile acid receptor agonist. Nevertheless, hepatocytes from male mice were not responsive. This study suggests that enterohepatic circulation of bile acids might be one of the factors that can affect sex dimorphism in NAFLD development, which underlines the importance of including female models in the NAFLD research field. NEW & NOTEWORTHY This work provides new insight into the use of high-fat diet as a model to induce nonalcoholic fatty liver disease in wild-type and ob/ ob female mice. We show that high-fat diet induces steatosis only in ob/ ob mice while, surprisingly, several health indicators improve. Noteworthy, experiments with primary hepatocytes from male and female mice show that they express Takeda G protein-coupled bile acid receptor and that it and bile acid enterohepatic circulation might be accountable for sex dimorphism in nonalcoholic fatty liver disease development.

Adenosine: Direct and Indirect Actions on Gastric Acid Secretion.

Composed by a molecule of adenine and a molecule of ribose, adenosine is a paradigm of recyclable nucleoside with a multiplicity of functions that occupies a privileged position in the metabolic and regulatory contexts. Adenosine is formed continuously in intracellular and extracellular locations of all tissues. Extracellular adenosine is a signaling molecule, able to modulate a vast range of physiologic responses in many cells and organs, including digestive organs. The adenosine A1, A2A, A2B, and A3 receptors are P1 purinergic receptors, G protein-coupled proteins implicated in tissue protection. This review is focused on gastric acid secretion, a process centered on the parietal cell of the stomach, which contains large amounts of H+/K+-ATPase, the proton pump responsible for proton extrusion during acid secretion. Gastric acid secretion is regulated by an extensive collection of neural stimuli and endocrine and paracrine agents, which act either directly at membrane receptors of the parietal cell or indirectly through other regulatory cells of the gastric mucosa, as well as mechanic and chemic stimuli. In this review, after briefly introducing these points, we condense the current body of knowledge about the modulating action of adenosine on the pathophysiology of gastric acid secretion and update its significance based on recent findings in gastric mucosa and parietal cells in humans and animal models.

Expression of Adenosine A2B Receptor and Adenosine Deaminase in Rabbit Gastric Mucosa ECL Cells.

Adenosine is readily available to the glandular epithelium of the stomach. Formed continuously in intracellular and extracellular locations, it is notably produced from ATP released in enteric cotransmission. Adenosine analogs modulate chloride secretion in gastric glands and activate acid secretion in isolated parietal cells through A2B adenosine receptor (A2BR) binding. A functional link between surface A2BR and adenosine deaminase (ADA) was found in parietal cells, but whether this connection is a general feature of gastric mucosa cells is unknown. Here we examine whether A2BR is expressed at the membrane of histamine-producing enterochromaffin-like (ECL) cells, the major endocrine cell type in the oxyntic mucosa, and if so, whether it has a vicinity relationship with ADA. We used a highly homogeneous population of rabbit ECL cells (size 7.5-10 µm) after purification by elutriation centrifugation. The surface expression of A2BR and ADA proteins was assessed by flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are partially coexpressed at the gastric ECL cell surface and that A2BR is functional, with regard to binding of adenosine analogs and adenylate cyclase activation. The physiological relevance of A2BR and ADA association in regulating histamine release is yet to be explained.

SND1 overexpression deregulates cholesterol homeostasis in hepatocellular carcinoma.

SND1 is a multifunctional protein participating, among others, in gene transcription and mRNA metabolism. SND1 is overexpressed in cancer cells and promotes viability and tumourigenicity of hepatocellular carcinoma cells. This study shows that cholesterol synthesis is increased in SND1-overexpressing hepatoma cells. Neither newly synthesised nor extracellularly supplied cholesterol are able to suppress this increase; however, inhibition of cholesterol esterification reverted the activated state of sterol-regulatory element-binding protein 2 (SREBP2) and cholesterogenesis. These results highlight SND1 as a potential regulator of cellular cholesterol distribution and homeostasis in hepatoma cells, and support the rationale for the therapeutic use of molecules that influence cholesterol management when SND1 is overexpressed.

Stimulation of gastric acid secretion by rabbit parietal cell A(2B) adenosine receptor activation.

Adenosine modulates different functional activities in many cells of the gastrointestinal tract; some of them are believed to be mediated by interaction with its four G protein-coupled receptors. The renewed interest in the adenosine A2B receptor (A2BR) subtype can be traced by studies in which the introduction of new genetic and chemical tools has widened the pharmacological and structural knowledge of this receptor as well as its potential therapeutic use in cancer and inflammation- or hypoxia-related pathologies. In the acid-secreting parietal cells of the gastric mucosa, the use of various radioligands for adenosine receptors suggested the presence of the A2 adenosine receptor subtype(s) on the cell surface. Recently, we confirmed A2BR expression in native, nontransformed parietal cells at rest by using flow cytometry and confocal microscopy. In this study, we show that A2BR is functional in primary rabbit gastric parietal cells, as indicated by the fact that agonist binding to A2BR increased adenylate cyclase activity and acid production. In addition, both acid production and radioligand binding of adenosine analogs to isolated cell membranes were potently blocked by selective A2BR antagonists, whereas ligands for A1, A2A, and A3 adenosine receptors failed to abolish activation. We conclude that rabbit gastric parietal cells possess functional A2BR proteins that are coupled to Gs and stimulate HCl production upon activation. Whether adenosine- and A2BR-mediated functional responses play a role in human gastric pathophysiology is yet to be elucidated.

Infection of primary hepatocytes with adenoviral vectors alters biliary lipid metabolism.

In the context of a study of the involvement of SND1 (also known as coactivator p100) in biliary lipid secretion by primary rat hepatocytes, first-generation adenoviral vectors were used to promote the overexpression and underexpression of the protein SND1. Although differential expression of SND1 did not result in significant changes in the processes studied, some effects of the adenoviral infection itself were observed. In particular, infected hepatocytes showed a higher intracellular taurocholate accumulation capacity. Additionally, small heterodimer partner (SHP) and farnesoid X receptor (FXR), which are nuclear receptors essential for the regulation of bile salt metabolism and transport, were underregulated at the mRNA level. Our results suggest that adenoviral vectors could be altering some important control mechanism and indicate that adenoviral vectors should be used with caution as transfection vectors for hepatocytes when biliary lipid metabolism is to be studied.