Mechanisms for and consequences of cellular lipid accumulation - Role of the Very Low Density Lipoprotein (VLDL) receptor

Lipid accumulation in non adipose tissue is associated with various cases of tissue dysfunction and tissue failure. Reduced availability of oxygen is known to cause intracellular lipid accumulation in cardiomyocytes as well as in hearts. Cardiac lipid accumulation has been shown to cause impaired cardiac function but it is not fully clear how the lipids accumulate in the hypoxic myocardium. We have studied a model of hypoxic/ischemic myocardium using HL-1 cardiomyocytes incubated in hypoxic condition as well as an in vivo model where mice were subjected to a myocardial infarction causing cardiac ischemia. We found that the Very low density lipoprotein receptor (VLDLr), a member of the low density lipoprotein receptor (LDLr) family suggested to be able to mediate uptake of lipids, was significantly upregulated in response to hypoxia and that this upregulation was mediated through hypoxic activation of transcription factor Hif-1α. The VLDLr induced an increase in intracellular triglycerides which were mediated not primarily through increased uptake of fatty acids but from an increased uptake of extracellular triglyceride-rich lipoproteins. The uptake of lipoproteins was rapid in response to hypoxia. The increase in intracellular lipids caused an accumulation of cardiotoxic ceramides in the cardiomyocytes which induced myocardial ER-stress. ER-stress initially induces a cardioprotective response but prolonged ER-stress cause apoptosis which was increased when the VLDLr was expressed. Ablation of the VLDLr reduced the ER-stress. The mice lacking VLDLr expression showed a reduced infarct size which could be dependent on a reduced amount of toxic ceramides and apoptosis. We could also show that it was possible to block the harmful actions of the VLDLr by using VLDLr specific antibodies. Treatment with these antibodies reduced the lipid accumulation, ER-stress and apoptosis otherwise following a myocardial infarction. The hypoxic VLDLr expression is not restricted to species or tissue. We could see that the VLDLr was increased in human ischemic myocardium compared to non-ischemic biopsies. We could also see that the VLDLr expression was increased in human clear-cell renal carcinoma where in this case the increased VLDLr expression was not due to hypoxia but on constitutive Hif-1α activation. Like in the myocardium the VLDLr caused an accumulation of intracellular triglyceride in the cancer, which already contained great amounts of cholesterol esters. These results indicate that the VLDLr is an important mediator of post-ischemic intramyocardial lipid accumulation and that the blocking of this lipid uptake improves survival

High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry

Technological advances in mass spectrometry and meticulous method development have produced several shotgun lipidomic approaches capable of characterizing lipid species by direct analysis of total lipid extracts. Shotgun lipidomics by hybrid quadrupole time-of-flight mass spectrometry allows the absolute quantification of hundreds of molecular glycerophospholipid species, glycerolipid species, sphingolipid species and sterol lipids. Future applications in clinical cohort studies demand detailed lipid molecule information and the application of high-throughput lipidomics platforms. In this review we describe a novel high-throughput shotgun lipidomic platform based on 96-well robot-assisted lipid extraction, automated sample infusion by mircofluidic-based nanoelectrospray ionization, and quantitative multiple precursor ion scanning analysis on a quadrupole time-of-flight mass spectrometer. Using this platform to compile comprehensive lipid arrays associated with metabolic dysfunctions is a powerful strategy for pinpointing the mechanistic details by which alterations in tissue-specific lipid metabolism are directly linked to the etiology of many lipid-mediated disorders

Increased expression of the very low-density lipoprotein receptor mediates lipid accumulation in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinoma (RCC) is, in most cases, caused by loss of function of the tumor suppressor gene von Hippel-Lindau, resulting in constitutive activation of hypoxia-inducible factor (HIF)-1α and expression of hypoxia-induced genes in normoxic conditions. Clear-cell RCC cells are characterized histologically by accumulation of cholesterol, mainly in its ester form. The origin of the increased cholesterol remains unclear, but it is likely explained by an HIF-1α-driven imbalance between cholesterol uptake and excretion. Here, we showed that expression of the very low-density lipoprotein receptor (VLDL-R) was significantly increased in clear-cell RCC human biopsies compared with normal kidney tissue. Partial knockdown of HIF-1α in clear-cell RCC cells significantly reduced the VLDL-R expression, and knockdown of either HIF-1α or VLDL-R reduced the increased lipid accumulation observed in these cells. We also showed increased uptake of fluorescently labeled lipoproteins in clear-cell RCC cells, which was significantly reduced by knockdown of HIF-1α or VLDL-R. Taken together, our results support the concept that the pathological increase of HIF-1α in clear-cell RCC cells upregulates VLDL-R, which mediates increased uptake and accumulation of lipids. These results explain the morphological characteristics of clear-cell RCC, and open up novel possibilities for detection and treatment of clear-cell RCC

Cholesteryl Esters Accumulate in the Heart in a Porcine Model of Ischemia and Reperfusion

Myocardial ischemia is associated with intracellular accumulation of lipids and increased depots of myocardial lipids are linked to decreased heart function. Despite investigations in cell culture and animal models, there is little data available on where in the heart the lipids accumulate after myocardial ischemia and which lipid species that accumulate. The aim of this study was to investigate derangements of lipid metabolism that are associated with myocardial ischemia in a porcine model of ischemia and reperfusion. The large pig heart enables the separation of the infarct area with irreversible injury from the area at risk with reversible injury and the unaffected control area. The surviving myocardium bordering the infarct is exposed to mild ischemia and is stressed, but remains viable. We found that cholesteryl esters accumulated in the infarct area as well as in the bordering myocardium. In addition, we found that expression of the low density lipoprotein receptor (LDLr) and the low density lipoprotein receptor-related protein 1 (LRP1) was up-regulated, suggesting that choleteryl ester uptake is mediated via these receptors. Furthermore, we found increased ceramide accumulation, inflammation and endoplasmatic reticulum (ER) stress in the infarcted area of the pig heart. In addition, we found increased levels of inflammation and ER stress in the myocardium bordering the infarct area. Our results indicate that lipid accumulation in the heart is one of the metabolic derangements remaining after ischemia, even in the myocardium bordering the infarct area. Normalizing lipid levels in the myocardium after ischemia would likely improve myocardial function and should therefore be considered as a target for treatment.Funding Agencies|Swedish Research CouncilSwedish Research CouncilEuropean Commission; Swedish Foundation for Strategic ResearchSwedish Foundation for Strategic Research; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation; Sahlgrenska University Hospital ALF; Novo Nordisk FondenNovo Nordisk Foundation [NNF13OC0004973] Funding Source: researchfish</p

Imaging of Intracellular and Extracellular ROS Levels in Atherosclerotic Mouse Aortas Ex Vivo: Effects of Lipid Lowering by Diet or Atorvastatin

Objective The first objective was to investigate if intracellular and extracellular levels of reactive oxygen species (ROS) within the mouse aorta increase before or after diet-induced lesion formation. The second objective was to investigate if intracellular and extracellular ROS correlates to cell composition in atherosclerotic lesions. The third objective was to investigate if intracellular and extracellular ROS levels within established atherosclerotic lesions can be reduced by lipid lowering by diet or atorvastatin. To address our objectives, we established a new imaging technique to visualize and quantify intracellular and extracellular ROS levels within intact mouse aortas ex vivo. Using this technique, we found that intracellular, but not extracellular, ROS levels increased prior to lesion formation in mouse aortas. Both intracellular and extracellular ROS levels were increased in advanced lesions. Intracellular ROS correlated with lesion content of macrophages. Extracellular ROS correlated with lesion content of smooth muscle cells. The high levels of ROS in advanced lesions were reduced by 5 days high dose atorvastatin treatment but not by lipid lowering by diet. Atorvastatin treatment did not affect lesion inflammation (aortic arch mRNA levels of CXCL 1, ICAM-1, MCP-1, TNF-alpha, VCAM, IL-6, and IL-1 beta) or cellular composition (smooth muscle cell, macrophage, and T-cell content). Aortic levels of intracellular ROS increase prior to lesion formation and may be important in initiation of atherosclerosis. Our results suggest that within lesions, macrophages produce mainly intracellular ROS whereas smooth muscle cells produce extracellular ROS. Short term atorvastatin treatment, but not lipid lowering by diet, decreases ROS levels within established advanced lesions; this may help explain the lesion stabilizing and anti-inflammatory effects of long term statin treatment

Lipid classes in human tissue sections from normal kidney tissue and clear-cell RCC tissue (CCRCC).

<p>All values are nmol/mg. Data are mean ± SEM.</p

VLDL-R overexpression in clear-cell RCC cells is mediated by HIF-1α, and promotes increased lipid accumulation through increased lipid uptake.

<p>(<b>A</b>) Quantification of VLDL-R mRNA normalized to 18S mRNA from cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue treated with siRNA against HIF-1α or VLDL-R (<i>n</i> = 10, *<i>p</i>≤0.05 vs. control siRNA normal cells, †<i>p</i>≤0.05 vs. control siRNA clear-cell RCC cells). (<b>B</b>) Quantification of immunoblot against VLDL-R with β-actin as loading control from cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue treated with siRNA against HIF-1α or VLDL-R (<i>n</i> = 10, *<i>p</i>≤0.05 vs. control siRNA normal cells, †<i>p</i>≤0.05 vs. control siRNA clear-cell RCC cells). (<b>C</b>) Quantification of Oil Red O staining of cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue treated with siRNA against HIF-1α or VLDL-R (<i>n</i> = 10, *<i>p</i>≤0.001 vs. control siRNA normal cells, †<i>p</i>≤0.05 vs. control siRNA clear-cell RCC cells). (<b>D</b>) Quantification of fluorescently internalized DiI-labeled lipoproteins in cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue treated with siRNA against HIF-1α or VLDL-R (<i>n</i> = 5, *<i>p</i>≤0.05 vs. control siRNA normal cells, †<i>p</i>≤0.05 vs. control siRNA clear-cell RCC cells). Data are shown as mean ± SEM.</p

VLDL-R expression is increased in clear-cell RCC.

<p>(<b>A</b>) Oil Red O staining of human tissue sections from normal kidney tissue (upper) and clear-cell RCC tissue (CCRCC; lower). (<b>B</b>) VLDL-R immunostaining (left) and quantification of immunostaining (right) of human tissue sections from normal kidney tissue (upper) and clear-cell RCC tissue (lower) (<i>n</i> = 6, *<i>p</i> = 0.0022). (<b>C</b>) Oil Red O staining (left) and quantification of staining (right) of cultured human cells isolated from healthy kidney tissue (upper) and clear-cell RCC tissue (lower) (<i>n</i> = 10, *<i>p</i> = 0.0058). (<b>D</b>) Quantification of VLDL-R mRNA normalized to 18S mRNA from cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue (<i>n</i> = 6, *<i>p</i> = 0.004). (<b>E</b>) Quantification of immunoblot against VLDL-R with β-actin as loading control from cultured human cells isolated from healthy kidney tissue and clear-cell RCC tissue (<i>n</i> = 6, *<i>p</i> = 0.0002). Data are shown as mean ± SEM.</p